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I.AdviceandGuidelines......................................................................................................................61.RotationStudentInformation......................................................................................................7Expectations.................................................................................................................................................................7BasicInformation.......................................................................................................................................................7ToDoinYourFirstWeek........................................................................................................................................8FindingThings.............................................................................................................................................................8LabCitizenship............................................................................................................................................................8RecordKeeping...........................................................................................................................................................9BeforeYouLeave........................................................................................................................................................9DBBSRotationEvaluationForm.........................................................................................................................9

2.NewEmployeeChecklist...............................................................................................................103.HowtoKeepaGoodLabNotebook...........................................................................................114.LabMeeting.......................................................................................................................................12WhatisLabMeetingFor?....................................................................................................................................12GeneralSuggestions...............................................................................................................................................12OutlineforaTypicalLabMeeting:..................................................................................................................12

5.LabJournalClub..............................................................................................................................136.LabAuthorshipPolicies................................................................................................................147.LabDatabase....................................................................................................................................15Oligos............................................................................................................................................................................15Plasmids......................................................................................................................................................................15Bacterial(andYeast)Strains.............................................................................................................................15Seeds.............................................................................................................................................................................16

8.SendingandReceivingDNAandSeedStocks........................................................................17PreparingPlasmidsforShipment....................................................................................................................17WhenYouReceiveaDNAStock........................................................................................................................17HowtoHandleRequestsforSeedStocks……………………………………………………………………18

9.Ordering.............................................................................................................................................19StandardLabItems(likeplasticware,gloves)...........................................................................................19CommonReagentsandSupplies(likechemicals,antibiotics).............................................................19SpecialtyItems(likeantibodies,cellulase,kits--thingsthatonlyonepersonuses)...................19Media............................................................................................................................................................................19GeneralPrinciples...................................................................................................................................................19SpecialNotes.............................................................................................................................................................20

10.LabJobs............................................................................................................................................21DutiesoftheLabSafetyOfficer.........................................................................................................................22

11.Dishwashing...................................................................................................................................23ForDishGenerators...............................................................................................................................................23FortheDishwasher................................................................................................................................................23ToSterilizePestles..................................................................................................................................................23

II.ProtocolsandProcedures...........................................................................................................24

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1.PlantGrowth.....................................................................................................................................25HandlingSeedBestPractices.............................................................................................................................26

2.AgrobacteriumCompetentCells&Transformation............................................................30COMPETENTCELLPREPARATION.................................................................................................................30TRANSFORMATION................................................................................................................................................30

3.ArabidopsisTransformation.......................................................................................................31NOTES..........................................................................................................................................................................31PROCEDURE..............................................................................................................................................................31

4.SelectionforBastaResistanceonSoil......................................................................................335.FindingHomozygousT3Plants..................................................................................................346.SeedSterilization............................................................................................................................35WETAPPROACH,about10minutes................................................................................................................35DRYAPPROACH,5-6hours.................................................................................................................................35

7.PlantDNAExtraction.....................................................................................................................368.PCRGenotypingforSALKLines..................................................................................................37

9.T-DNAGenotypingPrimerSelection........................................................................................3810.T-DNAGenotyping........................................................................................................................3911.GenotypingCommonAlleles.....................................................................................................4312.E.coliCompetentCells................................................................................................................44E.coliElectrocompetemtCells..........................................................................................................................45

13.RestrictionEnzymeDigest........................................................................................................4614.E.coliHeatShockTransformation..........................................................................................47

15.MembraneProteinIsolation.....................................................................................................48E.coliElectrocompetemtCells..........................................................................................................................49

16.ProteinExpressionviaFluorescenceImaging...................................................................50

17.StandardPCR.................................................................................................................................51

18.FusionPCR......................................................................................................................................5219.GelPurificationofDNAFragments.........................................................................................5320.In-FusionCloning..........................................................................................................................5421.Site-DirectedMutagenesis.........................................................................................................5622.ColonyPCR......................................................................................................................................58

23.GatewayCloning...........................................................................................................................5924.PlasmidPrep/GelExtraction/PCRcleanup(EpochLifeScience)................................60

25.ArchivingPlasmidsasDNA&BacterialStocks..................................................................63PREPARATIONOFDNASTOCKS.......................................................................................................................63PREPARATIONOFGLYCEROLSTOCKS..........................................................................................................63

26.DNASequencing............................................................................................................................6527.RNAExtractionwithTRIZOLReagent...................................................................................66

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28.Semi-QuantitativeRT-PCR.........................................................................................................6729.QuantitativeReverse-TranscriptasePCR.............................................................................68EXAMPLE....................................................................................................................................................................71

30.Mating-BasedYeastTwoHybrid.............................................................................................73MAKINGCOMPETENTCELLS............................................................................................................................73TRANSFORMATION................................................................................................................................................73MATING.......................................................................................................................................................................74INTERACTIONGROWTHTEST..........................................................................................................................75RECIPES.......................................................................................................................................................................75

31.SDS-PAGE.........................................................................................................................................7732.CoomassieStain............................................................................................................................8033.WesternBlotting...........................................................................................................................83STRIPPINGANDRE-PROBINGAWESTERNBLOT...................................................................................84

34.ProtoplastIsolation.....................................................................................................................85SpheroplastPreparation......................................................................................................................................86

35.ChloroplastIsolation&Immunostaining.............................................................................8736.AgrobacteriumInfiltrationofTobaccoLeaves...................................................................8937.BimolecularFluorescenceComplementation....................................................................9138.DNAParticleBombardment.....................................................................................................9339.ViabilityStaininginTobacco....................................................................................................9640.HechtianStrandProduction.....................................................................................................9741.GUSReporterStaining................................................................................................................9842.ProlineQuantification................................................................................................................9943.ABAGerminationAssay...........................................................................................................10244.SugarExtractionfromPlantCells........................................................................................10445.PollenViabilityAssay...............................................................................................................10646.InVivoPollenHydrationAssay............................................................................................10847.PollenGermination...................................................................................................................11048.XenopuslaevisOocyteRetrievalandInjection................................................................111EXPERIMENTALDESIGN..................................................................................................................................111TOBRINGTOANIMALFACILITY..................................................................................................................111SURGERY..................................................................................................................................................................111OOCYTEPREPARATION....................................................................................................................................112RNAPREPARATION............................................................................................................................................113OOCYTEINJECTION............................................................................................................................................113

49.PurificationofTaqPolymerase............................................................................................11650.HypoosmoticShockAssay......................................................................................................11851.PharmacologicalPolarizing/DepolarizingTreatmentsandDetectionwithaVoltageSensitiveDyeinArabidopsisRoots............................................................................................121

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51.ConfocalLaserScanningMicroscopy..................................................................................123TURNINGONTHEMICROSCOPE...................................................................................................................123VIEWINGSAMPLEWITHTRANSMITTEDLIGHT..................................................................................124EPIFLUORESCENCEIMAGING........................................................................................................................125CONFOCALIMAGING..........................................................................................................................................126XYIMAGEACQUISITION...................................................................................................................................130Z-SERIESOR3-DSTACKACQUISITION......................................................................................................131ADDINGASCALEBAR........................................................................................................................................131IMAGEANALYSIS..................................................................................................................................................132SAVINGANDVIEWINGFINALIMAGES......................................................................................................135CONFOCALMICROSCOPEQUICKREFERENCEGUIDE.........................................................................136

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I.AdviceandGuidelines

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1.RotationStudentInformationExpectationsAlaboratoryrotationisachanceforyoutocheckoutaparticularresearchgroupfromtheinsideandseewhetheritispersonallyandintellectuallyagoodmatch.Youmaywanttokeepinmindthatitisalsoachanceforthatgrouptogettoknowyouandtoevaluateyouasapossiblefuturememberof the lab. Anevaluation form filledoutby theadvisorat theendof the rotation isattachedbelow.--Itisnotimportantthatyoucompleteyourprojectduringyourrotation;thetimeisusuallytooshort formuch to be accomplished. However, you should be serious aboutmoving thingsforwardanddedicateenoughofyourtimetotheprojecttogiveanhonesteffort.

--In addition to an introduction to the techniquesused in the lab, the rotation isdesigned tointroduceyoutoourfieldofresearch,thelargerquestionsthatweareinterestedin,andtotheworkbeingdonebyotherlabmembers.Youshouldattendandbeengagedinlabmeetings,andshouldreadanypapersthatarerelevanttoyourproject.

--Thelabisaplaceforlabwork;itisnotaplacetodohomeworkorsurftheweb.Ifyouneedtostudyorwishtoplayontheinternet,pleaseusethestudentofficeortheBiologylibrary.

--Attheendofyourrotation,wewillscheduleatimeforyoutogiveatalkaboutyourproject.Please explain the question or questions youwere asking in your researchproject and theapproachusedtoaddressthem.Describethemethodsusedandtheresultsobtained.Proposeaseriesoffutureexperimentsandexplainnewquestionsposedbyyourresults.

BasicInformation1. ThemainHaswellLabincludesMcDonnell247,248,249,and250.Wealsouseseveralrooms

onthefirstandbasementfloorsofMcDonnell.2. Thephonenumberis935-96343. Liz’sofficeisMcDonnell221andthephonenumberis935-92234. Currentlabmembersare:

ElizabethHaswell,P.I.GrigoryMaksaev,PostdocDebaratiBasu,PostdocIvanRadin,PostdocYangbingWang,PostdocEricSchultz,PostdocEricHamilton,GraduateStudentAngelaSchlegel,GraduateStudentMattMixdorf,TechnicianRyanRichardson,Technician

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ToDoinYourFirstWeek1. Obtainkeysandalocker,ifyouwish.2. ReviewtheHaswellLabEmergencyProceduresfoundintheEnvironmentalHealth&Safety

binder,locatedabovethelabcomputer.ThedocumentisinSection2onthebluepages.3. PutacopyofyourEH&SsafetycertificateintheEH&Sbinder.

FindingThings1. Recipes–next to thechemical shelves.Thisbindercontains instructions for reagentsand

solutionsusedfrequentlyintheHaswellLabthatcanbecopiedforyourownbinder.Addnewrecipesasneeded.

2. HaswellLabProtocols-bookshelf.Makeyourowncopiesandfeelfreetoaddnotesifyouimproveaprotocol.Ifyoudevelopanewprotocolhere,pleaseaddittothebinder.

3. ReferenceBooks–bookshelf.BooksonMolecularBiology,Arabidopsis,andsoon.Thereisalsoabookentitled“AttheBench”whichcoversalotofthebasicsaboutbenchwork.

4. FreezerandRefrigeratorInventories-bookshelf.Beforeyouordersomething,lookheretoseeifwehaveit.Alsolookatthelistsonthefrontofthe-20.

5. Officesupplies,notebooks,and3-RingBinders–nexttothelabcomputer.

LabCitizenship1. Whenyouuseasupplythatisalmostgone,findouthowtoordermore.Donotputanempty

oralmostemptycontainerofanythingbackontheshelf.2. Ifyousetatimeronyourbenchdonotwalkaway.Ifyouhavetoleave,takethetimerwith

you.3. Thelabcomputerisshared;remembertologoffwhenyouarefinishedandtokeepyourfiles

organizedinyourownfolder.4. Muchofourdataaredigital images, and it isparticularly important tokeepcopiesof the

primarydatawithoutanymanipulation.Itismuchbettertotakeanotherpicturewiththecorrectgain,orrunanothergelwiththerightlanesthantofixthingsupinPhotoshop.

RecordKeeping1. Yournotebookandtheprimarydatathatyougeneratearethepropertyofthelab,andmust

notleaveMcDonnellHall.2. Wekeepboundnotebooks,withacross-referenced3-ringbinderthatisdesignedtoholddata

thatisnoteasilypastedintothenotebook,protocols,recipes,andanyotherinformationthatshouldbeassociatedwithyourboundnotebook.

3. Pleasewritewithapen,anddoallofyourcalculationsintheboundnotebook,notonloosepaper.Thisnotebookshouldalsocontainabriefdescriptionofyourprojectandconclusionsfromeachexperiment.Moredetailsaboutkeepingagoodlabnotebookareattached.

BeforeYouLeave1.Freezedownanystrains/plasmidsthatyou’vegeneratedandaddthemtothelabdatabase.2.Collectanyseed,discardplants.3.Discardordonatebuffersandsolutions.

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DBBSRotationEvaluationFormPhDPlantBiologyProgramDateStudent: RotationAdvisor:DateEnteredProgram: DateStartedRotation:Pleaseratestudentinthefollowingareasusing0through10points(0=lowest,5=average,10=highestperformance) Timeinlaboratory Abilitytobudgettimeeffectively Graspandapplicationofscientificprinciplesinpractice Readinganduseoftheliteratureinsolvingproblems Designofexperiments Evaluationofexperimentaldata Oralcommunicationability Writingability Generalattitudeandabilitytogetalongwithothers AttendanceandparticipationinjournalclubsPleasecommentonanyotherareasyoufeelarerelevantandexplainanylowgradesgivenabove.Pleaseindicatewhenstudentwillfinishthisrotation:ReturntoDBBS-Data@msnotes.wustl.edu,fax#362-3369orcampusbox8226.

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2.NewEmployeeChecklist¨ MeetwithHumanResources. KarenAlmstedt (salaried)or IvanaMedich (hourly) in312Rebstockwillhelpyoufillouttaxformsandbenefitformsandobtainanemployeenumber.MakeanoteonyourcalendarforyourHRorientationifyouwishtoattend.¨ GetanIDcardfromtheCCSin002Women’sBuilding.You’llneedyouremployeeIDnumberforthis.Allow2daysforyournameandnumbertobeinthesystemafterfillingoutformswithHR.¨ Getkeystothelab(B118E),sharedequipmentrooms(B94E),lunchroom(B191E),studentoffice (B185E), and theGreenhouse (B10E). Fill out a ‘key request’ form (found in the “LabOperations”binderabovethe labcomputers)andhave itsignedbyLiz(McDonnellkeys)andMikeDyer (Greenhouse key). Take the form to Gerry Rohde in the Biology Stockroom, 133Rebstock.NotethatyouwillalsoneedanIDcardforGreenhouseaccessafter5pm,andtoenteranyBiologybuildingafterabout6pmandontheweekends.¨ Getane-mailaddressandEthernetcable(ifneeded)fromFrancesThuet.Sendane-mailtoheratthuet@biology.wustl.eduandshewillsendyoualogin,passwordandinstructions.Thereisawirelessrouterlocatedabovethedesknexttotheseedbenches;thepasswordisindicatedontherouter.¨ AccessyourWashUaccountonline.CalltheHRMShelpdeskat5-5707togetatemporarypassword. You’llneedyouremployeeIDnumberwiththispasswordtoenterthesystemandaccessemployeeservices.YoucanusethesystemtotransferpointstoyourIDcardforuseinthefoodservices,registerforametropass,seeyourpaycheck,etc.¨ LookthroughtheblueEH&Smanualinthebinnexttothedishwashingsinkandfollowtheinstructionsinthefrontofthebinder(readthematerial,taketheexam,sign&datethetrainingrecord). Undergraduates access the online exam at http://ehs.wustl.edu/resources/EHSDocuments/Breeze_login_instructions.pdfanduseyourWUSTLkeytosignin.¨ Findaboundnotebookanda3-ringbinderinthedrawersunderthelabcomputers,andlabelbothwithyourname.Doallofyourworkinyournotebookandwritewithapen.Thebinderisforsupplementalmaterialordatathatdoesn’tfitinyourboundnotebook.¨ Findofficesuppliesinthetopdrawerofroom250.YoucanalsofindsuppliesintheBiologyStockroomin133RebstockorordersuppliesthroughStaples.¨ Checkoutthelabwebsite(http://pages.wustl.edu/haswell)fornewsandthelabcalendar.

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3.HowtoKeepaGoodLabNotebookByRamDixitYourresearchnotebookisoneofthemostimportantpiecesofdocuments!Itisarepositoryofyour ideas, experimental strategies, snags, and successes. It traces every project’s path frominception to fruition and therefore it isessential that youmaintain an honest and completerecordofyourexperiments.Thiswillnotonlyhelpyouorganizeyourworkandtotroubleshootyourexperimentsandwriteyourpublications,butwillalsoserveasausefulguideforotherswhowillcontinueyourwork.Betternotebookkeepingpractices:1. Leave the first fewpagesof yournotebookblank so thatyou can createan index for thatnotebookwhenitisfilled.2. Haveacleardateandheadingforeachdayandexperiment.Datesandtitleswillhelpyouorganizeyourworkandkeeptrackofyourexperimentsinthelongterm.3. It’sagoodideatowritedownyourobjectivefortheexperiment,soyouandotherscanfigureoutwhyyoudidsomethingmonths/yearslater.4. Planyourexperimentinadvanceandwritedownyourplan.Doyouhaveeverythingyouneedforeverystepoftheexperiment?5. Writedownyourexpectedresultsfortheexperiment.6. Writedowneverydetailduringorassoonasyoucompleteyourexperiment.Nodetailistootrivial!!Neverassumethatyouwillremembersomethingyoudidandnotwrite itdown!Themoredetailedyournotesarethemoreeasilyyou’llbeabletotroubleshootyourexperiments.7. Ifyouarefollowingastandardlabprotocol,mentionitandbesuretospecificallynotedownanydeviationsfromit.8. Paste all pictures, graphs, etc. next to your notes for that experiment. Be sure toannotate/labelthemsotheytheinformationtheycontainisclear.9. Writedownyouranalysis/interpretationoftheresultsordata.Didtheexperimentworkasexpected?Ifnot,whataresomepossiblereasons?10. Writedownyourplanofattackincasetheexperimentneedstroubleshooting.Whatdoyouplantododifferentlyandwhy?11. Andfinally,clearlyindicatetheendoftheexperimentunderaparticularheading.Leaveanyblankspacebelowthatheadingemptyandstartthenextexperimentonafreshpage.

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4.LabMeetingWhatisLabMeetingFor?• Tolettherestofthelabknowwhatyouareworkingon.• ToletLizknowhowyou’vebeenspendingyourtime.• Toconvinceusofyourinterpretationofyourresults.• Togetfeedbackfromtherestofthelab.• Togethelptroubleshootingorbrainstorming.• Tohelpyouorganizeyourthoughtsaboutyourproject(s).• Toprovideawaytomakefiguresfromyourrawdata,whichwillbeusefulinfuturetalksandinmanuscriptpreparation.

• Tomotivateexperimentsbyprovidingadeadlineforreportingresults.• Topracticepresentingyourworkinoralformat.

GeneralSuggestions• Startpreparingyourslidesearly.Donotwaituntilthehourbeforelabmeetingtogetyourdataoffanothercomputer,scanagelimage,ortoputtheintroductiontogether.

• Graduatestudentsandpostdocsshouldplantotalkforatleast30minutes.Ifyoudon’thavemuchdata,youwillhavemoretimefortroubleshooting,brainstorming,ordiscussionoffuturework.

OutlineforaTypicalLabMeeting:1. Introduction. Don’tskimponthis; there issure tobesomeonenewwhodoesn’tknoworsomeoneoldwhodoesn’trememberwhatyouaredoing.Don’tworrythatotherswillbeboredinthissection!Youwanttoputyourworkintothecontextoftheentirefield,aswellasthelab.Letusknowwhatisknownalreadyaboutyourtopic.Itisimportanttoconcludebyindicatingthequestionsyouarehopingtoanswer,orthehypothesisyouaretestingwithyourresearch.2. Approach andMethods. Describe the experiments you are doingwith enoughdetail thatotherscanunderstandwhatyou’vedone.Pictures,flow-charts,andtextareallrecommended.Relevantinformationshouldbeincluded:Howmanyplantsdidyoulookat?WhatconditionsdidyouuseforPCR?HowlongdidyouinducewithIPTG?3.Results.ThisshouldincludeRAWDATAaswellascharts,figures,andimages.Ifyouonlyhaveresultsthatarenottoyourliking(negativeresults,uglypictures),theyshouldstillbeincludedinthispartofthetalk.Yourdatashouldbereadyforpublication:labeledproperlyandincludingscalebars,titles,andlegends.4.Discussion.Whatdoyourresultsmean?Whatconclusiondoyoudraw?Thisisnotalwaysobvioustoyouraudience,whohaven’tbeenthinkingabouttheprojectaslongasyouhave,sostateexplicitlywhatyourresultsindicate.5.FutureDirections.Describetheworkyouwillbedoinginthenextfewmonths.

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5.LabJournalClubLabjournalclubisdesignedtodoseveralthingsatonce:

1.Keepyouuptodateoncurrentliterature2.Introduceyoutoclassicpapers,newtopicsortechniques3.Practicecriticalevaluationofscientificliterature

Criticalreviewofscientificliteratureinvolveslookingatthedatapresentedinjournalarticlesandevaluatingthestrengthoftheevidencewhenitispresentedobjectively,apartfromtheopinionoftheauthorsofthearticles.Donotbesurprisedifyoudonotreachthesameconclusionsastheauthorsafterlookingcarefullyatthedata.Ifyouaregivingjournalclub,pleasesendoutaPDFofthepaperyouhavechosenat least24hoursinadvancetothelabgroup.FeelfreetorunitpastLiz,butthisisnotnecessary.Pleaseincorporateintoyourjournalclubthefollowing.Andpleasedonotcopyanythingbutthefiguresandthefigurelegendsfromthepaperitself,especiallytheconclusions.Background.Whatisthemainquestionaddressedbytheresearch,andhowdoesitfitintothecurrent literature? What is the hypothesis being tested? What are possible alternativehypotheses?Reviewofdata,panelbypanel.Doeseachanalysisperformedaddressthequestionsposed?Werethedatapresentedinanunderstandableandclearway?Dotheresultsruleoutorsupportanyalternativehypotheses?Whatwerethecontrolsandwereanyleftout?Articlecritique.Whatdo theauthorsclaimtheir resultsmean? Does thedatasupport theirconclusions?Havetheystretchedorover-interpretedtheresults,oraretheyconservativeintheirinterpretationofthefindings?Arethereanyalternativeexplanationsfortheresultsobtained?Futuredirections.Howwouldyouproceedifyouwerethedirectorof thisresearchproject?Whatexperimentswouldyoudotoextendtheresultsandtoadvanceknowledgeinthisfield?Canyouraisesomenewquestionsorthinkofexperimentsthattheycoulddotomaketheirpointmorestrongly?Ifyouareintheaudience,pleasereadthepaperinadvance,andcometolabmeetingwithatleastonequestionorcommentforthepresenter.Thequestioncanbeasimpleoneofclarification,orcanbemorecomplexregardingtheinterpretationofdata.

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6.LabAuthorshipPoliciesIdeally,thefirstauthorwritesthepaperandhandlesmuchofthesubmissionduties,includingfigurepreparation,letterstotheeditor,andpermissions.However,thesedutiesareoftensharedbyLiz.ThecorrespondingauthorisalmostalwaysLiz.Anexceptionmightapplywhenapostdocknowsthemostaboutthepaper,oristakingmostofthereagentstohisorherownlab.Ifafirstauthorleavesthelabbeforeapaperiscompletelywritten,thesecondauthorisofferedfirstauthorshipdependentonwritingthepaper,andactingasfirstauthorforallnecessaryfirstauthorduties.Whatissufficientforauthorship?Thisneedstobediscussedforeachpaperindependently,butone of the following contributions are most likely required for authorship by postdocs orgraduatestudents:• Independentlydesigninganexperimentincludedinthepaper• Producingafigureforthepaper• Writingpartofthepaper• ProducingareagentorassayessentialtotheresearchinthepaperThese rules don’t apply to technicians, as theywork full time tomake reagents and providesupportforprojects.Techniciansareauthorsonanypaperswheretheycontributedresearchtotheproject,whetherornottheyproduceda figureor independentlydesignedanexperiment.Undergraduatesorrotationstudentsmightbeexceptionsaswell,iftheydidsomegroundworkthatlaidthefoundationforfurtherresearch,thoughtheiroriginaldatadoesn’tmakeitintothefinalfigures.

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7.LabDatabaseAllthesedatabasesarekeptinFilemakerProonthelabcomputer.Oligos

Whenyoureceiveanoligo,pleaseenteritintotheoligodatabase.Giveitaninformativenameand carefully type in the sequence. Add any important notes about the sequence (what is itdesignedfor,doesitaddrestrictionsites,pointmutations,etc.).TheprogramwillautomaticallyassigntheoligoaLHO(LizHaswellOligo)number.

WritetheLHOnumberatthetopoftheinformationsheetthatcamewithyouroligoandaddthesheettotheoligo3-ringbinderfoundinthebookcaseacrossfromthe37oCincubator.

Lookattheinformationsheettofindthetotalnumberofpmolssynthesized,andresuspendyouroligosinsterilewatertoaconcentrationof100pmol/µl.Theyshoulddissolvequicklywithavortex.LabelthetopofthetubewiththeLHOnumberandplacethestockinthe-20oCintheappropriatebox.A1:10dilutionofthisstockwithwaterorTE(to10pmol/µl)istheworkingdilutionforPCRandsequencing.

Plasmids

DetailedmapsandsequenceofplasmidsthataremadeinthelabarekeptinVectorNTI.TomakeaDNAmolecule,importsequencefromApeoratextfile.Addfeatures(genes,promoters,etc.byclickingonthe“AddFeature”button(6thfromleftonbottomtoolbar).Besuretousethemanysubcloningtoolsincludingprimeranalysis,sequencealignment,readingABIfiles,andsoon. Once you’ve finished the clone, sequenced it, and are ready to enter it into the plasmiddatabase,followthenotesbelowandgetaLHPnumber.YoucanaddthistotheVectorNTIfilebyclickingon“generaldescriptionfolder”,then“userfields”.

Whenyougenerateaplasmidconstruct,receiveaplasmidfromanotherlab,orpurchaseonefromavendor,youmustenteritintotheplasmiddatabase.Addanyimportantnotesabouttheplasmid(whatisitdesignedfor,whatgenesareencoded,whatistheselectablemarker)intothedatabasefile.TheprogramwillassigntheplasmidaLHP(LizHaswellPlasmid)number.IfthereisnomapinVectorNTI,pleaseattacharestrictionmapfromanothersource.OnceyouhaveaLHPnumber,pleasedotwothings:1. PutastockofDNAintheDNAstockboxinthe-20oC.2. MakeaglycerolstockandputitintheLHPglycerolstockboxinthe-80oC.Aprotocolfordoingbothofthesecanbefoundintheprotocolsection.Bacterial(andYeast)Strains

Bacterial strains (not DH5a carrying different plasmids, but different strains of bacteria,includingbothE.coliandAgrobacterium)arekeptinthebacterialstraindatabase.EnteralltherelevantinformationintotheFilemakerprogram,getanLHBnumber,andfreezedownaglycerolstock.

Seeds

Stocksofanyseedswegetfromotherlaboratories,fromtheSALK,andanypublishedlinesgenerated in the labmust be backed up in the lab seed database. Enter all the relevant

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informationintotheFilemakerprogram,giveyourseedanumber,andplaceitinaneppendorftubeinthecardboardboxesintheseedcabinet.ClearlylabelthetubewiththeLHSnumberanddatethattheseedwascollected.Ifpossible,pleaseincludeco-growncontrols(ifwildtype,thesedonotneedtheirownLHSnumber).Includeatleast50µlofhealthy,dryseed;ifyouneedtobulkuptoprovidethismuch,pleasedoso.

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8.SendingandReceivingDNAandSeedStocksPreparingPlasmidsforShipmentUsingsteriletechniqueandsterilesupplies:1. Drawacircleonapieceofsterilewhatmanpaperwithapencil.2. Place~500ngofplasmidin1-2uLofTEinthecenterofthecircleandletdry.3. Placethecircleinasterileeppendorftubeorwrapinplasticwrap/aluminumfoilandmail

withacopyofBiotechniques(1990)8:509(extracopiesareinfilecabinet,MCD247).WhenYouReceiveaDNAStock1. TransformaportionoftheDNAyoureceivedintotheproperE.colistrain(checkthis!Gatewaydestinationvectorsmustbepropagated inDB3.1,whileothersare find inDH5aor inNEBTurbo)andsavetherestinyourfreezerbox,incasesomethinggoeswrong.

2. As soon as is reasonable, prepare a lab DNA stock and a lab glycerol stock and enter theplasmidintothePlasmidDatabase.Includeamap,informationabouttheproviderofthestock,andreferencestothestockintheliterature.

HowtoHandleRequestsforSeedStocksUponreceivingseedrequestsfromanylaborindividualoutsideourgroup,atearliestconvenience,gatherseedandship.

• AllseedthatissentshouldbefromtakenfromtheverifiedLizHaswellco-growndatabaseformainlinesfromourlab.OtherlinesshouldbetakenfromtheLizHaswellseeddatabase.

• Noseedmaybesentorsharedwithotherlabsifithasnotbeengenotypedandcomesfromlabstocks.DONOTsendorshareyourprivatestocks.

• Addseedtoeppendorftubesandwraplidswithparafilmtotightlyseal.• Putinsideofabubblewrappedenvelopeandtapetoensurethatadhesiveof

envelopedoesn’topenduringshipping• ShipmentsarebestdonethroughFedexatthestockroom,butshort-distance

shipments(instate)canalsobedonewiththeUSpostalservicesentfromthedepartmentalmailroom.

• Assoonasarequestcomesin,recordallpertinentinformationinthelaboratoryseedrequestsspreadsheet,foundintheLabGoogleDrivefolder,.Whenyousendtheseed,emailtherecipients,makingsuretocc:Liz.

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9.Ordering StandardLabItems(likeplasticware,gloves)• Stocksarefoundontheshelvesacrossfromthegelbench.• ManyitemscanberestockedfromMcDonnell220(pipets,15and50mltubes,etc.).• OtheritemsarealwayskeptinMcDonnell220(Petriplates,multi-wellplates,cuvettes,etc.).• Someitemsarepurchasedfromthestockroom(gloves,1.5mltubes,PCRtubes,pipettips,

etc.).• EmmaisresponsibleforregularrestockingoftheshelvesandMcDonnell220.• However,YOUareresponsibleformonitoringthelevelsofourstocks. Ifyoutakethelast

item,pleasegetityourselffromMcDonnell220,runtothestockroom,ororderitimmediately.Ifyounoticethatwearerunninglowforanyitem,getitfromMcDonnell220,orwriteitupontheboardfoundonthe-20.

CommonReagentsandSupplies(likechemicals,antibiotics)• Emma is responsible forregularorderingofcommon labchemicals,except forstockroom

items.• However,YOUareresponsibleformonitoringthelevelsofcommonlyusedreagents,andfor

restocking items from the Biology stockroom. If you notice that we are running low onanything,pleasegetityourselforwriteitupontheboardonthe-20.Ifyoutakethelastitem,YOUMUSTgetityourselfortellEmmainpersontoorderit.

SpecialtyItems(likeantibodies,cellulase,kits--thingsthatonlyonepersonuses).• YOUareresponsiblefororderingspecialtyitems.Gregcanshowyouhowtoorder.Lizwill

needtosigntheorderformandenterthegrantcode.Media• Awork-studystudentisresponsibleformakingmoststockmedia.Ifyouneeditquickly,or

youaregoingtousealargevolume(morethan0.5L),makeityourself.Ifamediaisrunningloworyouwantsomethinginafewdays,writeitontheboardonthe-20sothestudentcanmakemorewhenheorshearrives.

GeneralPrinciples• Nevertakethelastofanythingwithouttakingsomeaction.• Forthatmatter,nevertakethesecond-to-lastofanythingwithouttakingsomeaction.• Pleasetakeyourownboxes/bagsofplasticwaretoyourbench.Allthesuppliesonthestock

shelvesshouldbeun-opened.• Takeyourownfrozenstockofantibiotics,IPTG,etc.• IfyouuseaLOTofanything,pleaserestockityourself.Emmaistheretokeepthingsrunning-

-butnottobeyourpersonalshopper.SpecialNotes• Whenyouorderoligos,pleaseemailtherestofthelabincaseyoucancombineorders,tosave

onshippingcosts.• WhenyouorderseedsorconstructsfromtheABRC,makesurethattheshippingandbilling

addressesareaswrittenonthegreenpurchasingsheetsweuseforothersupplies.

18

10.LabJobs

JOB DUTIES PERSON

1 Safety officer (biohazard, sharps, glass, and ethBr waste)

see lab manual Matt

2 Dish washer see instructions by sink Workstudy

3 Media making/Common Stocks/Sterile Beads

see recipes binder Ryan

4 Lab Reagents see recipes binder Ryan

5 Ordering and Supplies see lab manual Ryan

6 Pestle and Bead Sterilization

see instuctions on desposal container Matt

7 Minus 80oC Monitor

clean, keep racks organized, keep updated list of contents on the front, contact person for problems 5 racks labeled 1) LHP (plasmid stocks); 2) bacterial and yeast strain stocks 3) lcompetent cells and other lab stocks 4 and 5) personal stocks will need to oversee a lab-wide clean up for this one

Debarati

8 Minus 20oC Monitor clean, defrost occasionally, keep updated list of contents and assigned space on the front, monitor overuse of eppendorf racks

Debarati

9 Plasmid database, DNA and glycerol stocks

make sure that database is up to date, that maps are made, and that stocks are present for everything in the database. This will include bugging lab members when they leave.

Ivan

19

10 Bacterial, yeast strain, and oligo databases

make sure that database is up to date and stocks are present for everything in the databases. This will include bugging lab members when they leave.

Ryan

11 Seed database supervisor

need to start up a new database for seed, then stock from old seed and establish protocols for getting new seed in there (do we have a separate database just for SALK lines, or do we mix them in with ours etc)

Eric

12 Lab computer back up the lab computers on a monthly basis, contact Michael or Frances for help when trouble arise

Grigory

13 Lab party Motivator organize get-togethers for departing lab members, for post-quals and publication celebrations

Angela

14 Lab Meeting organization

check schedules of all, find a good time, schedule the talks and journal clubs, and reserve room each semester

Eric

15 Microscope room Monitor

keep area clean, bug people who leave crap around, monitor oil and lens paper levels, slides and coverslips move this stuff into microscope room from the lab and order bulk slides and coverslips to store in there--oversee confocal calendar

Ivan

16 Protocols, Lab Manual organize, oversee binding, update yearly, store online

Ryan

17 Common Lab Area Clean-up

Keep common stations cleaned, organized, and stocked. (gel loading, ph meter, weigh area, etc).

Ryan

18 Lab Chemical List keep online database current, check that things are up to date

Matt

19 Equipment Monitor Troubleshoot lab equipment that is malfunctioning/order replacements

Matt

20

20 Cold Room Monitor Keep area clean and bug people who leave a mess.

Matt

21 Restocking Shelves Keep shelves in MCD 248 stocked with tubes, gloves, etc.

Eric

22 Seeding Seed/Plasmind Send stocks requested by fellow researchers. Everyone

JOB DUTIES PERSON

1 Safety officer (biohazard, sharps, glass, and ethBr waste)

see lab manual Matt

2 Dish washer see instructions by sink Workstudy

3 Media making/Common Stocks/Sterile Beads

see recipes binder Ryan

4 Lab Reagents see recipes binder Ryan

5 Ordering and Supplies see lab manual Ryan

6 Pestle and Bead Sterilization

see instuctions on desposal container Matt

7 Minus 80oC Monitor

clean, keep racks organized, keep updated list of contents on the front, contact person for problems 5 racks labeled 1) LHP (plasmid stocks); 2) bacterial and yeast strain stocks 3) lcompetent cells and other lab stocks 4 and 5) personal stocks will need to oversee a lab-wide clean up for this one

Debarati

8 Minus 20oC Monitor clean, defrost occasionally, keep updated list of contents and assigned space on the front, monitor overuse of eppendorf racks

Debarati

21

9 Plasmid database, DNA and glycerol stocks

make sure that database is up to date, that maps are made, and that stocks are present for everything in the database. This will include bugging lab members when they leave.

Ivan

10 Bacterial, yeast strain, and oligo databases

make sure that database is up to date and stocks are present for everything in the databases. This will include bugging lab members when they leave.

Ryan

11 Seed database supervisor

need to start up a new database for seed, then stock from old seed and establish protocols for getting new seed in there (do we have a separate database just for SALK lines, or do we mix them in with ours etc)

Eric

12 Lab computer back up the lab computers on a monthly basis, contact Michael or Frances for help when trouble arise

Grigory

13 Lab party Motivator organize get-togethers for departing lab members, for post-quals and publication celebrations

Angela

14 Lab Meeting organization

check schedules of all, find a good time, schedule the talks and journal clubs, and reserve room each semester

Eric

15 Microscope room Monitor

keep area clean, bug people who leave crap around, monitor oil and lens paper levels, slides and coverslips move this stuff into microscope room from the lab and order bulk slides and coverslips to store in there--oversee confocal calendar

Ivan

16 Protocols, Lab Manual organize, oversee binding, update yearly, store online

Ryan

17 Common Lab Area Clean-up

Keep common stations cleaned, organized, and stocked. (gel loading, ph meter, weigh area, etc).

Ryan

22

18 Lab Chemical List keep online database current, check that things are up to date

Matt

19 Equipment Monitor Troubleshoot lab equipment that is malfunctioning/order replacements

Matt

20 Cold Room Monitor Keep area clean and bug people who leave a mess.

Matt

21 Restocking Shelves Keep shelves in MCD 248 stocked with tubes, gloves, etc.

Eric

22 Seeding Seed/Plasmind Send stocks requested by fellow researchers. Everyone

JOB DUTIES PERSON

1Safetyofficer(biohazard,sharps,glass,andEthBr

waste)seelabmanual Matt

2 Dishwasher seeinstructionsbysink Workstudy,Matt

3 Mediamaking/CommonStocks/SterileBeads seerecipesbinder Ryan

4 LabReagents seerecipesbinder Ryan

5 OrderingandSupplies seelabmanual Ryan

6 PestleandBeadSterilization seeinstuctionsondesposalcontainer Matt

7 Minus80oCMonitor

clean,keepracksorganized,keepupdatedlistofcontentsonthefront,contactpersonforproblems5rackslabeled1)LHP(plasmidstocks);2)bacterialandyeaststrainstocks3)

Debarati

23

lcompetentcellsandotherlabstocks4and5)personalstockswillneedto

overseealab-widecleanupforthisone

8 Minus20oCMonitor

clean,defrostoccasionally,keepupdatedlistofcontentsandassignedspaceonthefront,monitoroveruseofeppendorf

racks

Debarati

9 Plasmiddatabase,DNAandglycerolstocks

makesurethatdatabaseisuptodate,thatmapsaremade,andthatstocksarepresentforeverythinginthedatabase.Thiswillincludebugginglabmembers

whentheyleave.

Ivan

10 Bacterial,yeaststrain,andoligodatabases

makesurethatdatabaseisuptodateandstocksarepresentforeverythinginthedatabases.Thiswillincludebugging

labmemberswhentheyleave.

Ryan

11 Seeddatabasesupervisor

needtostartupanewdatabaseforseed,thenstockfromoldseedandestablishprotocolsforgettingnewseedinthere(dowehaveaseparatedatabasejustforSALKlines,ordowemixtheminwith

oursetc)

Eric

12 Labcomputerbackupthelabcomputersonamonthlybasis,contactMichaelorFrancesfor

helpwhentroubleariseGrigory

13 LabpartyMotivatororganizeget-togethersfordepartinglabmembers,forpost-qualsandpublication

celebrationsAngela

14 LabMeetingorganization

checkschedulesofall,findagoodtime,schedulethetalksandjournalclubs,and

reserveroomeachsemesterEric

15 MicroscoperoomMonitor

keepareaclean,bugpeoplewholeavecraparound,monitoroilandlenspaperlevels,slidesandcoverslipsmovethisstuffintomicroscoperoomfromthelabandorderbulkslidesandcoverslipstostoreinthere--overseeconfocalcalendar

Ivan

16 Protocols,LabManual organize,overseebinding,updateyearly,storeonline Ryan

24

17 CommonLabAreaClean-up

Keepcommonstationscleaned,organized,andstocked.(gelloading,ph

meter,weigharea,etc).Ryan

18 LabChemicalList keeponlinedatabasecurrent,checkthatthingsareuptodate Matt

19 EquipmentMonitor Troubleshootlabequipmentthatismalfunctioning/orderreplacements Matt

20 ColdRoomMonitor Keepareacleanandbugpeoplewholeaveamess. Matt

21 RestockingShelves KeepshelvesinMCD248stockedwithtubes,gloves,etc. Eric

22 SeedingSeed/Plasmind Sendstocksrequestedbyfellowresearchers. Everyone

DutiesoftheLabSafetyOfficer1.KeepEH&SnotebookuptodatewithrecentmemosfromEH&S.2.Eyewashinspectiononceamonth–signanddatetherecord.3.Fireextinguishersinspectiononceamonth–signanddatetherecord.4.Checkwastecontainersinthefumehoodsonceamonth.Iftheyaregettingclosetofullor

expiration(6months)thenscheduleapick-upusingtheonlinerequestform.5.Annualupdateoftheonlinechemicalinventorysystem(thisisanewdevelopment).6.Organizealab-specificsafetymeetingeveryfall,tocoordinatewitheveryone’sannualEH&S

training.Probablywe’lldothisonlabclean-upday.Thiscanbeonourownoralongwithsafetypersonnel.Shouldcoveranychangesinpolicyoverthelastyearandareviewofmostcritical safety concerns for the lab (ethidiumbromide, proper disposal, transgenic plants,bacteria,etc.).

7.Priortolabinspections(weusuallygetawarning),checktheInspectionFormandComment

Sheetinsection2,tomakesureweareincompliance.

25

8.Drop-offbiohazardouswastecontainer(groundfloorofpsychologybuilding,northentrance).Submittheweightofthewasteinthebinderbythescale.Bringanewbiohazardcontainerandbagbacktothelab.

26

11.DishwashingForDishGenerators--Pleaseremoveanytapeandrinseyourglasswareandspatulasbeforeplacinginthebin(alargeplasticbinthatisusuallyonthelargecarttotheleftofthedishwashingsink).Thinkaboutthedishwasher:s/hehasnoideawhatmighthavebeeninyourglasswareorstucktoyourspatula!

FortheDishwasher--UsetheAlconoxdetergentfoundunderthesink.Dilutethedetergent1:100(thatis,1gin100mlwater)intheorangesquirtbottle,thenuseafewsquirtsinyourdishwashingbucket.

--Rinsetheglasswarethreetimesindistilledwaterafterwashing.--Oncedry,glasswareshouldbeputawaypromptly;gethelpwiththisifnecessary.ToSterilizePestles1. Soakforafewhourstoovernightinbeakerof~0.1MHCl

Preferablyshakinggentlyatroomtemp2. WashinddH2O3. Autoclaveinsmallbeakers

27

II.ProtocolsandProcedures

28

1.PlantGrowthGENERALPOINTS• Trynottoleaveyourplantsinthelabovernight.Theshiftsintemperatureandlightlevelsare

verystressfultotheplantsandcancauseleafbleachingandinfertility.• Be careful about transferring plants from the McDonnell chambers to the Greenhouse

facilities.Wedon’twanttoberesponsibleforcontaminatingtheirchambers.• BeawareofthedifferentwateringrequirementsforMcDonnellchambers.Insome,yourtrays

willrequirewateringeveryotherday;inothersyouwillseemoldifyoudon’tletthesoildrywell.

Checkyourplantsonaregularbasis--optimallyeveryday.Youshould:• Monitorthegerminationandgeneralhealthofyourplants.• Thinseedlingsattheproperstage(usuallywhentheyhave4-6leavesisbest).• Lookatanydevelopingphenotypes.• Staketheplantsbeforetheyaretooentangled.• Movethemoutofthechambersoncetheybegintodrydown.• DONOTEXPECTTHEGREENHOUSESTAFFTOCAREASMUCHABOUTYOURPLANTSASYOU

DO!• Ifthereareanyproblems,reportthemimmediatelytoMikeDyerandtoLiz.PLANTINGPROCEDURE1. In the Greenhouse, select clean trays without holes in their bottoms. Fill pots with

“Arabidopsismix” andplace in the tray. (Alternatively,placea traywithholes intoa traywithout, holes, and fill the entire tray with soil). Pick out any large clumps of soil orvermiculiteonthesurface.Add2litersofwaterandwetthesurfaceofthesoil.Plantseedsonthesoilsurface,andifnecessary,markeachpotswithtapeoraplasticmarker.Labelthetraywithyourname,thedate,and“HaswellLab”,andcoverwithaplasticdome.Youmightalsowanttoaddaremindertoyourselfaboutthefinalpurposeoftheplants.Stratifyfor4daysinthecoldroom.

2. Fortransplantingseedlingsfromplates,prepareaflatasabove,makingsuretothoroughly

saturatethesoilwithwater.Removeseedlingsfromtheplates.Itcanbehelpfultousetheflatsideoftweezersto“mash”theagarbeforeremovingtheseedling—otherwisetherootmaysnapoff.Pokeholesinthemoistsoilandplantyourseedlings.Makesuretheentirerootisunder the soil and no air pockets are formed. Cover the flat with a dome and label“transplantedseedlings”.Afteraweek,oroncetheseedlingsaremakingnewleaves,removethedomeasdescribedbelow.

3. Putthetrayinagrowthchamber.Thereshouldbeonlyasmallamountofstandingwaterin

thetrayatthispoint.Aftertheseedshavegerminatedandtrueleavesarestartingtoshow(approx. 1 week), crack the dome for one day, then remove it. If your plants are in theGreenhousefacility,thestaffwillremovethedomeandtakecareofwateringandfertilizing.ForplantsintheMcDonnellchambers,allowtheflattoalmostdryoutbeforewateringwell

29

(shouldbeasmallamountofstandingwater)withwater+asmallamountofMiracleGro(watershouldbefaintlyblue).Youcanfertilizeeveryotherwateringifyoulike.

4. Stakeyourplantsassoonastheyaretallenough.Thisisimportantforyou,sothatyoucan

keeptheseedfromindependentlinesseparate,andalsokeepsyourplantsfromfloppingontoandcontaminatingotherflatsinthechamber.Youcanuselabtapeortwist-tiestoaffixthestems to wooden stakes. Be careful to collect inflorescences from only one plant duringstaking(unlessyouarepooling),becauseitwillbemuchhardertoseparateplantsaftertheyaredrieddown.Forinfertilelines(likemsl2-1;msl3-1),bagplantsassoonastheinflorescenceistallenough,sothattheyarelesssusceptibletocontaminationwithair-bornepollen.Youcanusefabricbagsorplasticcylinderstokeepplantsseparated.

5. Oncetheplantsarenolongermakingflowers,stopwatering.Onceafewsiliquesaredrying,

removethe tray fromthechamberandput it in thegreenhouseora labbenchtodryoutcompletelybeforecollectingseed.Donotallowplantstoshedseedinthegrowthchambers—thiscontaminatesotherflats,thechamberitself,andcostsusmoney.

6. Collectseedintoplastictubesorglassineenvelopes.Ifyouusetubes,punchaholeinthetop

withasyringeneedle.Arabidopsisseedsmustbestoreddry—anywaterwillpromotefungalgrowthandkilltheseeds.Payattentionasyouworksothatyoudonotcollectseedfrommorethanoneplant(unlessyouarepooling).Labelyourseedstockwiththegenotype,generation(F2,T3,etc.)andthedate(includingtheyear!).Drytheseed1weekbeforeplanting.

Greg Jensen

30

SeedHandlingBestPracticesTherearethreedifferenttypesofseedstocksthatexistinthelab:1)GeneralSeedDatabase,2)Co-GrownSetSeedDatabase,3)personalstocks.Eachofthesehasadifferentpurpose.GeneralSeedDatabase:1 Thisdatabasecontainslinesfromoutsidesources(i.e.ordersfromresourcecentersor

seedsfromotherlabs),backupstocksoflines,andotherrandomstocksthatweremaybemadebysomeoneinthelabthattheywanttokeepormakeavailablegenerally.It’sanicewaytoorganizeseedinthelab.

2 Informationaboutwherethesestocksoriginated(e.g.lab,ABRC)andpublicationsassociatedwiththemshouldbeincludedforeachentry.

3 Alllinestakenfromthisdatabaseshouldbeverifiedthroughgenotyping(orothermeanswhenappropriate)beforeperformingexperiments.

4 Donotuseupalloftheseedwithoutaplanofhowtoreplenishit.

5 Makepersonalstocksforyourselfforfutureexperiments(seebelow).Co-GrownSetSeedDatabase:

1 Thisisthedatabasewherepersonalstocksofcommonlyusedgeneticbackgroundsshouldoriginatefrom(e.g.MSLmutants).Itcontains“sets”ofco-grownstocksthathavebeenverifiedthroughgenotyping.Genotypeverificationisespeciallyimportantforbackgroundswithnovisiblephenotypes.Thegenotypinginformation,includingpicturesofgelsandadescriptionoftheprimersused,mustbeincludedwitheachentrywhenappropriate.Verificationmaybedifferentforothertypesoflines(overexpressionlines,RNAilines,visiblephenotypes,etc.).

2 Allgeneticbackgroundsthathavebeenpublishedonshouldbeaddedtothisdatabase.

3 Whensuchseedisrequestedfromotherlabs,sendasmallaliquotfromthesestocks.Lettherecipientknowthatthestockhasbeenverifiedandco-grown.

4 Whenonememberofasetisrunningloworthesetisgettingold(5-10years),itmustbereplacedwithanewset.Donotthrowouttheoldstock,justmakeanewentry.Replacingstocks:

1 Plantareasonablenumberofseedsforeachstock,dependingontheexpectedgerminationrate.Itisimportanttohaveenoughplants,butnotsomanythatyouwouldmisscontaminationinthepot.5plantsineachof2largepotsshouldbeenoughmostofthetime.YoumaywanttobulkuptwiceasmuchWTfromagivenset,asit

31

tendstobeusedthemost.Youcouldalsoputtheseedsonplatesandtransplantifgerminationrateisanissue.

2 Ifthisisanewsetandreliablegenotypinginformationisnotavailablefortheseedstockyouaretakingfrom,youmustgenotypetheseindividualsbeforecollectingseed.ThisisagoodideaincaseswhentheinformationISavailabletoo,asitcouldsavetimedowntheroad,butmaynotbestrictlynecessary.

3 Staketheplantswhentheyboltandaretallenough.Donotwaittoolong,asitbecomeseasiertomistaketheoriginofbranchesandendupwithcross-contamination.Ideally,plantswouldbestaked2-3timesbeforedryingandcollecting,astheywillcontinuetogrowandsendoutnewsidebranches.

4 Removeplantsfromchambersoncetheyseemtohavestoppedgrowing.Somesignsofthisincludetheyellowingandlossofrosetteleavesandsiliquesstartingtoyellow.Brown,dryplantsinchambersarejusttakingupspace.

5 Waitforthesiliquestoturncompletelybrownbeforecollectingtheseed.Ifthereisalot,youcancollectinto15mltubes.Youcanaliquottheseinto1.5mltubeslater.

6 Genotypetheseseedsforthedatabase.Putapproximately10seedsoutonplatesorsoilforgenotyping.Youcanpoolthesesamples.TestthisDNAforallallelesthatweregownintheset.Makethegelpicturesniceandclear.Thereisnoneedtowaitbetweencollectionandplantingifthesiliquesaredry,butyoucanwaitaweekorsoifyouwant.

7 Enterinformationintodatabase.Recordtherequiredinformationincludinggenotypingandthefulldate.Donotthrowoutanyseedleftoverfromtheoldset.Itcouldbeneededoneday.Personalseedstocks:

1 Theseareyourseeds.Ifyouneedtoreplenishthemandtheyaremutantsfoundinthedatabases(e.g.mslmutants),takeasmallaliquotfromthedatabasestocks.Donotpropagatefromyourownstocks.Thegoalhereistobeusingseedfromasfewgenerationsaspossibleforexperimentsinthelabaswellastoavoidmix-ups.

2 Neversendpersonalstocksunlessthatiswhatisbeingrequestedspecifically.

3 Ifyoumadeaparticularline,goodforyou!Consideraddingittothegeneraldatabaseifyouthinksomeonemaywantitoneday.

4 Ifyouareworkingwithsomethingthatwassentfromanoutsidesource,verifyitbeforedoinganyexperimentsandaddittothegeneraldatabase.Thiscansaveyoumonthsofheadache.

5 Usetheadvicegivenabovewhenbulkingupyourownseed.

32

2.AgrobacteriumCompetentCells&TransformationCOMPETENTCELLPREPARATION

1. FromaglycerolstockoftheAgrobacteriumstrainyouwanttousee.g.GV3101(LHS54),makeafreshstreakontoaLB-Rifampicin-Gentamycinplate.Incubateat28°Cfor2days.

2. Meanwhile,autoclaveanempty125mlflaskand500mlofLBina1-literflask.3. Preparestocksof20mMCaCl2and150mMNaCl,0.2µm-filter,andstoreat4°C.4. Choose4coloniesfromyourstreakplateandinoculate20mlofLB-Rif-Gentinthe125ml

flask.Shakeovernightat28°C.5. AddRif-Genttothe500mlofLBandinoculatewith5ml(1:100)oftheovernightculture.

Shakeat28°Cfor4-6hoursuntiltheO.D.reaches~0.5.6. Transferthecultureto4x250mlbottlesandspinfor15minutesat3000rpmand4°C.7. Decantpellets,chillcellsonicefor~10minutes.8. Resuspendpelletsinatotalof100mlof150mMNaClandcombineinasinglebottle.Spin

for15minutesat3000rpmand4°C.9. Resuspendpelletin10mlof20mMCaCl2.10. Makealiquotsin1.5mltubese.g.40x250µlor100x100µlorsomecombination.11. Freezein(N2)liquid.Storelong-termat-80°C.

TRANSFORMATION

1. ThawcompetentAgrobacteriumonice.Use100µlofcellsperDNAsample.2. Add1µgofDNApertube.Keeponicefor~30minutes.3. Freezein(N2)liquidfor1minute.4. Thawat37°Cforafewminutes.5. Add900µlofLBpertubeandshakeat28°Cfor1-2hours.6. Spinat10,000rpmfor2minutes.7. Decantmostofthevolume,resuspendintheremaining~100µl.8. Plateontotripleantibioticplates:LB-Rif-Gent+yourplasmidDNAselection.9. Incubateat28°Cfor2days.10. Expect100-200colonies.

Greg Jensen

33

3.ArabidopsisTransformation*Adaptedfrom“Floraldip:asimplifiedmethodforAgrobacterium-mediatedtransformationofArabidopsisthaliana,ThePlantJournal(1998)Vol16(6)pages735-743,StevenJ.CloughandAndrewF.Bent.”NOTES• Usegloves,protectyourbench,andcleanupwellwhenusingAgrobacterium.Agrobacterium

is capable of transforming human cells (PNAS 13:98(4):1871-6) 2001. GenetictransformationofHeLacellsbyAgrobacterium).

• ProtectyourbenchwithbenchpaperorunderpadsandautoclaveorbleachallmaterialsthatareincontactwithAgro.

• Aftertransformation,filtertheAgroandsoilthroughapapertowel-linedfunnelandintoaflaskcontainingbleach.Disposeoffilteredmaterialinthebiohazardouswastebin.

PROCEDURE1. GrowhealthyArabidopsisplantsuntiltheyareflowering.Typicallyoneflatperconstructis

transformed,thoughahalf-flatmaybesufficient.Ifyouareusingasemi-sterilelinesuchasmsl2-1;msl3-1youwilldefinitelywantanentireflat.

2. OPTIONAL:Oncetheboltshavereached5-10cminheight,clipthemtostimulatethegrowth

ofsecondaryinflorescences.Dothisoneweekbeforeyouwanttotransformthem.3. Prepare the Agrobacterium strain by transforming it with your plasmid and plating on

selectablemedia.TheGV3101strainiscurrentlyinvogue.Tubesofcompetentcellscanbefoundinthe-80°Cfreezer.Thisstrainshouldbegrownon50µg/mlRifampicinand30µg/mlGentamycin for the helper plasmids. A third antibiotic e.g. Kanamycin, Spectinomycin isrequiredforselectionofyourT-DNAplasmid.(Seepreviousprotocol).Dothis4daysbeforeyouwanttotransformplants.

4. Growa2mlovernightcultureofAgrobacteriumharboringyourplasmidat28°C inLB+3

antibiotics.5. Inoculatea500mlculturewith0.5mlofyourovernightculture(1:100)andgrowovernight

at28°CinLB+3antibiotics.6. SplittheAgrobacteriumculturebetween3x250mlcentrifugebottlesandspinfor15minutes

at3000rpmand4°C.7. Decantthesupernatantintoaflaskcontainingbleach.8. Prepare1literof5%sucrosesolutionperculture(1literissufficienttotransform30pots).

Add100mltoeachcentrifugebottleandletsitfor15-30minutes,periodicallyswirlingtoresuspendtheculture.

34

9. Combinethe300mlofresuspendedcultureinarectangularglassbakingdish.Add200µlofSilwet L-77 (0.02% final concentration) to the dish along with the remaining 700 ml ofsucrosesolution.

10. Dipthepotsintothedishacoupleatatime.Leaveinvertedforupto30seconds.Theplants

shouldbecompletelysaturated.11. Setthepotsontheirsides–onthecornersofthedish–todrain.12. Laythepotsontheirsidesinatraylinedwithpapertowels–5potspertray.13. Coverwithanemptytrayandleaveonthebenchoronacart–avoidleavingtheminbright

light.14. Crackthelidsafter24hrs.15. Removethelidsafter48hrsandsetthepotsupright. Recombinepotsto10pertrayand

returntochamber.16. Allowthesoiltodrywellbeforewateringforthefirsttime.

Greg Jensen

35

4.SelectionforBastaResistanceonSoil1. Fillflatswithsoilbyoneofthesemethods:

A.Chooseatraywithoutholes.Fill10potswithsoilandplaceinsidethetray.B.Chooseatraywithholesandplaceinsideatraywithoutholes.Filltheentiretraywith

soil.2. Waterflatsbymaking3-4passeswiththewatersprayer;thesoilshouldbefullywetted,but

toomuchwatermaycausemoldproblemsdowntheroad.3. Measureout1mlof(T1)seedfromyourtransformationsinto1.5mlmicrocentrifugetubes.4. Emptyatubeofseedontoafolded3”x5”card.Dispenseseedbygentlytappingthesideof

thecardasyoumoveacrosstheflatinasystematicmanner.5. Covertheflatswithclearplasticdomesandplaceinacoldroomfor4-5days.6. Transferflatstoa16-24hrlightchamberandgrowplantsfor1-2weeksuntilthefirsttrue

leavesappear.7. PrepareaworkingsolutionofBASTAinaspraybottlebydilutingFinale1:1000inwatere.g.

500µlFinaleinto500mlwater.8. Placeaflatofplantsintoasink.Weargloves.SprayBASTAliberallyacrosstheflat.Return

tochamber.9. Continueselectionbysprayingeveryotherdayforaweeke.g.Mon-Wed-Fri-Mon.Remember

tospraytheedgesoftheflat/pots.10. Asthesurvivorsdistinguishthemselvesoverthenext1-2weeks,givethemroomtogrowby

removinganynon-survivorsfromtheflat.11. Agoodselectionwillyield50-100survivorsperflat.

Liz Haswell

36

5.FindingHomozygousT3Plants

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#that#th

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Liz Haswell

37

6.SeedSterilizationWETAPPROACH,about10minutes--Solutions:

sterilewatersterile0.1%agarose50%bleachsolutionwithadropofTween-20

--Allinasterilehood,usingsteriletechnique:

1.Putseedsina1.7mltube.2.Add1mlbleach/Tweensolution.Vortex,andletstandnomorethan5minutes.Over-bleachingcankillseeds;notenoughwillnotsterilizesufficiently.3.Washwith1mlsterilewater3times.4.Add0.5mlsterileagaroseandresuspendseed.Dropontoplateswithapipetmanandsterilepipettip.5.Drytheplateforafewminuteswiththelidoff;thenaddthelidandwrapwithfiltertape.6.Incubatetheplatesinthefridge2days,thenputintheincubatortogrow.

DRYAPPROACH,5-6hours

1.Put100to500µlseedintoalabeledeppendorftube.(AVWRmarkerisbest).2.Placethetubeinarackinsideasealableplasticcontainerinthefumehood.3.Wearingappropriatesafetygear,add4mlconcentratedHClto100mlbleachina250mlglassbeakersetinsidethecontainer.4.Quicklysealthecontainerandletitsitinthehoodfor5-6hours.5.Ventinthefumehoodandclosethetubes.Nowtheyarereadytoplaceonplatesinthelaminarflowhood.

38

7.PlantDNAExtraction*DesignedforPCRanalysis*AsmallleafdiscissufficienttoisolategenomicDNA*Whenusinglesstissue(onecotyledon)allvolumesshouldbereducedby50%*BasedonthemethoddescribedbyEdwardsetal.(1991)THINGSTODOBEFORESTARTING

§ NeedextractionbufferandTE(pH7.5)PROCEDURE

1. Puthealthyleaftissueintoanepitube2. Usesmallpestletogrindleafmaterialintube3. Add300µlextractionbuffer

a. Finishgrindingb. Vortex5secc. Samplesmay be kept at room temp for <1 hour until all samples have been

extracted4. Centrifugeatfullspeedfor5mintopelletdebris5. Decantsupernatantintonew1.5epitube

a. Avoidtakingdebrisfrompellet6. Add300µlisopropanol

a. Mixandincubateatroomtempfor2min7. Centrifugeatfullspeedfor5mintopelletDNA

a. Decantsupernatantb. Drypellet(letsitupsidedownonpapertowelatroomtemp)

8. Add100µlTEa. Vortextodissolvepellet/scrapesidewithpipettetip

SOLUTIONSStocks ExtractionBuffer50mLof1MTris-Cl(ph7.5-8) 200mMTris-HCL(pH7.5-8)12.5mLof5MNaCl 250mMNaCl12.5mLof0.5MEDTA 250mMEDTA6.25mLof20%SDS 0.5%SDSH2Oupto250mLTE10mMTris(pH7.5)1mMEDTA

39

8.PCRGenotypingforSALKLinesEachReaction 2µL 10Xbuffer 0.4µL 10mMdNTPs 0.4µL Fprimer(10pmol/ul) 0.4µL Rprimer(10pmol/ul) 0.2µL Taq 2µL DNA 14.6µL H2O 20µL total Primerstouse MSL forWTallele formutantallele 4(At1g53470) 4.F1B,4.R1 Lba,4.R1 5(At3g14810) 5.F1,5.R4b Lba,5.R4b 6(At1g78610) 6.F1,6.R5 6.F1,Lba 9(At5g19520) 9.F3,9.R5 Lba,9.R5 10(At5g12080) 10.F1,10.R1 Lba,10.R1 4.F1B LHO672 caccatggccgttgattcaactgatc or4.F8 LHO1039 ggatcgacggtgaagacaatg 4.R1 LHO630 ctttaacagacggagaaagcgtg 5.F1 LHO631 atggcggctgtcgattcaac 5.R4b LHO741 ccatttccacaaatccagcttcc 6.F1 LHO633 gagagacggaagctacgatttttgg 6.R5 LHO789 gttcccagagtttcttcttacgcaag 9.F3 LHO696 caaggggttcttttgacagg 9.R5 LHO784 tccaagcaatcagaaccaga 10.F1 LHO623 atggcagaacaaaagagtagtaacg 10.R1 LHO634 cttactgcgcatctctctgttcag Lba LHO500 tggttcacgtagtgggccatcg PCRconditions 94degrees 5min 1X 94degrees 30sec 55degrees 30sec 30X 72degrees 1min 4degrees forever 1X

Kelsey Kropp

40

9.T-DNAGenotypingPrimerSelection*ToidentifyplantscontainingtheT-DNAinsertanddetermineT-DNAmutantgenotypesTHINGSTODOBEFORESTARTING1) LookuptheSalknumberontheSalksignalT-DNAexpresswebsite

a) http://signal.salk.edu/cgi-bin/tdnaexpress

2) InputSalknumberin“Query”(ex:SALK_003004)3) DeterminelocationanddirectionofT-DNAinsertingene

a) Thedirectionofthegene(green)isdesignatedbythegreenarrowb) Thedirectionoftheinsert(pink)isdesignatedbythepinkarrow

4) Clickontheappropriateinsertindicatedby3b,thenclickon“Vector”a) ThisgivesyouthenameofthevectorusedtoinserttheT-DNAsequenceintoyour

geneofinterestandwillbeusedtodeterminetheappropriateT-DNAspecificprimer(BP)instep7c

5) Decidewhichprimers touse that span the insert and theappropriateT-DNAspecificprimersa) http://signal.salk.edu/tdnaprimers.2.html

2

3b

3a

41

6) Insertsalknumberinsearchboxandclick“Submit”7) ProductlengthforWTprimersisindicated

a) WTLPandTMareindicated

b) WTRPandTMareindicated

c) T-DNAspecificprimersandapproximateproductlengthareindicated

i) note:lengthsvarydependingonthelengthfromtheinsertionsitetotheflankingsequence (designated “N”; 0-300bp difference); to calculate more specifically,downloadgenesequence,determineinsertlocation,andcalculatesequencesizebetweeninsertandRP

ii) thismeansusetheWTRPasindicatedin7bandaBP(referbacktosalksignalt-dnaprimerwebsitein5atodeterminethespecificBPtouse.

6

42

iii) BP=LB(1) Selecttheprimerthatcorrespondstothevectorinformationobtainedinstep

4a(2) generally,LBb1.3canbeused;ifusingLBa1insteadofLBb1.3theproductsize

willbe200bplargerthanapproximatedin7.c.ii.

Liz Haswell

43

10.T-DNAGenotyping*Theprotocolbelowisforthemsl2-1andmsl3-1alleles,butcanbeadaptedtoanyT-DNAinsertion.(SeequickreferencechartinProtocol#10,andhowtodesignyourownoligosinProtocol#8).jl-202jl-202 2.F4 3.F8MSL2MSL32.3’ 3.3’A.2.F4+2.3’ C.3.F8+3.3’WTgives~1kbproduct. WTgives~700bpproduct.Mutantgivesnoproduct Mutantgivesnoproduct.(inserttoolargetobemade (inserttoolargetobemadewithonly1minextension). withonly1minextension).B.JL-202+2.3’ D.JL-202+3.3’WTgivesnoproduct WTgivesnoproduct(thereisnoinsert). (thereisnoinsert). Mutantgives~1kbproduct. Mutantgives~700bpproduct PROCEDURE

1. ExtractDNAfromeachplanttogenotype(Seeprotocol#7above)

2. RunPCR/Gelonallplantsampleswithprimersdeterminedabove

3. DetermineifaWTcopyofgeneispresent

4. DetermineifT-DNAinsertispresenta. IfhitwithWTandnoT-DNA:HomozygousWTb. IfhitwithWTandT-DNA:Heterozygousc. IfnohitwithWTandhitwithT-DNA:HomozygousMutantd. *MaybeabletosetannealingtimelongertogetabandacrossentireopenT-

DNAinsert.PCRREACTIONPREPARATIONPrepare‘templatemix’for(n+2)reactionswhere‘n’isthetotalnumberofreactions.Prepare‘primermix’for(n+1)reactionswhere‘n’isthenumberofDNAtemplates.Templatemix:4µl5xbuffer(with10mMMgCl2)0.4µl10mMdNTPs

44

0.2µlTaqpolymerase9.4µlwater14µlPrimermix:2µl5µMforwardprimer:2.F4(1055)orJL-202(1056)2µl5µMreverseprimer:2.3’(1057)4µlEXAMPLEYouhave20DNAsamplestogenotype.Youhave2primersets(AandB).Thetotalnumberofreactionsis40.Prepareenoughtemplatemixfor42reactions.Prepareenoughofeachprimermixfor21templates. Aliquot½ofthetemplatemixtoeachoftheprimermixes.Aliquot18µlofthismastermixto0.2mltubes.Add2µlofDNAtemplateforatotalreactionvolumeof20µl.PCRprogram:94°C5min94°C30sec55°C30sec30cycles72°C30sec

Greg Jensen

45

11.GenotypingCommonAlleles

Gene WTprimer MUTprimer REVprimer T-annealing Ext.Time Enzyme

1 msl2-1 2.F4(1055) JL-202(1056) 2.3'(1057) 55 1min x wt=1kb mut=1kb

2 msl2-1 2.F4(1055) JL-202(1056) MS2g.3'(856) 55 1min15sec x (2g) wt=1140bp mut=1140bp

3 msl2-3 MSL2BF.Sal(852) LB-GABI(1059) msl2-R4(1110) 55 1min x wt=900bp mut=350bp

4 msl2-3 2g.5'(1077) LB-GABI(1059) msl2-R4(1110) 55 2min30sec x (2g) wt=2300bp mut=350bp

5 msl3-1 3.F8(1061) JL-202(1056) 3.3'(1058) 55 45sec x wt=700bp mut=700bp

6 FtsZKO AtFtsZ1-1.R(1119) LBb1.F(1120) AtFtsZ1-1.F(1118) 55 1min x wt=900bp mut=350bp

7 #6-4-2 At4g29830.F(1265) LBb1.F(1120) At4g29830.R(1266) 60 30sec x wt=400bp mut=500bp

8 SALK_083364 At4g29830.F2(1288) LBb1.F(1120) At4g29830.R2(1289) 55 30sec x wt=400bp mut=300bp

9 arc3 arc3DCAPS.F2(1121) x arc3DCAPS.R6(1176) *50/55 30sec EcoRV wt=290bp mut=270bp

10 arc11 arc11DCAPS.F(956) x arc11DCAPS.R(957) *50/55 30sec SphI wt=280bp mut=300bp

11 arc12 arc12DCAPS.F(1115) x arc12DCAPS.R(1116) *50/55 30sec NcoI wt=275bp mut=300bp

12 pgm-1 pgm.3(162) x pgm-11(186) 50 30secBsrI(65°)

wt=200bp+300bp mut=500bp

PairWTprimerwithREVprimerorMUTprimerwithREVprimer. Productsizesarelistedbeloweachprimer. *arcmutantgenotypingmayworkbetterusing5cyclesatthefirsttempfollowedby25cyclesatthesecondtemp.

46

12.E.coliCompetentCells*For50mlE.colicultureinZymoBrothorSOBmedium THINGSTODOBEFORESTARTING§ Grow0.5mlovernightE.coliLBculture(usedinstep1)§ Getice§ Pre-chill201.5mlsterilemicrofugetubes(usedinstep6)§ Preparebuffersandiceuntilreadytouse(usedinsteps3,4,&5)PROCEDURE

1. Ina500mlflask,inoculate50mlofZymoBrothorSOBwith0.5mlFRESH,overnightE.coliculturegrowninLB

a. Shakeat250rpmat28oCuntilO.D.reaches0.4-0.6(~4-6hours)

2. Prepare 5ml each of 1xwash buffer and 1x competent buffer by adding 2.5mldilutionbufferto2.5mlofthe2xstocks

a. Storebuffersoniceuntilreadytouse

3. Transferculturefromstep1toicea. Incubateonicefor10minb. Centrifugeat2,500xgfor10minat4oC

4. Removesupernatanta. Resuspendcellsin5mlice-cold1xwashbufferb. Centrifugeat2,500xgfor10minat4oC

5. Removesupernatanta. Resuspendcellsin5mlcold1xcompetentbuffer

6. Aliquot250µlofcellsinto20pre-chilled,1.5mlsterilemicrofugetubesa. CellsarereadyfortransformationwithDNAb. FreezeinliquidN2andstoreat-80oCforfutureuse

47

E.coliElectrocompetentCells THINGSTODOBEFORESTARTING§ Grow5mlovernightE.coliLBculture(usedinstep1)§ Getice§ Pre-chill501.5mlsterilemicrofugetubes(usedinstep6)§ Preparebuffersandiceuntilreadytouse(usedinsteps3,4,&5)PROCEDURE

1. Ina500mlflask,inoculate100mlofLBwith5mlFRESH,overnightE.coliculturegrowninLB

a. Shakeat250rpmat37oCuntilO.D.reaches0.4.

2. WhentheO.D.reaches0.4,rapidlytransferflasktoanice-waterbathfor15-30min.Swirlthecultureoccasionallytoensurecoolingoccursevenly.

3. Splitthecultureintotwo,50mLconicaltubes.Harvestthecellsbycentrifugationat1000gfor15min.at4C.Decantsupernatantandresuspendthepelletin100mLice-coldpurewater(50mLpertube).

4. Harvestthecellsbycentrifugationat1000gfor15min.at4C.Decantthesupernatantandresuspendin50mLice-cold10%glycerol(25mLpertube).

5. Harvestthecellsbycentrifugationat1000gfor15min.at4C.Decantthesupernatantandresuspendin20mLice-cold10%glycerol(10mLpertube).

6. Resuspendpelletin1mLice-coldGYTmedium.

7. Transfer 50 microliters to a chilled electroporation cuvette (1mm gap) and testwhetherarcingoccurs.Ifarcingoccurs,washtheremainderofcellsoncemorewithGYTmedium.

8. Aliquot100microlitersintoeachofthe50pre-chilledtubes.Flashfreezeinliquidnitrogenandstoreat-80C.

48

13.RestrictionEnzymeDigest

RestrictionenzymedigestiontakesadvantageofnaturallyoccurringenzymesthatcleaveDNAatspecificsequences.Digestsarecommonlyusedformolecularcloningorplasmiddiagnostics.AllrestrictionenzymescanbefoundinEnzymeBox#3inthe-20°CinMcDonnell249.WestockenzymesfromThermo,Promega,andNEB.Procedure

1) Selectarestrictionenzymethatwillcutyourplasmid.2) Determineanappropriatereactionbufferbyreadingtheinstructionsforyour

enzyme.(Thisinformationcanbefoundonmanufactureswebsite)

3) Ina0.2mlPCRtubecombine:• DNA• RestrictionEnzyme(s)• Buffer• BSAifneeded• H2Oto20µl

*TheamountofDNAthatyoucutdependsonyourapplication.Diagnosticdigeststypicallyinvolve~500ngofDNA,whilemolecularcloningoftenrequires1ugofDNA.*Atypicalreactionin20µLwilllooklike: 5µLDNA(500ng) 2µLBuffer 1µlRestrictionEnzyme 12µlH2O

4) Spindowntubesfor5seconds.

5) Incubateat37°Cfor30mintoovernightdependingonapplicationand

manufacturer’sinstructions.

6) Runreactiononanagarosegeltovisualizeresults.

49

14.E.coliHeatShockTransformation

CompetentstrainsofE.coliarelocatedinthe-80°CfreezerinMcDonnellroom105.IfwearerunninglowoncompetentcellspleasenotifyEmmaorGregsomorecanbemade.Procedure

1) Thawcompetentcelltubeonice.Everytubecontains~200µLofcells,use50µlto

100µlpertransformation.

2) Add1µLofDNAto50-100µLofE.coliandincubateonicefor10minutes.

3) HeatshockE.coliat42°Cfor1minute.

4) Placebackoniceforanadditional10minutes.

5) Add900µLofLB.

6) Incubatein37°Cshakerfor1hour.

7) Spindownbacterialpelletfor1minuteat15000rpm.

8) Decantsupernatant,leaving~100µLofLB.

9) Re-suspendpelletandspreadontoLBplatewithappropriateantibiotic.

10) Incubateplatesat37°Covernight.

50

15.MembraneProteinFractionation

Day1:-TransformBL21cellswithdesiredconstructcontaining6xHisTag.Day2:-Pick3coloniesoftransformedcellsandaddto3mLLB+3μLCarb.-GrowuntilOD600=0.5.-Add2mLOD600=0.5equivalentsto100mLLB+1mLCarb.-GrowuntilOD600=0.5.(Take2.5mLofuninducedfraction,spindown,resuspendin100μL2xSB.)-Induce with 100 mM IPTG for 90min. (Take 2.5 ml of induced fFraction, spin down,resuspendin100μL2xSB.)-Splittheremainingcultureevenlyintotwo50mLconicaltubes.-Spindownat2500xg,4oC,10min.Removesupernatantandstorepelletsat-80oC.Day3:-Thaw pellets on ice. Resuspend in 1x Solubilization buffer (dissolve 1 tablet proteaseinhibitorin10mLwaterusedtodiluteSolubilizationBuffer10xto1x)+1%Fos-Choline-14.-Addlysozyme(200μg/mLfinal),DNase(25μg/mLfinal),andPMSF(100μMfinal),increasefinalvolumeto1mL.-Rocktheresuspendedpelletinthe50mLconicaltubeat4Cfor60min.-Transfersolutionintoa1.7mLEppendorftubeandsonicatewithamicrotip(BlankenshipLab),oniceandinthecoldroom.Sonicationsettings:20%dutycycle,output=1,10secpulsefollowedby20secrestrepeated6timesforatotalof3minutes/sample.-Spinsamplesdowninthecoldroom:11,000rpm,4C,5min.-Transfer supernatant to a new tube. (Take 15μL supernatant and add 15μL 2x SB.Resuspendpelletin500μL2xSB).-Storeallfractionsat-80C.-Runallfractionson10%SDS-PAGEgels,75Vfor2handproceedtoCoomassiestaining: Uninducedfraction–load10μL,0.25%startingmaterial Inducedfraction–load10μL,0.25%startingmaterial Supernatant–load5μL,0.5%startingmaterial Pellet–load2.5μL,0.5%startingmaterial10xSolubilizationBuffer(SB?)200mMTris,pH7.5300mMImidazole,pH7.5200mMNaCl40mMMgCl2

51

HisPurNi-NTAColumnProteinPurification

-RemovebottomtabsfromHisPurcolumnsandplacecolumninemptyEppendorftube.-Spinat700xgfor2mintoremovestoragebuffer.-Equilibratecolumnresinwith0.5mLequilibrationbuffer.-Load0.5mLsupernatant(fromisolationprotocol)ontothecolumn.Capbothendsofthecolumnandrockat4Cfor30min.-Placecolumnintoa2mL“flowthru”tubeandspinat700xgfor2mintoremoveflowthrufromcolumn.-Washthecolumnwith0.5mLwashbuffer.Spinat700xgfor2mintocollectwashes.Collectatotalof3washfractions.-Elutetheproteinwith100μLelutionbuffer.Spinat700xgfor2mintocolleteluate.Collectatotalof5elutionfractions.-Runallfractionsona10%SDS-PAGEgel,75vfor2handproceedtoCoomassiestaining:-Load5μL(+5μL2xSB)offlowthruandwashfractions,1%startingmaterial.-Load1μL(+1μL2xSb)ofelutionfractions,1%startingmaterial.EquilibrationBuffer20mMTris,pH7.530mMImidazole,pH7.5500mMNaCl10%glycerol

WashBuffer20mMTris,pH7.560mMImidazole,pH7.5500mMNaCl10%glycerol

ElutionBuffer20mMTris,pH7.5500mMImidazole,pH7.510%glycerol

52

16.ProteinExpressionviaFluorescenceImaging

Day1:Transformation

1.ThawBL21cellsonice.Setheatblockto42C.Aliquite100μLcellsintoone

tube/constructandplaceonice. 2.Add1μLDNAtoeachtubeofcells. 3.Heatshockcellsat42Cfor1min.Returncellstoice. 4.Add900μLLBtoeachtubeofcells.SoinatR/T,12,000rpmfor1min. 5.Decantallexcept~100μLsupernatantandresuspendcellsinremainingsupernatant.

PlateallcellsonLB+CarbplatesandgrowO/Nat37C.Day2:Microscopy

1.Inoculate2mLLBwith2μL1000xCarb,and10colonies.Shakeat37C,250rpmfor

30-60minuntilOD=~0.2.Duringgrowthperiod,labeltubesforcellpellets. 2.Save50μLuninducedcellculture.Induceexpressionbyadditionof20μL0.1MIPTG

andshakeculturesat37C,250rpmfor1hour.Placetubesoniceuntilreadytouse.CheckOD600ofcultureanddonotreturntotube.Turnontheconfocalscopeatthistime.

3.Prepslideswithagarosepads(make2xthanactualneeded). a.makeup1%agaroseina50mLflask,20mLwater+0.2gLEagarose b.microwaveuntilboilingandagarosedissolved,letcooluntiltouch c.place15μLhotagaroseontocleanslideandquicklybutgentlylayanotherglass

slideontopofagaroseandpressflat. d.allowagarosetosetfor~5min. e.carefullypeelslidesapartandadd2μLculturetotopoftheagarosepadandtiltto

spreadthedrop.Allowdroptobeabsorbedfor2-3minuntilmostoftheliquidisabsorbedintothepad.

4.Imagethecells: a.makesuretheprismissetto100xobjective.Adjustcondenserthatitiscentered

andinfocuswiththe100xobjective. b.focusontheedgeofagarosepadandmoveobjectivetofindcellsimmobilizedby

thepad.Makesurethereisascalebarpresent. c.switchtofullcamerasetting.Takebrightfieldimageofcells,manual

focus/exposuresetting,averagingthreetimesforeachimage,ISO200,whitebalanceblank(nocells)area.Adjustangleofincominglightasneeded.

d.switchtoGFPfluorescence.Setblackbalanceareawithoutcellsasidentifiedinbrightfield.Adjustexposuretomaximumrequiredtoseesignalwithoutlotsofbackground.Takeandsaveanimage.

e.takeimagesofthreeotherpartsoftheagarosepad,roughlythesamedensityofcellsineacharea.¾images=1036x1024,¼imagefornegativecontrol->takeGFPimageatlongestexposuretimeusedforallsamples

4.afterimaging,spindownremainingcultureinmicrofugetubeanddecantallsupernatant.

53

17.StandardPCR

1)Youwillneedtodeterminewhichpolymeraseisneededforyourreaction.Ifyouarestilloptimizingpleaseuseourhomemadetaqpolymerase.Iferrorfreeamplificationisneededusetheproofreadingpfxpolymerase.2)Setupthereaction.Eachreactionwillbe20µl.*Commonset-upsHomemadeTaq4µL5xPCRBuffer1µLDNA(~50-100ng)1µLdNTPS1µLprimermix12µLH2O1µLTaqPfx2µL10xBuffer1µLDNA(~50-100ng)1µLdNTPs1µLprimermix0.4µLMgSO414.1µLH2O0.5µLPfx3)Chooseyourreactionconditions:Denature-95°Cfor30seconds.Anneal–oligoTmfor30seconds.Elongation–72°CifusingTaqand68°Cifusingpfx.Runfor1minute/kb.

54

18.FusionPCR

Tomaketwofragments,A(amplifiedwith forwardoligoAFandreverseoligoAR)andB(amplifiedwithBFandBR):1. Designyouroligos:

i. Designallfouroligostohaveaninitialannealingtemperatureof50oCandafinalannealingtemperatureof55oC.

ii. EncodeoverlappingsequenceintheARandBFoligosthathasameltingtemperatureof50oC.

2. UsePfxtoamplifyfragmentsAandBusing5cyclesthatuseanannealingtemperatureof50oCfollowedby25cyclesthatuseanannealingtemperatureof55oC.

3. Column-purifythePCRproducts(orgel-purifyifnecessary).

4. Makethefollowingmix:10µl10Xbuffer5µlPCRproductA5μlPCRproductB72µlwater(oramountthatwillbringyourPCRreactioninstep5to100µl)Annealthetwofragmentsbyboilingandthenallowingthetubetoreturnslowlytoroomtemperature;thenchillonice.

5. AdddNTPs,AFandBRoligos,andPfx

6. Incubatefor1minute(orappropriatetimeforthelengthofproduct)at72oC,then

runanormalPCRprogramtoamplifythefusionproduct.

7. Checkforproduct,clone,andsequence.

Kelsey Kropp

55

19.GelPurificationofDNAFragments

THINGSTODOBEFORESTARTING

1. EnsureEtOH(96-100%)hasbeenaddedtoWashBuffer2. CheckcolorofDNABindingBuffer;yellowindicatesapHof7.53. Setheatblockto55oC4. All spin columncaphinges shouldbepointingoutward in the centrifugeduringall

spinstoachievemaxyield

PROCEDURE

1. ExciseDNAfragmentfromagarosegel,addtocleanmicrofugetubea. Minimizegelsizebytrimmingoffexcessagarose

2. Weighgelslice;add3volumesofDNABindingBufferto1volumeofgela. Forexample:add300µlDNABindingBufferto100mggelsliceb. Forgels>300mg,usemorethanonespincolumnc. ForPCRclean-up,add3volumesofDNABindingBufferto1volumePCR

product.Skipoverstep2.

3. Incubateat55oCfor15minoruntilcompletelymelteda. Gently vortex tube2-3 timesduring incubation to ensure gel is completely

dissolved4. Optional:Add1volumeisopropanolandmix

a. Increasesyieldforfragments<500bpor>4000bp5. Addmixturetospincolumn

a. Centrifugeat13,000rpmfor1minb. Ifvolumeis>700µl,repeatspinc. Discardflow-through;placespincolumnbackincollectiontube

6. Add500ulWashBuffertocolumna. Centrifugeat13,000rpmfor1minb. Discardflow-throughandplacecolumnbackincollectiontube

7. Centrifugeat13,000rpmfor2mina. Transfercolumntoclean1.5mlmicrofugetube

8. Add50µlddH2Odirectlytocenterofcolumna. Letstandfor1minb. Centrifugeat13,000rpmfor1minc. Toincreaseconcentration,elutein30µlddH2O

9. StorepurifiedDNAat-20oC

56

20.In-Fusion“Gibson”Cloning

*Thepurposeofthiskitistoeasilydirectionallycloneanyfragmentintoanylinearizedvector

bymeansofaproprietaryenzymethatfacilitateshomologousrecombination.Thus,primerdesignisthemostcriticalpartofthisprotocol.

*SeefullIn-FusionmanualfromClontechfordetailsonlineorprintedlabcopy.OVERVIEW1. SelectvectorandlinearizewithrestrictionenzymeorPCRamplification2. Designprimersforinsertwithappropriate15bpofhomologytovector3. Amplifyinsertandpurifybygelelectrophoresisandworkup4. SetupInFusionreactionandincubate15minat50°C5. TransformcellsPROCEDURE1.LinearizetheVector• The vector can be linearized either by restriction digest or PCR. Linearization by PCR

involvesdesigningprimerssuchthatyouamplifythevectorwithspacesforyourinsertasdesired.

• TheFORWARDprimerbeginsimmediatelyDOWNSTREAMoftheinsertsite.• TheREVERSEprimerbeginsimmediatelyUPSTREAMoftheinsertsite.• ThisdesigncreatesadoublestrandedPCRproductofyourlinearvectorwithanyspace/gap

asneeded.• Identifyandpurifybygelelectrophoresis(Protocol#13).Useahigh-fidelityenzyme.

2.Designprimersforinsertwith15bphomology• This is thekeyof theprocedureandmustbedonecarefully(seepg.7-8of the In-Fusion

manualfordetails—especiallyfornon-bluntproductsgeneratedbyrestrictionenzymes).• Theinsertmustbeamplifiedwith15bphomologoustothevectoratthesiteofinsertion.• The5’endoftheprimeris15bphomologoustothelinearizedvector.• The3’endoftheprimerisinsert-specific,ideally18-25bp,GCcontent40-60%andTm58-

65°C.• Meetingallofthesespecificationsisnotalwayspossible.Theprimercanbelonger.• Onlyalterthe3’portionoftheprimer,keepexactly15bphomologoustothevectoratthe

5’end.Onlythe3’portionneedstomeettheseprimerspecifications(orgetclosetothem).3.Amplifyinsertandpurify• Amplifyyourinsertwithahigh-fidelitypolymerase.Duetousingabizarreandlongprimer,

troubleshootingwithaprocessiveandcheapenzymelikeTaqmayberequired.• Confirmsizebygelelectrophoresisandcutoutandpurify(seeProtocol#13).

4.SetupIn-FusionReaction(seepg.9ofprotocolfordetails)• There is a balancebetween themolar equivalents of the vector and insert (seepg. 9 for

details).

57

• Insert:10-200ng(10-100ngifbelow10kb).• Vector:50-200ng(50-100nglessthan10kb;50-200ngforgreaterthan10kb).• 2µl5xEnzymemix• H2Oto10µl• Incubate15minat50°Candplaceonice.• Youcantryahalf-reactionof5µlifyourDNAisconcentratedenough.

5.TransformCells• ThekitcomeswithasetofStellarcompetentcells.Anycompetentcell likeDH5αshould

workfine.• Transform50µlofcellswithhalfofthereactionmixture.• AddDNAandincubateonicefor30min.• Heatshockcellsfor45sat42°C.• Placeonicefor1-2min.• AddLBtoavolumeof250µlandincubatefor1hrat37°C.• Plateonselectivemedium.

Grigory Maksaev

58

21.Site-DirectedMutagenesis

*Takenfrom:Stratagenekit#200521PROCEDURE

1)Primerdesign

• The desiredmutation should be in themiddle of the primerwith 10-15 bases ofcorrectsequenceoneitherside.

• Bothmutagenicprimersmustcontainthemutationandannealtothesamesequenceonoppositestrandsoftheplasmid.

• Idealprimer specificationsare25-45bases in length,Tm≥780C (usually it ismuchlower),GCcontentofatleast40%(thisisimportant)andterminationinoneormoreCorGbases.

• Checkprimersforself-dimerization:(http://www.idtdna.com/analyzer/applications/oligoanalyzer/Default.aspx)

2)PCRreaction Example Perreaction sameprimers sametemplate 2μl10xPfubuffer x5= 10μl 10μl 2μltemplate(5-25ng/μl) - 10μl 0.5μl10μM“forward”primer 2.5μl - 0.5μl10μM“reverse”primer 2.5μl - 0.5μl10mMdNTPs 2.5μl 2.5μl 14μlwater 70μl 70μl 0.5μlPfu 2.5μl 2.5μlTotal: 20μl 90μl 95μl

Aliquot18μlofenzymemixtotubescontaining2μloftemplateor19μlofenzymemixtotubescontaining1μlofprimers,dependingonifyouhavemultiplesampleswiththesametemplateorwiththesameprimerpair.RunPCRprogram“PTMUT”: Step1 950C 1min Step2* 950C 50sec Step3 600C** 50sec Step4 680C xmin(1min/kbofplasmid) Step5 18-25cycles (steps2-4) Step6 680C 7min

*TofacilitateannealingthefollowingstepsmaybeincludedbetweenStep1andStep2: Ramp950Cto250Cat0.10C/sec(minimalpossiblespeed) 680Cfor6min**Itmayhelptolowerthistemperature,55–600Cisagoodstartingpoint

59

3)Digestion.Add1μlofDpnIenzymetoeachreaction,thoroughlymix,spindownandincubateat370Cfor1hour.ThiswilllinearizethemethylatedparentsupercoileddsDNA(plasmid),leavingthenickedmutantplasmidintact.Inactivateresidualenzymefor20minat800C.4)Transformation.Mix5μlofthedigestedmutantplasmidwith50-100μlofcompetentE.colicells.Icefor5min.Heatshockat420Cfor1min.Icebriefly.Add900μlofpre-heatedLBandshakeat370Cfor1hour.Spincellsfor2minat10,000rpm.DecantLB,resuspendinremaining~100μl,andspreadontoplateswiththeappropriateantibiotic.Incubateovernightat370C.Mini-prepandsequencetoconfirmmutation.

Liz Haswell

60

22.ColonyPCR

1. Eachreactionwillbe20µl.2. Multiply the followingrecipeby thenumberofcoloniesyou’d like to test, includinga

negativecontrol(nocolonyadded)andapositivecontrol(plasmidDNA,ifavailable),andanextrareactiontomakeupforlostsolutionduringpipetting:

4µL5XTaqbuffer0.4µl10mMdNTPs0.4µl10pmol/µlforwardprimer0.4µl10pmol/µlreverseprimer0.2µLTaqpolymerase14.6µlwater

3. Aliquot20µlintoeachPCRtube.4. DividetheappropriateLBplateintoagridwithapenontheoutside,oneboxpersample.5. Pickacolonyfromyourtransformationplatewithasterilepipettetipandpipetupand

downtodislodgethematerialintothePCRreactionsolution.Leave1-2µlinthepipettetipandspotontotherightgridonthefreshLBplate.Incubatethisplateat37°C.Onceyouhaveidentifiedpositiveclones,youcanusethisplatetostartovernightculturesforminipreps.Evenifthedotisbarelyvisible,therewillusuallybeenoughtheretoinoculateaculture.

6. PCRamplifyasindicatedbytheoligomeltingtemperatureandthesizeoftheexpected

productandcheckonanagarosegel.

Kira Veley

61

23.GatewayCloning

*RefertotheGatewayTechnologymanualfromInvitrogen/LifeTechnologiesforadditionalinformationPROCEDURE

1. GatewayDestinationvectorscontain theccdb gene,whichkillsmoststrainsofE.coli.This is removed through recombination during the Clonase reaction. In order topropagatethese‘empty”vectors,aresistantstrainmustbeused.WeuseDB3.1.

2. Before doing an LR reaction, check that the antibiotic resistance of your Entry and

Destinationvectorsaredifferent.Iftheyarethesame,youneedtopre-digestyourEntryconstructtocompletionwithanenzymethatdoesnotcutyourgeneoranythingwithinthegatewayregion(MluIfrequentlyworksforthis).Afterthisdigestion,heat-inactivatetheenzymeunlessyouknowFORSUREthatitdoesnotcutthedestinationvector.

3. ForLR reaction, I usually try for a1:1molar ratio forEntry andDestination vectors.

However,thisisusuallylimitedbytheDestinationvectorifitisabinaryvector(usuallyforexpressioninplants),giventheirlargesizeandlowyield.Therefore,Iusuallyuse0.5-1.0µlofEntry(ordigestedEntry)andasmuchasIcanfortheDestinationvector(3.0-3.5µl).Use1.0µlofLRClonase.Thefinalreactionvolumeis5.0µl.

4. ItypicallylettheLRreactiongoalldayorovernightatroomtemperature,dependingon

whatisconvenient.Smallerproducts(<1kb)arefinetojustletgoanhourorso,butthereactionismuchlessefficientforlargerproducts.

5. The Gateway protocol says to stop the LR reaction with Proteinase K. This is not

necessary.Justtransformthereactiondirectly.6. Transform theentireLR reaction into~100µlDH5αcells. Somecell strains thatwe

frequentlyuseinthelab(specificallyNEBTurbo)haveacquiredsomelevelofimmunitytoccdbandwillgiveyouincreasedbackgroundifused.DH5αworkswell.PlateallofthecellsfromthetransformationonanantibiotictheselectsfortheDestinationplasmid.

7. CheckthatyourfinalconstructiscorrectwithacombinationofPCRanddigestionofthe

mini-preppedproduct. YoucoulddothiswithcolonyPCRaswell. TheidealtestistoPCRamplifyusingapairofprimerswhereoneisspecificforyourinsert/geneandtheotherisspecificforthedestinationvector(ex:35Spromoter,ataglikeGFP,oranattBsite). Then, tobeextrasure,digest thisproductwithanenzymethatcan informyouaboutorientationiftheprimersyouchosedonot(ex:onethatcutsyourgene+tagintotwodifferentlysizedproducts).

Kelsey Kropp

62

24.PlasmidPrep/GelExtraction/PCRcleanup(EpochLifeScience)

REAGENTS

PrepareBuffersP1,P2,N3,PB,PE,QG,andEBaccordingtothelabrecipebinder.PLASMIDPREP

1.Harvestpelletfrom1-5mlofLBovernightculturebycentrifugingat15,000rpmfor1-2minutes.2.Discardsupernatantandresuspendpelletin250μlP1buffer.3.Add250μlP2bufferandgentlyinvertthetube5timestomix.IncubateatRTfor5minutes.4.Add350μlN3bufferandinvertthetube5timestomix.Centrifugeat15,000rpmfor10-15minutes.5.Transferthesupernatanttoaspincolumnandletsitfor15minutes.6.Centrifugeat4,000rpmfor1minute.Discardtheflow-through.7.Add500μlPBbuffertothecolumn.Centrifugeat12,000rpmfor1minute.Discardtheflow-through.8.Add750μlPEbuffertothecolumn.Centrifugeat12,000rpmfor1minute.Discardtheflow-through.Centrifugeat12,000rpmfor2minutestoremoveresidualethanol.9.ElutetheDNAbyadding50μlEBbuffertothecolumn.Incubateat50-65°Cfor15minutes.Centrifugeat12,000rpmfor1minute.GELEXTRACTION

1.Excisegelandadd3volumesQGbuffer.Incubateat55-65°Cfor10minutesuntildissolved.2.Transferthesupernatanttoaspincolumnandcentrifugeat4,000rpmfor1minute.Discardtheflow-through.3.Proceedtothewashstepsinstep7above.PCRCLEAN-UP

1.AddwatertothePCRproductupto100μl.Add500μlQGbufferandmixwell.2.Transferthesupernatanttoaspincolumnandcentrifugeat4,000rpmfor1minute.Discardtheflow-through.3.Proceedtothewashstepsinstep7above.

Emma January

63

25.ArchivingPlasmidsasDNA&BacterialStocks

*TheplasmiddatabasecanbefoundintheFileMakerProfolder(titledLHPlasmids)Tosearchforaplasmid:

1. Click “records” then “show all”. Thiswill insure that other labmembers have notpreviouslysortedthedatabase.

2. Clicksmallmagnifyingglassinupperlefthandcorner.Plasmidscanbesearchedbytheirnumber,name,datecreated,gene,orpurpose.

Ifenteringanewplasmid:

3. Click“records”then“newrecord”.4. Makesuretoincludeplasmidname,gene,map(ifpossible),purpose,andanyother

searchablenotesthatmaybeusefultofutureusers.DNAandGlycerolStocks

*TheDNAandglycerolstocksgohandinhandwiththeplasmiddatabase.Makecertainthatplasmidsareassignedaplasmidnumberandthatthenumberisrecordedonthetopofeachstocksample.Thesideofeachstockshouldbe labeledwithplasmidname, thedate,andcreator’sname.PREPARATIONOFDNASTOCKS

1. Inoculate2mLLBculturefromsinglebacterialcolony.2. GrowculturesinLBsupplementedwiththeappropriateantibiotic(1µL/mLofLB),

andshakeat37Cfor12-18hrs.3. Collectplasmidusingstandardmini-prepprocedure.4. Label 1.5 mL tube with plasmid number (top), and name of plasmid, date, and

creator’sname(side).Transferatleast20µLDNA(~200ng/µL).5. StoreintheLHPboxinthe-20CfreezerinMcDonnell249.

PREPARATIONOFGLYCEROLSTOCKS

1. GetDH5acompetentE.colicellsfromthe-80CfreezerinMcDonnell105.2. Add50µLofbacteriatolabeledmicrofugetube.3. Whiletubesareonice,add1µLofplasmidDNA.Leaveonicefor10min.4. Placemicrofugetubesin42Chotwaterbathfor1min.Afterthatminute,placetubes

backonicetorest(~5-10min).5. Spinoutbacterialpelletat10,000rpmfor1.5mins.6. Decantsupernatant,thenre-suspendpellet.7. Transfer100 µLofbacteriatoplatecontaininganantibioticspecifictotheplasmid’s

selectablemarker.Spreadwithsterilespreader.8. Incubateovernightat37C.9. Inoculate2mLLBculturefromsinglebacterialcolony.10. GrowculturesinLBsupplementedwiththeappropriateantibiotic(1µL/mLofLB),

andshakeat37Cfor12-18hrs.

64

11. Label Nalgene tube with plasmid number (top), and name of plasmid, date, andcreator’sname(side).

12. Add350µLofovernightLBcultureto700µLglycerolsolution.13. FlashfreezetubesinliquidnitrogenandstoreintheLHPboxinthe-80Cfreezerin

McDonnell105.SOLUTIONSGlycerolStorageBuffer65%glycerol0.1 MMgSO40.2 20mMTris,pH7.5-8

Eric Hamilton

65

26.DNASequencing

*BigDyeisfoundintopshelffreezerboxof-20°C*5XSeq.Bufferaliquotscanbefoundinthewalk-in,topdrawerontheright.*BigDyeisboughtontheMedCampus,PNACL,3rdFloorNorthBuildingroom3833.

CyclingConditions

94°C 1min.94°C 30s 55°C 30s 30Cycles 72°C 90s72°C 10min.16°C ForeverSequencing

• BringPCRsampleto-20°CfreezerinMcDonnell112.• FilloutformspecificingV3.1BigDyeandaskingto“pleaseclean”.• Putappropriategrantnumber.• Submitby10amforafternoonresults.• Resultsfoundonlabcomputerunder“Sequencing”network.

Ø Sequences>Haswell

SOLUTIONS

5XSequencingBufferTris-HCl1M9.0 400mlMgCl2 1M 10mlH2O 590ml*Stirfor1hr,filterthroughfilterpaperandaliquotinto15mlconicaltubes.Canbestoredat4°CorRT.

Components Volume(µl)Primer(5pmol/µl) 1.0BigDyeTerminatorV3.1 2.0TemplateDNA ~80ng/kbplasmid5XSeq.Buffer 1.5 µlH2O To11µl

Maggie Wilson

66

27.RNAExtractionwithTRIZOLReagent

*UsebarriertipsandRNase-freewatertoavoiddegradation.*Onlyusereagentsdesignatedfor“RNAUSEONLY”.NOTES

§ 260/280=RNAtoDNAratio.Wantascloseto2aspossible.Lowerthan1.8meansalotofDNApresent

§ 260/230=Measureofno-nucleicacidcontaminants(phenols,redpigments).Wantvaluesof2orgreater.Lowerthan2meanscontaminationpresent

PROCEDURE

1. Grindbetween40-100mgfreshorfrozentissueinliquidNitrogenintoafinewhitishpowder.2. Transferto1.5mltube,add1mlTRIZOLreagent,vortex,incubateatroomtemperature(RT)

for5min.3. Add0.2mlchloroform,shakebyhandvigorouslyfor15sec,andincubateatRTfor2-3min.4. Centrifugeat12,000xgfor15minat4°C.5. RNAprecipitation:TransferupperlayertonewRNase-free1.5mltube.NOTE:Transferno

morethan450μlofupperlayer,takingspecialcarenottodisruptthelayerofcelldebris.6. Optional:Repeatsteps2-4toincreasepurityofRNA(Thiscanbehelpfulwhenworkingwith

difficulttissueandtheendgoalisquantitativeRT-PCR).7. Add0.5mlisopropanol,incubateatRTfor10min.8. Centrifugeat12,000xgfor10minat4°C–canoftenseepellet.9. RNAwash:removethesupernatantadd1mlof75%EtOH(RNase-free),vortex,centrifugeat

12,000xgfor5minat4°C.10. Cleanup:re-dissolveRNAin100μlRNase-freewater(vortex).11. Re-precipitateRNAbyadding250μl100%EtOH(RNase-free)and10μl3Msodiumacetate

(pH5.2).12. Incubateat-20°Cforatleast1hr.(Increaseincubationtimewillimproveyieldandpurity.)13. Centrifuge12,000xgfor10minat4°,removesupernatant,washwith75%EtOH,andlet

pelletdryonbench.14. Resuspendin30μlofRNase-freewater.Quantifywithspec.Recordconcentrationandquality

measurements.

Kira Veley

67

28.Semi-QuantitativeRT-PCR

1.DNaseTREATMENT0.5-5µgRNA1µl10XDNasebuffer1µlDNaseAddRNase-freewaterfor10µltotalreactionvolume

1. Incubateat37°Cfor30min2. Add1ul50mMEDTA

a. Incubateat75°Cfor10mintoinactivateenzyme2.cDNASYNTHESIS11µlRNA(DNasetreated)1 µl500mMprimer(usuallyoligodT)

*Incubateat70°Cfor10min,chillonice1-5minThen,makemastermix:4µl5Xreversetranscriptasebuffer(M-MLVbufferalreadycontainsDTT)1µl10mMdNTPs0.5µlreversetranscriptase(M-MLV)0.75µlRNAsin2.75µlRNase-freeH2O20µltotal.

*Incubateat42°Cfor2hours.Storeat-20°C.3.Semi-quantitativeRT-PCRIusuallyuse1µlofcDNAper20µlreaction(homemadeTaq).Docontrolfirsttocheckthatallreactionshavethesameamountofstartingmaterial(usuallyActin,25cycles).Iftheyareeven,moveontoothergenes.Ifnot,re-quantifyandrecheckqualityratios(260/280and260/230).Ifthisisagenethathasnotbeentestedbefore,Iusuallystartwith30cycles,andthenadjustaccordingly. Ialsousuallyrunmyproductsoutona2%geltogetnice, tightbands.Primeradvice: ThebestprimersforRT-PCRmakesmallproducts(lessthan500bp)andamplifyaregionneartheendofthegene(becausethat’swheretheRTenzymestarts).Also,ideallyatleastoneoftheprimerswillspananintron.ThenextbestthingistohaveanintronincludedinyourproductsothatyouwillbeabletovisualizeanyDNAcontamination.

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68

29.QuantitativeReverse-TranscriptasePCR

PrimerDesign:

Length~20Bases.Primersshouldgeneratea100-140bpproduct.EachprimershouldhaveaTmbetween58-60°C.Thefivenucleotidesatthe3’endshouldhave<3GorCbases.Both5’and3’endsshouldbeaGorC.Avoidrunsofidenticalnucleotides,especiallyGs.GCcontent45-55%.Ampliconshouldspanoneortwointrons.Template:

cDNAtemplateshouldbereversetranscribedfromRNAtreatedwithDNAse.GeneralPCRsetup:

ThePCRreactionsarerunonoptical96-wellreactionplates.Setplateuponseparateplasticbaseonice.Toorganizeyoursamplesusethereactionplatediagramandassignreactionstoeachposition.Eachsampleshouldberuninsupplicateortriplicate,thereshouldbeatleast4non-templatecontrols(NTC–negatives)andifaprimersethasnotbeenusedbeforethenyouhavetorunaseriesof5orsodilutionstogetabestfitlinesothatyoucancheck

theefficiencyoyourprimers.Oncereactionsaresetup,sealplatewithopticalfilm.Useakimwipetopressaroundtherimofeachwelltoensureagoodseal.*Alwaysweargloveswhenhandlingopticalplatesandfilm.Preliminarytestreactions:

Therearetwotypesofteststhatneedtobedoneeitherinparallelorprior(recommended)toyourexperimentalsample.

1. DissociationcurveanalysisforNTCs.2. Dilutionseriesofstandardtemplate.

ThedissociationcurvedescribesthemeltingpropertiesofanyDNAinyoursampleafterthePCR is complete. Any change in fluorescence (caused by dye interacting with doublestrandedDNA)isplottedagainsttemperature.Primerdimmershaveacharacteristicpeakoffluorescenceofaround75°CwhereasampliconpeakshaveahigherTmofaround80.5°C.IdeallyiftherearenoprimerdimmersthereshouldbenolargesinglefluorescencepeakinthedissociationplotfortheNTCs.Totesttheefficiencyofyourprimersrelativetothecontrolgene,youwillneedtomakea5Xdilutionseriesoftemplateeachdiluted1:10.Real-timePCRreactionsarethensetupusingthesedilutionsastemplatewithastandardprimerconcentrationofaround5pmolesperreactionforbothcontrolandtestprimers.

69

Note:Ifithasbeendeterminedthattheexpressionofthecontrolgenehasatypicalefficiency(1.9)overthedilutionrangealready)thenthedilutionseriesisonlynecessaryforyourtestgeneprimer.InthiscasetheCT(Yaxis)isthenplottedagainstthedilution(loggraph)andthebestfitcurveshouldbelinearwithaslopeof3.5±0.5.OtherwisetheCTforbothcontroland test genes over the dilution series should be obtained and the ΔCT plotted againsttemplatedilutions.Iftheslopeofthiscurveisclosetozerothenthecontrolgeneisvalidforyourexperiment.PCR/PlateSetup:

A) Primermix=2.5μMeachprimer(Forward&Reverse){½5μMF,½5μMR}ForActin=½5μMF(#1358) 1/65μMR1(#1359) R2(#1360) R3(#1361)B) cDNADilutionSeriesmixcDNAsfromallsamples,dilute(1:5or1:10)à1:4à1:4à1:4*Need10μl/tubeXeachprimerset.C) Primer/SYBRGreenSolutionTheSYBRgreenmastermixcontainseverythingexceptprimersandtemplate.12.5μlSYBRGreen0.5μlH2O2.0μlPrimers(A)15/tube*Don’tforgettoincludedilutioncurvesandnotemplatecontrols.D) cDNAMixForeachreaction: 0.25μlcDNA 9.75μlH2O 10/tube*Fordilutioncurves,use10μleachdilution*Thisisagenericexampleandeachtemplateshouldbetitratedouttofindasuitabledilution

forthetemplatetogiveagoodlinearrangearound20cycles(forex.If1:100givesyoualinearrangeat15cyclesyoumayconsiderdilutingtemplate1:1000.10foldmore(2^3)shouldmoveyouto18/20cyclesforthelinearrange).

Aliquot(B),(C),and(D)

Onceplateissealedwithopticalfilm,turnovertomix.Thenbrieflyspinreactionplateincentrifuge(~500Xgfor2-3minutes).

MachineSetup:

Turnonmachine30minutesinadvance,AppliedBiosystemsintheBlankenshipLab.

70

Password:Ch1orosomes! StepOnesoftwarev2.1 LoginasGUEST

71

EXAMPLE

3genotypesand3primersets A Col

(1)Col(2)

Col(3)

msl2(1)

msl2(2)

msl2(3)

msl2-3msl3-1(1)

msl2-3msl3-1(2)

msl2-3msl3-1(3)

Primer2

1 2 3 4 5 6 7 8 9 10 11 12

ACTIN

A Col(1)

Col(1)

Col(1)

B Col(2)

Col(2)

Col(2)

C Col(3)

Col(3)

Col(3)

Primer1

D msl2-3(1)

msl2-3(1)

msl2-3(1)

E msl2-3(2)

msl2-3(2)

msl2-3(2)

F msl2-3(3)

msl2-3(3)

msl2-3(3)

G D1 D2 D3 D4 Neg D1 D2 D3 D4 msl2-33-1(1)

msl2-33-1(1)

msl2-33-1(1)

H D1 D2 D3 D4 Neg Neg msl2-3;3-1(3)

msl2-3;3-1(3)

msl2-3;3-1(3)

msl2-33-1(2)

msl2-33-1(2)

msl2-33-1(2)

cDNA

Dilutions

1:10

0.5 0.5 1

0.5 12.5μL 12.5μL 12.5μL 0.5 1 + 37.5μL + 37.5μL + 37.5μL

0.5 0.5

5μl

50μL 50μL 50μL

+ 45μl

50 D1 D2 D3 D4 Samples

ßμLcDNA*PoolcDNAfromsamplestomakedilution

72

0.25μLcDNA )X 1

0

2.5

9.75μLH2O

97.5

100 10μLperwell

Primer/SYBRGreenMix

12.5μL

SYBRGreen )

X

413

0.5μLH2O

3316.5

2.0μLPrimers

66

495 15μLperwell

Actin

ulcdna log std.Curve

1 0.00 21.22283745

0.25 -0.60 23.06932259

0.0625 -1.20 25.08555222

0.015625 -1.81 28.1104641

0 32.77287292

P5CS1

ulcdna log std.Curve

1 0.00 22.96528053

0.25 -0.60 24.03655243

0.0625 -1.20 25.98813629

0.015625 -1.81 28.892416

0 Undetermined

ProDH

ulcdna log std.Curve

1 0.00 22.73629761

0.25 -0.60 24.39732552

0.0625 -1.20 26.25531769

0.015625 -1.81 29.38565063

0 35.65597916

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Actin WT1 Wt2 WT3 msl2-3 msl2-3 msl2-3

22.09156 21.24307 20.7212 21.27322 21.63375 21.5933

22.2055 21.22785 20.86473 21.35755 21.64237 22.23039

22.23131 21.14853 20.85077 21.36083 21.68712 21.71881

Ctaverage 22.18 21.21 20.81 21.33 21.65 21.85

-0.32 -0.06 0.04 -0.10 -0.18 -0.23

relative[RNA] 0.48 0.87 1.10 0.80 0.66 0.58

P5CS1 WT1 Wt2 WT3 msl2-3 msl2-3 msl2-3

23.89656 23.68434 23.03528 23.00382 23.87647 23.66715

23.86546 23.6953 23.06692 23.11202 23.85345 23.62286

23.94906 23.67931 23.10585 22.92048 23.87579 23.62503

Ctaverage 23.90 23.69 23.07 23.01 23.87 23.64

-0.42 -0.36 -0.17 -0.15 -0.41 -0.34

relative[RNA] 0.38 0.44 0.68 0.70 0.39 0.45

WT1 Wt2 WT3 msl2-3 msl2-3 msl2-3

[b]/[Actin] 0.79 0.51 0.61 0.64 0.88 0.59 0.77 0.75 Average

Norm.to1 1.00 1.00 1.00 0.08 1.12 1.16 1.26 0.09 StErr

0.141 0.148 StDev

TTEST 0.402

y=-3.7669x+20.97R²=0.98535

0

10

20

30

-2.00 -1.50 -1.00 -0.50 0.00

Actin

y=-3.2776x+22.511R²=0.95863

0

20

40

-2.00 -1.50 -1.00 -0.50 0.00

P5CS1

y=-3.6219x+22.423R²=0.97548

0

20

40

-2.00 -1.50 -1.00 -0.50 0.00

ProDH

74

ProDH WT1 Wt2 WT3 msl2-3 msl2-3 msl2-3

22.74219 21.84295 21.3024 23.15631 23.23879 23.63514

22.9172 21.97053 21.44917 23.301 23.81059 23.25305

22.80419 21.8642 21.34826 23.2237 23.24454 23.1734

Ctaverage 22.82 21.89 21.37 23.23 23.43 23.35

-0.11 0.15 0.29 6.19 -0.22 -0.28 -0.26

relative[RNA] 0.78 1.40 1.96 0.60 0.53 0.55

WT1 Wt2 WT3 msl2-3 msl2-3 ml2-3

[c]/[Actin] 1.62 1.62 1.78 1.67 0.75 0.80 0.95 0.83 Average

Norm.to1 1.00 1.00 1.00 0.05 0.46 0.49 0.53 0.06 StErr

0.090 0.103 StDev TTEST 0.000

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73

30.Mating-BasedYeastTwoHybrid

*LiActransformationofyeast.*AdaptedfromGietz&Schiestl,1995.MAKINGCOMPETENTCELLS

1. Themorningbefore,incubateonecolonyofTHY.AP4andTHY.AP5,eachshakingin5mlYPADat28°C,for24hours.Place2flaskswith100mlYPADinincubator.

2. Earlyinthemorning:Inoculate100mlpre-warmedYPADwiththepre-culturetoan

OD600of0.09.Thisusuallytakesapprox.0.5ml.

3. Incubateflasksinshakerat28°CuntilOD600reaches0.5-0.6.(4-5hours)

4. Whileflasksareincubating,makesolutions:a. 10mlLiAc/TE

1ml10xTE1ml1MLiAc8mlddH2O

b. 5mlPEG/LiAc0.5ml10xTE0.5ml1MLiAc4.0ml50%PEG

c.100μl1xTE(dependingonDNAconcentrations—seestep9)

5. Placecellsinsteriletubes(FALCON),centrifugeat2,500xgfor5min(20°C).

6. Removethemedium,re-suspendpelletsin5mlsterileddH2Oeachandre-centrifugeasdescribedabove.

7. Remove ddH2O, re-suspend each pellet in 2 ml LiAc/TE, pool the suspensions

togetherandmixcarefully.Re-centrifugeasdescribedabove.

8. Removethesupernatantandre-suspendthepelletcarefullyin0.5-0.8mlLiAc/TE,letthe suspension incubate for 30 min at room temperature. While the cells areincubating,preparethemixturesbelow.Ifyouusethecellslaterthan30minafterthisstep,thenkeepthemat4°C.

TRANSFORMATION

*Beforestarting,boiltheSSDNAfor3minandchilloniceimmediately

9. Foreachtransformation,addinthefollowingorder,mixingwellaftereachstep:- 20μlcarrier-SSDNA(5or10mg/ml)- 20μlDNA-mix=1xTEbufferandDNA

either: plasmidalone(0.1-10μg)

74

or: linearplasmid(100ng)plusinsert(>100ng),molecularratioofvectorvs.insertatleast1:10

- 4.5μl1MLiAc- 50μlcompetentcells- 300μlPEG/LiAcmix

10. Incubateshakingfor20minat30°C.

11. Heat-shockin42°Cwaterbathfor20min(keepthetime!!!)

12. Centrifugeat7000rpmfor1minandcarefullyremovethesupernatantwithapipette

13. Re-suspendthepelletcarefullyin100μlsteriledH2Owithpipette.

14. Streak the transformations on appropriate selectivemedia (make sure plates are

dry!)ForAP4Cubconstructs: SC/+AHTUForAP5Nubconstructs: SC/+AHL

15. Growcoloniesfor2-4daysat28°C.

MbSUSTestsMATING

1. Themorningbeforemating,growcellsovernighttothestationaryphase

- GrowCubcoloniesin5mlSC/+AHTU- GrowNubcoloniesin5mlSC/+AHL- Wipepipettordownwith95%ethanol tomaintainsterility,collectcolonieson

pipettetip,thenejecttipsintoculturetubes.

2. Thenextmorning,for13orfewercrossesperconstruct,concentrate1mlofculturesbycentrifugation(1minat7,000rpm)inafinalvolumeof200μlYPD.Doublethesevolumesforhighernumberofcrosses.

3. BegindryingYPDplates.

4. Mix15μloftheappropriatematingtypesforeachcross.

5. Inhood,drop4μlofthemixedsuspensionsontheYPDplate.Makeatleasttwodropsforeachcross.Matethecellsfor6-8hoursat28°C.

6. ReplicaplateonSC/+AHplatesforselectionofdiploidcells

7. Incubate2-3daysat28°C.

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INTERACTIONGROWTHTEST

1. Replicaplatecellsonto4differentplates,inthefollowingorder,SD+a. +0mMmethionineb. +75mMmethioninec. +150mMmethionined. +400mMmethionine

2. Recordgrowthafter2daysat30°C.

RECIPES

YPADliquid--TransformationCultures 5g yeastextract 10g peptone 50mgadeninesulfatepHà 6-6.5(HCl) H2O à475ml,autoclave,add25ml40%glucoseYPADplates 1g yeastextract 2g peptone 10mgadeninesulfatepHà 6-6.5(HCl) H2O à95ml,add2gbacto-agar,autoclave,add5ml40%glucoseYPDliquid--Matingre-suspension 0.5g yeastextract 1g peptone 1g glucosepHà 6-6.5(HCl) H2O à50mlYPDplates--Matingcolonies 2.5g yeastextract 5g peptone 5g glucosepHà 6-6.5(HCl) H2O à250ml 5g agarSyntheticDeficient(SD)plates--TestingforInteractions 0.68g yeastnitrogenbase 2.0g ammoniumsulfate 8g glucose In100mleach…pHà 6-6.3 0mlM+2gagar H2O à400ml------------ 0.11mlM+2gagar 0.22mlM+2gagar

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0.59mlM+2gagarSyntheticComplete(SC)liquid 0.17g yeastnitrogenbase 0.5g ammoniumsulfate 0.15g dropout 2g glucose-SC/+AHTU

1.0mlA0.2mlH0.2mlT1.0mlUpHà6-6.3(NaOH)H2Oà100ml2gagar

- SC/+AHL1.0mlA0.2mlH1.0mlLpHà6-6.3(NaOH)H2Oà100ml2gagar

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31.SDS-PAGE

Reagents:30%AcrylamideMix(BioRad) 1.5MTris(pH8.8)1MTris(pH6.8)10%SDS10%ammoniumpersulfateTEMED(BioRad)2XSampleBuffer(seebelow)5XPAGEBuffer(seebelow)1XPAGEBuffer

2XSampleBuffer(10ml)

1ml1MTris-HClpH6.8 2ml20%SDS2ml1%bromophenolblue4ml50%glycerol1mlwater*aliquot10x1mlandstoreat-20C**add20ulbeta-mercaptoethanolto1mljustpriortouse5XPAGEbuffer(1L)

15.1gTRISbase94gGlycine25ml20%SDSwaterupto1liter

Procedure:

1.PreparegelsaccordingtoTableA8-9(Sambrook&Russell,CSHLPress,3rdedition).Forexample,asingle,0.75mmthickgelrequires5mlofresolvingsolutionand1-2mlofstackingsolution(AddTEMEDjustbeforepouringeachgel).Choosea%acrylamidethatsuitstheproteinsizesyouareinterestedin:

AcrylamideConcentration(%) LinearRangeofSeparation(kD) 15 10-43

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12 12-60 10 20-80 7.5 36-94 5.0 57-212

fromTableA8-8(Sambrook&Russell)2.Chooseglassplatesandacorrespondingcombaccordingtothethicknessofgelyouwanttopour.Forexample,a0.75mmthick,15-wellcombprovides10ulofvolumeperwell,anda1.5mmthick,10-wellcomballowsyoutoload50ulperwell.Cleantheplateswith70%ethanol,ifnecessary.Assembleplatesingel-pouringapparatus(BioRad).AddTEMEDtoresolvingsolutionandmixwell.Pipetthesolutionbetweentheglassplatestowithin2-3cmofthetop.Carefullyoverlaywithwatertoensurepolymerization(~15minutes).3.Decantthewateranddabtheremainderwithakimwipe.AddTEMEDtothestackingsolution,mixwell,andpipetasabove.Insertthecomb.Allow~15minutestopolymerize.4.Meanwhile,prepareyoursamplesforloading.Forbacterialpellets:Spindown5mlofOD600=0.5culture,removeallofthemedium,add50ulof2Xsamplebuffer(withbeta-mercaptoethanol),andmix/vortexwell.Transferextractsto1.5mltubes,boil5minutes,cooloniceforafewminutes,andspinfor1minuteatmaximum.Keepsamplesatroomtemperatureuntilreadytoload.5.Removethegelfromthepouringapparatusandremovethecombfromthegel.Assemblethegelintheproteinelectrophoresisapparatus(BioRad).Ifyouareonlyrunningonegel,usetheBioRadBufferDamontheoppositeside.Pressdownontheinner(electrode)compartmentasyouclosetheclampsonthefront.Thiswillensureagoodseal.6.Prepare500mlof1XPAGEbuffer.Filltheinnerchamberwith1XPAGEbufferuptothetop.Waitafewminutestomakesurethebufferisn’tleakingintotheouterchamber.Filltheouterchamberwiththeremainingbuffer.7.Chooseasetofproteinstandardstorunalongsideyoursamplese.g.BioRadPrecisionPlusProteinDualColor(161-0374).Letwarmtoroomtemperature.Load10ulperlane.8.Load10-50ulofyoursamples(dependingongelthicknessandthesizeofthewells).9.Runat75Vfor~30minutes–untilthesampleshaveclearedthestackinggel.Runat100Vforanadditional~90minutes–untilthebromophenoldyehasruntothebottom.10.Disassembleelectrophoreseapparatusandpryaparttheplatesusingarazorblade.ProceedeithertoCoomassieStainorWesternBlotprocedure.

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80

32.CoomassieStain

Reagents

DestainSolution(1L)500mlmethanol400mlwater100mlaceticacidStainingSolution(200ml)0.5gCoomassieR-250powder200mldetainsolutionProcedure

1.Pour~100mlcoomassiestainintoaglassbakingdish.CarefullytransfertheSDS-PAGEgelfromtheglassplatetothedish.Coverwithsaranwrap.Microwave~45sec.Letcoolslightlyfor5minutes.Microwave~30sec.Placedishonarotatingplatformfor~30min.2.Reclaimcoomassiestain.Rinsegel1-2timeswith~50mlofdestainsolutionandcollectinawastecontainer.Add100mlofdestainsolution.Coverwithsaranwrap.Microwave~45sec.Letcoolslightlyfor5minutes.Microwave~30sec.Placedishonarotatingplatformfor~30min.3.Decantdestainintowastecontainer.Addafresh100mlofdestainsolution,alongwithacoupleoftwirled-upkimwipestoabsorbthestain.Returntorotatingplatformuntilcompletelydestained.Forthingels(0.75mm)thisisacoupleofhoursatmost,butthickgels(1.5mm)requiredestainingovernight.4.Collectthedestainsolutioninawastercontainer.Add~100mlofwaterandshakefor~15minutes.5.Drythegelbetweentwosheetsofcellophaneandmountonaglassplatewithspacersandbinderclips.Cutoutpiecesofcellophanethatareslightlylargerthantheglassplate,soakbrieflyinwater,andassemblegelasfollows:glassplate,onesheetofcellophane,gel,secondsheetofcellophane,4spacers,andgelclips.Besuretoforceanybubblesoutbeforeattachingthefinalspacerandclip.Letdryovernight.

Liz Haswell

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83

33.WesternBlotting

1. Whileyourgelisrunning,prepareTransferBufferandchillonice.2. Wetpre-cutPVDFmembranein100%methanol.3. Equilibrate thePVDF, twopiecesofWhatmanpaper, twosponges,andyourgel in

TransferBufferfor5-10minutes.Assembleaccordingtodiagrambelow.4. Transfergel toPVDF for1hourat100Volts. Usea stirbarand the icepackand

transferinthecoldroomtokeepthebufferfromoverheating.5. Incubateonarockerfor30minutesatRTin5%nonfatmilkinTBST,orovernightin

thecoldroom.6. Add primary antibody at the appropriate dilution in 5% nonfat milk in TBST.

Incubatefor1houratRT.7. Washblot3timesfor10minuteseachinTBST,withrocking.8. Add secondary antibody at the appropriate dilution in 5% nonfat milk in TBST.

Incubatefor1houratRT.9. Washblot3timesfor10minuteseachinTBST,withrocking.10. Incubatewithequalvolumesofappropriatedetectionreagentanddetectonfilm.

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STRIPPINGANDRE-PROBINGAWESTERNBLOT

1. Incubate blot in 100mL Stripping Buffer at 50oC for 30minuteswith occasional

agitation.Mustbesetupinthehoodbecauseofthetoxicb-MEfumes.2. Washin100mlTBST3timesfor10minuteseachatroomtemperature3. Blockwith5%nonfatmilk inTBSTforat leastanhourbeforeaddingtheprimary

antibody.TransferBuffer(for10liters)30.3gTrisbase144gGlycine1.5LMethanolTBST(for10liters)121gTrisbase88gNaClDissolvein2litersandpHto7.5Waterupto10litersAdd10mlTween20StrippingBuffer62.5mMTris-HCl,pH6.72%SDS100mMb-mercaptoethanol,addedjustbeforeuse100mlStrippingBuffer:6.25mL1MTris,pH6.710mL20%SDSH20to100mLAdd716uLb-ME

Eric Hamilton

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34.ProtoplastIsolation

Buffers: Otherreagents:ProtoplastBuffer(0.2µMfiltered) cellulase(Sigma)400mMsorbitol pectinase(Sigma)20mMMES/KOHpH5.2 0.5mMCaCl2 MSB(0.2µMfiltered) 0.5Msucrose 30mMPIPESpH6.8 10mMEDTA 5mMMgCl2 Add25mgof cellulose and8mgof pectinaseper10mlprotoplast buffer (3mlusedpersample).Cut5-10Arabidopsisleaves(~5forWT,asneededifsmaller).Useanewrazorblade,cleanlyslicetheleavesintosmallpieces(~1-2mmsquare).Placefragmentsintowellsofa6-wellplatewith3mlofenzyme-addedprotoplastbuffer.Incubatefor1hrat37°Cwithoccasionalswirling.Filter solution through 100 µm cell strainer (BD-Falcon) and collect filtrate in 50mlcentrifugetube.Rinsefilterwithadditional1mlofMSB.Spin5min.at1500rpm.Discardsupernatantbypipetting.Washprotoplastswith2mlMSB.Spin5min.at1500rpm.

Maggie Wilson

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SpheroplastPreparation

CellElongation1.Take200μLofO/Ncultureandadditto20mLLBina100MLflask.Addappropriateselectionantibiotics.2.Setup54mLLBculturein250mLflaskandplaceitinashakerincubatorat42Ctobepre-warmedandreadyforusewhentheODofthemainculturehasreachedtherequiredvolume.3.Growthemaincultureat37CwithshakinguntilOD600isapproximately0.4-0.5.4.Makefresh0.22μmfiltered10mg/mlcephalexinsolution.5.Add6mLofthemainculturetothepre-warmed54mLLB.Optoutofusingantibiotics,keepsolutionssterile.6.Initiatetheelongationgrowthofsnakesbyadding360μLofthecephalexinsolution(60μg/mlfinal).Settherpmoftheshakerto180rpm.Duringthistime,makefresh5mg/mllysozyme(120μL)and5mg/mlDNase(100μL)solutions.7.Atintervals,take20μLaliquotstoviewunderalightmicrosope.Continuethegrowthofsnakesuntiltheyreach100-150μm.Thelengthcanbe~25%lessifthenextstepbelowisrequired.8.Inducetheexpressionsystemwith0.1MIPTG.Shake30-60minutes.Spheroplasting1.Splitthesnakecultureevenlyintwo50mlFalcontubes.2.Centrifugefor5minat3000xg,discardsupernatant.3.Resuspendpelletwith2.5mlof1Msucrosebyswirlingonly,nopipetting.Repeatingthecentrifugationandresuspensionstepcanimprovespheroplastsquality.4.Add150μLof1MTris-HClpH2.7toeachresuspendedpelletsolution.Swirl.5.Add120μLofthelysozymesolutiontoeachresuspendedpellet.Swirl.6.Add50μLoftheDNasesolutiontoeachresuspendedpellet.Swirl.7.Add150μLof0.125MEDTAtoeachresuspendedpellet.Swirl.8.ThetimeaftertheadditionofEDTAiscritical.Takea15μLsampletoobservespheroplastsformationunderthephase-contrastmicroscopeeveryminute.ThishelpsdetermineifspheroplastsformationisoccurringfasterorslowerthanexpectedandthustheStopSolutionmaybeaddedatdifferenttimesthansuggested.9.FiveminafterEDTAaddition,add1mlStopSolutiontoonetubeandswirlimmediately.SevenandahalfminutesafterEDTAadditionadd1mlStopSolutiontotheremainingtubeandswirlimmediately.10.Choosethebestsample(5or7.5min)andaliquot50μLinto200μLPCRtubes(inarackonice)tothenumberofaliquotsdesired.Spheroplastsarereadyforuse.Keeponiceduringexperiments.Oncethawed,spheroplastswillneedtobediscarded.

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35.ChloroplastIsolation&Immunostaining*Adaptedfrom:Strawn,M.A.,etal,JBiolChem282,5919-5933

DAY1Prepareprotoplastsasinpreviousprotocol.Whilesamplesareincubating,prepare3%paraformaldehydeinMSB.Need2mlpersample.PreheatMSBto65°C(10secinmicrowave).InHood:PlaceMSBinsideabeakerofhotwater.Per10mlMSB,add50μlof5MNaOHand0.3gparaformaldehyde.Mixandcool.Add10μlHCL.Filterthrougha100μmcellstrainer(BD-Falcon)andcollectfiltratein50mlcentrifugetubes.Rinse leaf fragments with an additional 1ml of MSB, filter, and pool. Spin 5 minutes at1500rpminaswingingbucketrotor.Discardsupernatant.Washprotoplastsoncewith2mlofMSB.Spin5minutesat1500rpm.Discardsupernatant.Resuspendin2mlof3%paraformaldehyde/MSBtofixsamples.LetsitatRTfor30minutes.Meanwhile,preparesomemicroscopeslidesfortheprotoplasts.Useslideswithtwoetchedrings(GoldSeal#3032).Add10μlofpoly-L-lysinesolutionperringandletsitfor5minutes.Rinseusingasquirtbottleofwaterandairdry.Spin the fixed protoplasts 5 minutes at 1500rpm. Dispose of the supernatant in theparaformaldehydeliquidwaste.Washoncewith2mlofMSB,discardthewastesimilarly,andspin5minutesat1500rpm.Resuspendprotoplasts inavolumeof50-100μl.Pipet50μlontothepoly-L-lysinetreatedslidesandallow10minutestosediment.Carefullyremovetheexcessliquidbypipet.Addacoverslipandtapfirmlywithforcepstopoptheprotoplasts,releasingchloroplasts.Removethecoverslipandletairdrycompletely.Placetheslidesinaglassdishandincubate15minutesinPBS/0.05%triton(200mlPBS+100μlTritonX-100)atRT.TransferslidestoafreshdishofPBS(200ml)withouttritonandincubateforanother15minutes.RemovetheslidesfromdishandremoveasmuchoftheexcessPBSaspossible,usingatissuetowipearoundtheedgeofthering.Add50μlofPBS/5%BSA(Blockingsolution)toeachsampleandblockinahumidchamber(IuseabakingdishwithwetKimwipes,sealedwithplasticwrap)at37°Cfor20minutesonly(over-blockingwillcauselossofsignal,soIsetmytimerfor18minutes).

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Taptheslidesonapapertoweltoremovetheblockingbuffer.Add50μlof1°antibodyinPBS/5% BSA to each sample. We use affinity-purified anti-FtsZ antibody raised againstrecombinantBacillussubtilisFtsZ(Levin,P.A.,Losick,R.,1996.GenesDev10,478-488)atadilutionof1:5000.Placetheslidesinahumidchamberagainandincubateovernightat4°C(makesureyourchamberisveryhumidtopreventyourslidesfromdryingout).

DAY2Remove1°antibodyandplace slides inaglassdishand incubate inPBS for15minutes.TransfertoafreshdishofPBSandincubateanother15minutes.RemovetheslidesfromtheglassdishandremoveexcessPBS.BlocksamplesinPBS/5%BSAasbefore–20minutesinahumidchamberat37°C.Taptheslidesonapapertowel.Prepare2°antibodyinPBS/5%BSAandadd50μltoeachsample (αRabbit-FITC1:160.Allmanipulationswith the2°antibodyshouldbedonewiththeoverheadlightsoff.Placeslidesinhumidchamberandincubatefor1hourat37°Cinthedark.Rinseslides2x15minuteswithPBS.RemoveexcessPBS,asbefore.MounttheslidesusingtheSlowFadeAnitfadekit.Add1-2dropsofcomponent ‘C’ topre-equilibrate.Letsit10minutesthenremove.Add1-2dropsofcomponent‘A’.AddacoverslipanddabtheedgeswithaKimwipetoremovetheexcessbuffer.Visualizebyfluorescentmicroscopy.Storeslidesindarkenvironment.

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BUFFERS:Phosphate-bufferedSaline(PBS)137mMNaCl2.7mMKCl10mMNa2HPO42mMKH2PO4pH7.4withHCl

OTHERREAGENTS:

Cellulase(Sigmaproduct#C0615-1G)Pectinase(Sigmaproduct#P2401-1KU)3%paraformaldehydeinMSB0.1%(w/v)poly-L-lysine(Sigmaproduct#P8920)PBS0.05%TritoninPBS5%BSAinPBSPrimaryantibodySecondaryantibody(Sigma,product#F0382)SlowFadeAntifadeKit(Invitrogen,product#S2828)

Kira Veley

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89

36.AgrobacteriumInfiltrationofTobaccoLeaves

*Plants:N.benthamianaleaves,4-5weeksold.*Leaves:usuallyuse1cotyledonandfirst2or3trueleaves.* Adapted from Waadt, R., and Kudla, J. (2008). In Planta Visualization of ProteinInteractionsUsingBimolecularFluorescenceComplementation(BiFC).CSHProtoc2008.http://cshprotocols.cshlp.org/content/2008/4/pdb.prot4995.full#R11PROCEDURE1. Inoculate10mLculturesforeachexpressionconstructtransformedintoA.tumefaciens

(strainGV3101,Rif,Gentresistant,plusantibioticfortheexpressionconstruct)andofp19(strainAgl1,useKanforthep19andCarbforthestrain).Makeone10mlovernightculture forevery3 injections thatyouplan touse theculture for. Growovernight inshakerat28°C.Uselargecolonies.Note:p19isasuppressorofgenesilencingfromthetomatobushystuntvirusandmustbeco-infiltratedforpropergeneexpression.

2. Measure theOD600 of the overnight cultures. Thiswill be used to calculate dilutions

duringspin.3. Centrifugethemixturesat3000gfor15minatroomtemperaturein50mlFalcontubes.

Makeactivationbuffer.4. Discard the supernatant and resuspend the mixtures to the appropriate OD600 with

ActivationBuffer.ExpressionconstructsshouldbeusedatanOD600of0.5andp19shouldbe0.3.Iusuallymake10mlcultures,but5mlisokay.

5. Incubate themixtures for at least 2 hours at room temperature in falcon tubeswith

gentileshaking.6. Infiltrate themixturesusing a1mL syringewithout aneedle into the abaxial sideof

tobaccoleaves.Incubatetheplantsinthegreenhousefor3-6d,dependingonthelevelofproteinexpression(Iusuallyused5days,butifdoingforthefirsttime,checkat3and7days also. I also found that Iwould seepoor expression fromplants grown in thegreenhouse during hot summer months. Growth in a growth chamber seems to benecessaryatthattime.

SOLUTIONS

ActivationBuffer(100ml)–makefresheverytime1ml 1MMES/KOH(pH5.6),filter-sterilized1ml 1MMgCl2,autoclaved

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100μl 150 mM Acetosyringone (3′,5′-Dimethoxy-4′-hydroxyacetophenone) in DMSO,storedat−20°C

Kira Veley

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37.BimolecularFluorescenceComplementation

*Adapted from Waadt, R., and Kudla, J. (2008). In Planta Visualization of ProteinInteractionsUsingBimolecularFluorescenceComplementation(BiFC).CSHProtoc2008.http://cshprotocols.cshlp.org/content/2008/4/pdb.prot4995.full#R11* Vectors from Gehl, C., Waadt, R., Kudla, J., Mendel, R.R., and Hansch, R. (2009). NewGATEWAY vectors for high throughput analyses of protein-protein interactions bybimolecularfluorescencecomplementation.MolPlant2,1051-1058.*DrawbacktoBiFCandsuggestedcontrols:Lalonde,S.,Ehrhardt,D.W.,Loque,D.,Chen,J.,Rhee,S.Y.,andFrommer,W.B.(2008).Molecularandcellularapproachesforthedetectionofprotein-protein interactions: latest techniquesandcurrent limitations.Plant J53,610-635.VECTORS:G1:pDEST-(gateway)-MYC-VYNEG2:pDEST-VYNE-MYC-(gateway)G3:pDEST-(gateway)-HA-VYCEG4:pDEST-VYCE-HA-(gateway)G#=shorthandusedtorefertothevectors(gateway)=wheregeneisinsertedafterLRreactionVYNE=N-terminalhalfofVenustagVYCE=C-terminalhalfofVenustagHA=HAtagforwesternsandpull-downsMYC=MYCtagforwesternsandpull-downsG1andG2harborabacterialKANresistancegeneandaplantHYGresistancegeneG3andG4harborabacterialandaplantKANresistancegeneCONTROLS:G15:pEXP-VYNE-CNX6,KANresistantinbacteria,HYGresistantinplantsG16:pEXP-VYCE-CNX6,KANresistantinbothbacteriaandplants

PROCEDURECheckthatyour finalconstruct iscorrectwithacombinationofPCRanddigestionof themini-preppedproduct.YoucoulddothiswithcolonyPCRaswell.TheidealtestistoPCRamplifyusingapairofprimerswhereoneisspecificforyourinsert/geneandtheotherisspecificforthedestinationvector(ex:35Spromoter,GFP-UP(bindsVYCE),NOSterminator,oranattBsite(createdafterLRreaction)).Thendigestthisproducttomakesureitiswhatyouthinkitis. Ihadtroublewiththisinthepast. Also,lowyieldafterminiprepistobeexpected,asthesevectorsarequitelarge.

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*Expressconstructsusingtransientexpressionintobacco(see“Agrobacteriuminfiltrationoftobaccoleaves”).Iusuallycheckforinteraction5daysaftertobaccoinjections.Confocalsettings(eYFP):excitewith515nmlaserandcollectwith535-565nmband-passfilter.

Greg Jensen

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38.DNAParticleBombardment

PREPAREMICROCARRIERS

1. Weighout15mgofgoldmicroparticles.Thismakesalotbutthebeadscanbestoredat4°Cfollowingstep6.

2. Add1mlof70%ethanol,vortexwell.3. Incubatefor15minutes,vortexperiodically.4. Spinfor5seconds.5. Removeliquid,washpelletwith1mlwater–vortex,sonicate,letsettle,spin2seconds.Do

this3times.6. Add500μlof50%glycerol,vortextomix.Storeat4°C.

COATMICROCARRIERS

1. Rinsecarrierdiscswith100%ethanoltoremovedebris.Dryonapapertowel.Insertintometalcarriagesandsealtheedgesusingtheredplastictool.

2. Coatparticles:Haveallreagentsreadyandmovequicklythroughthenextsteps.Volumeslistedbelowarepersample,butifusingthesameDNAformultiplesamplesyoucanprepareitinbulk:---vortexgoldmicrocarriers,sonicate–makesuretheyarecompletelysuspended.---aliquot9μlofparticlestoatube.---vortex,sonicatetheremainingparticles,aliquot9μltothenexttube,etc.---vortex,thenadd2μlof0.5mg/mlDNA.---vortex,thenadd9µlof2.5MCaCl2.---vortex,thenadd4µlof0.1Mspermidine.---vortexfor1-2minutes.3.Allowmicroparticlestosettlefor1minute.4.Spinfor2seconds.5.Removesupernatant,washwith125µl70%ethanol–vortex,sonicatebriefly,letsettle,spinfor2seconds–thenwith125µl100%ethanol.6.Resuspendin10µlof100%ethanol–vortex,sonicatebriefly.7.Pipetall10µlontothecenterofacarrierdisc(discsideUP).8.Letdryonthebenchtopfor~15minutes.

BOMBARD

1. PlaceleavesonMSplates,clusteredaroundthecenter.Trytochooseleaveswithminimalcurling.Pressdownwithyourfingersothattheysticktothemedia

2. Bombardusing450psipressurediscs.(forleekandonionepidermis,use900psipressurediscs.PeelofftheinnerepidermisandplaceinsideoftheinnerlayerfacinguponMSplates

3. Sealwithparafilmandincubateonthebenchtop,preferablyinthedark.Cansometimesseesignalin4-6hours,definitelybyovernight.

USINGTHEBIOLISTICBOMBARDMENTMACHINE

1.Unscrewtheholderatthetopofthechamberanduseforcepstoplaceapressurediscintotheverybottomoftheholder.Tapitonthetableuntilthediscsettlesintothebottom.Screw

94

theholderbackintothetopofthechamberandtightenwithatorquewrench(inthedrawerbelow).Tightenbeyondthetorquepoint.Itmustbeverytightorthechamberwillnotpressurize.

2.Unscrewthemetalcaponthetopofthewhiteremovableshelf.Placeastoppingscreenabove the hole in the center, and flip themetal piece containing theDNA-coated carrierupsidedownoverthescreen.Screwthemetalcapbackontotheassembly.3.Slidethiswhiteshelf,notchestotheback,intothetopshelfholder(thecloserthisistothepressuredisc,thegreatertheforcethatblastsintothecarrierwiththeDNA).RemovethelidfromtheMSplateandputtheplateontheclearshelf. Placetheshelftwonotchesbelowthewhiteshelf.Closethedoortothechamber.4.Opentheheliumtanklineusingthevalveonthetopofthetank.Itshouldread>=1500psi;ifthelevelisbelowthisthenthetankneedstobereplaced.5.Turnonthebiolisticmachine(leftmostredswitch).6.Turnonthevacuumpump(blackswitchtowardstheback).7.Flipthemiddleredswitchonthebiolisticmachinetothetopmostsetting("VAC").Watchthemetertotheleftofthechamber-theneedleshouldstartturningcounter-clockwise.8.Whentheneedlehits25-27,flipthemiddleredswitchtothebottomsetting("HOLD")andthenflipandholdtherightmostredswitch("FIRE").Afterafewsecondstheneedleinthemeterabovethechambershouldstartclimbing.Youshouldhearapopwhenitreachesthepsiatwhichthepressurediscruptures,andtheneedleshoulddropbacktozeroagain.Ifyoudonothearapop,andthemeterhasnotreachedthepsiofthepressuredisc,you

needtogobacktostep1andtightentheholder.

9.Releasethe"FIRE"switchandflipthemiddleswitchtothe“MIDDLE”settingtoletthevacuumdissipate.Turnthevacuumpumpoff(orleaveitonifyouenjoythenoise/smell).10.Oncethepressureinthechamberreturnstozero,openthedoorandmakesurethat(1)thepressuredischashaditscenterblownout,and(2)theDNAcarrierhassmashedintothestopping screen;both the stopping screenand theDNAcarrier shouldbedeformed, andthereshouldn'tbeanyDNAleftonthecarrier.11.Afterthebombardmentiscomplete,bleedtheheliumlineasfollows:

(a)Closethevalveontheheliumtankcompletely.(b)Turnonthevacuumpump(andkeepthebiolisticmachineon).(c)Switchthemiddleswitchtothe"VAC"position(up),letitpullavacuumto~20

psi,andholdthe"FIRE"switchon.Waittillthemetersontheheliumtankbothdroptozeroandthenreleasethe"FIRE"switch.

(d)Turnthemiddleswitchoff(middleposition),turnthevacuumpumpoff,turnthebiolisticmachineoff.

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39.ViabilityStaininginTobacco

* Express constructs in using transient expression in tobacco (see transient expressionprotocol).Doaninjectionofp19onitsownforanegativecontrol.* If possible: it’s good to randomize your samples after injection and only check theiridentityaftercountingiscomplete.*Adapted From: Chaves, I., Regalado, A.P., Chen, M., Ricardo, C.P., and Showalter, A.M.(2002). Programmed cell death induced by (β-d-galactosyl)3 Yariv reagent in NicotianatabacumBY-2suspension-culturedcells.PhysiologiaPlantarum116,548-553.ADDITIONALINFOA dual staining with Fluorescein diacetate (FDA) and propidium iodide (PI). FDA is afluorescent indicatorofcellviability,asonly livingcellsprocessnon-fluorescentFDAandproducethefluorescentcompound(signalexcitedat488nmandemissionscollectedwitha505-to525-nmband-passfilter).PIpreferentiallypenetratescellswithdamagedorleakingcellmembranes,indicatingdyingordeadcells,andotherwisestainstheplasmamembrane(signalexcitedat543nmandemissionscollectedwitha560-to5660-nmband-passfilter).

PROCEDURE

1. Exciseleafsamplesandimmerseinthestainingsolution(500μg/mlFDA(5mg/mlstockdissolvedinacetone)plus1.25μg/mlPI(2.5mg/mlstockdissolvedinwater))for20minutesbeforerinsinginwaterandimaging.

a. usea60Xlensinordertoclearlyvisualizethecells.

2. Forquantification,IopentheimagesinPhotoshopandputlarge,greendotsonlivecellsandreddotsondeadcellssothatIhavearecordofwhatwasscored“live”and“dead”

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40.HechtianStrandProduction

*NaClconcentrationdependsonthicknessoftissueTHINGSTODOBEFORESTARTING§ Make5MNaCl(thickertissue)or1MNaCl(moreporoustissue)PROCEDUREIliketomountthetissueonaslidedirectlyin5MNaClthenwatchHectianstrandformationhappentogetthebestpictures.Notethetimeittakesforreportinglater.Ifmountingin5MNaClistoofastorcausestoomuchdamage,switchto1MNaCl.

Liz Haswell

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41.GUSReporterStaining

*AdaptedfromMeyerowitzlabprotocol:STOCKSOLUTIONS

• X-Gluc(cyclohexylammoniumsalt)Makeup50mg/mLinDMF.Besttomakeupfresheachtime,justbeforeaddingtotheassaybuffer,butyoucanfreezeitat-20.Whenitturnsred,it’sgonebad.

• 200mMNaP04,pH7Mixsolutionsof200mMNa2HPO4and200mMNaH2PO4untilthesolutionreachespH7.

• 100mMK3Fe(CN)6potassiumferricyanide

• 100mMK4Fe(CN)6potassiumferricyanide

• icecold90%acetone(optional)

STAININGSOLUTION

100mMNaP04pH75mMK3Fe(CN)65mMK4Fe(CN)610mMEDTA0.5mg/mLX-Gluc0.1%tritonX-100PROCEDURE

1.Immersetissueinice-cold90%acetone(ineppendorftubesonice)tofix.Thisstepisoptional,butwillhelpwithchlorophyllremovalandtissuepreservation.2.Placeseedlingsinthewellsofa24-wellplatecontainingapproximately0.5mLstainingsolution.3.Sealplatewithparafilmandwrapwithaluminumfoil.4.Incubateat37degreesforonehourtoseveraldays.Monitorsignalproductionasyougo.5.Ifdesired,performanethanolseriestodehydrate.Incubatethetissuein0.5mlof15%ethanol,30%,50%(orinsteadof50%usedFAAfixative,whichis50%ethanol,5%aceticacid,and3.7%formaldehyde),70%,85%,95%.

Maggie Wilson

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42.ProlineQuantification

*AdaptedfromAbrahametal.,2010PROCEDURE

1. Harvestthesamples,measuretheirfreshweight,andusebetween30-100mgforareaction.Keepthetubesonice.Note:UselesstissueforsamplesexpectedtocontainlargeamountsofPro.

2. Grindtissuewithpestlein500μl3%sulfosalicylicacid(CanmakeinadvanceandstoreatRTforupto2weeks).Keepthetubesoniceuntilfinishedwithallsamples.

3. Centrifugesamplesfor5minatroomtemperatureusingbenchtopcentrifugeatmaximumspeed.

4. Preparethereactionmixtureinaseparatetube:a. 100μl3%sulfosalicylicacidb. 200μlglacialaceticacidc. 200μlacidninhydrin(Seebelow)

Note:Itisbesttoprepareamastermixforthereactionmixture.

5. Addbetween25-100μlofsamplesupernatantto500μlofreactionmixture.Note:AddlesssupernatantforsamplesexpectedtocontainlargeamountsofPro.

6. Incubatetubesat96°Cfor60min.

7. Terminatereactionbyplacingthetubesonicefor5min.

8. Add 750 μl tolulene to extract samples, vortex, allow samples to sit on bench forapproximately5mintoalloworganicandwaterphasestoseparate.Note:Samplescanbestoredovernightat4°C.

9. Measuretheabsorbanceat520nmusingtolueneasreference.*Mustusequartzcuvettes,tolulenewillmeltdisposableplasticcuvettes.

10. Proconcentrationscanbedeterminedusingastandardconcentrationcurveandcalculatedonafreshweightbasis.

Pro (μmol/g FW) = (Absextract/slope)*(volume of reactionmixture/volume of plant

extract)*(1/sampleweight(g))

ProlineStandards

μM/mL

μltoaddof1mML-Proline

5 0.1 504 0.075 37.53 0.05 252 0.025 12.5

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1 0.012 6

AcidNinhydrin:

Tomake20mldissolve0.5gninhydrin(1,2,3-indantrionemonohydrate)in12mlglacialaceticacidbystirringat65°C.(solutionwillbedarkblue).Add8mlof6Morthophosphoricacid(solutionwillturnyellow).Canbestoredat4°Cforupto1week.

Maggie Wilson

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43.ABAGerminationAssay

PlatePreparation:

-Make750mlof½MurashigeandSkoog(MS)media+0.3%Sucrose

1.6125gMSsalts2.25gSucrosepHto5.7with1MKOHH2Oupto750ml,Mixwell

-Splitinto3flask(use500mlflask),250mleach-Add2gCaisson’smicroprapagationagar-Autoclave30min-AdddesiredconcentrationofABAtoeachflaskusinga10mMworkingsolutionofABAinPBS.Example:

Flask#

Concentration(μM) ABAsolution(μl)

1 0 02 1 253 3 75

-75mlperlargeroundplate-LetdryovernightPlatingSeed:

-Surfacesterilizeseedwith50%bleachsolution+Tween-20(seesterilizationprotocol)-UsingP-1000andsteriletipsplace50-100seedpergenotypeinindividualwedges.-3platesperconcentration.-Sealplateswithmicroporetapeandplaceat4°Cfor3days.Scoringgermination:

-Germination=Radicleemergence-Recordgerminationatdifferenttimepoints.-Calculategerminationrate #ofseedgerminated/Total#ofseed

Seed Coat Rupture

Endosperm Rupture =

Radicle emergence =

Germination

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Examplewildtype

era1-2(hypersensitivecontrol)abi4-1(insensitivecontrol)

mutant1

mutant2

complementedline

Kira Veley

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44.SugarExtractionfromPlantCells

*Adaptedfrom:Dumez,S.,etal.,MutantsofArabidopsislackingstarchbranchingenzymeIIsubstituteplastidialstarchsynthesisbycytoplasmicmaltoseaccumulation.PlantCell,2006.18(10):p.2694-709.THINGSTODOBEFORESTARTING

§ Collectrosetteleavesatendofdayandimmediatelyfreezeinliquidnitrogen.Storeat-80°Cuntiluse.

PROCEDURE1. Grindtissuewithmortarandpestleinliquidnitrogen.Weighout200-300mgoftissuefor

eachsampleina2mltube(3replicatesofeachtissuetypeisideal).

2. Add1mlof ice-cold16%trichloroaciticacid(powder,4°C) indiethylether(1.6g in10mlether–makefresh).Keepsamplesoniceatalltimes.

3. Add10µlof0.5MEGTAandgrindfurtherwithpestle.Incubateat4°Cfor2hours.

4. Spinat16,000Xgfor5minat4°Ctopelletdebris.Removeaqueousphase(bottomphase

minussolids)andtransfertoanewtube.

5. Washaqueousphasefourtimeswithdiethylether,centrifuging5minat16,000Xgat4°Cbetweeneachwashandremovingtheether(toplayer).

6. Transferaqueousphasetoanewtubeandneutralizewith10µlof5MKOH(50mMfinal)and

5µlof2Mtriethanolaminebufferstock(10mMfinal)andstoreat-80°C.SOLUTIONSTriethanolaminebuffer(TEA)2Mstocksolution–storeatroomtemperature10mlsolution2.65mlTriethanolamine(MW149.19g/mol,density1.124g/mLat25°C)(2M)0.2ml0.5MEDTA(MW372.24g/moldihydrate)(10mM)24.6mgMgSO4(MW246.48g/molhepta-hydrate)(10mM)10mgSodiumAzide(0.1%)pH7.4HCl

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EGTA0.5Mstocksolution–storeatroomtemperature,stable10mlsolution1.9gEGTA(MW380g/mol)ddH2Oto9mladjustpH7.5/8.0withsolidNaOH(>0.4g)adjustvolumeto10mlNote:EGTAwillnotgointosolutionwithoutNaOH.OncethepHhasbeenraisedsufficientlyitdissolvesquickly.ForpH7.5theexactamountrequiredisslightlyabove0.4g.Add0.35-0.4gimmediately,thenproceedcarefullynottoovershootthedesiredpH.

Greg Jensen

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45.PollenViabilityAssay

PROCEDURE1. GrowArabidopsisasusual:Putoutonepoteachofmsl8-1,msl8-4,Col-0,andLer;15-20seeds

intorectangularpots;stratifyinthecoldroomfor4-5days;transferto16-24hrlightchamberandgrow~3weeksuntilflowering.

2. Preparestocksolutionsforstainingandvisualizingpollen:Propidium Iodide (PI) – taken up by ‘compromised’ pollen and stains red. Prepare a 1mg/ml

solutioninwater.FluoresceinDiacetate(FDA)–enzymaticallyprocessedinthemembraneoflivecellsandstainslight

green.Preparea2mg/mlsolutioninacetone.§ Storebothsolutionsat4°C.3. Prepare2Xstainingsolution: 2µl1mg/mlPI 0.5µl2mg/mlFDA 1mlwater4. Preparehumidchambers:Fillanempty200µlpipettipboxwith~100mlofwater.Insert8pipettipssoastoprovidesupport

fortworectangularslides. Foldakimwipetissueandplacebetweenthepipettips. Wetthetissuecompletelywithwater

5. Spot50µlofwaterontoarectangularmicroscopeslide,oneslideperlinetobeanalyzed.Using

forceps,selectflowerswithpetalsthathavejustopened.Pinchthebaseoftheflowerwiththeforcepssothattheanthersareexposed.Invertthefloweranddabthepollenontothesurfaceofthewater.Do3-4flowersformsl8-4andLer,and5-6flowersformsl8-1andCol-0.Thepollenshouldlookdenseontheslide.

6. Inverttheslide(quickly,carefully)andplace,dropletdown.ontothepipettipsupportinthe

humid chamber. Close the lid. Remove the chamber to a location with a 21-22°C stabletemperaturee.g.themicroscoperoom.Formsl8-4andLerpollen,lettheslidessitfor30minutespriortoanalysis.Formsl8-1andCol-0pollen,lettheslidessitfor~2hrspriortoanalysis.

7. Remove the slide from the humid chamber and invert (quickly, carefully). Add 50 µl of 2X

stainingsolutionandmixbypipetting. Adda20mmx40mmcoversliptotheslide. Dabtheedgesoftheslidewithatissuetoremovetheexcesswater. Letsit for~15minutespriortoanalysis.

8. Analyzeusingthecamerasoftware:GFPfilterforFDA,RFPfilterforPI.Shootforn=100.Focus

onafieldof25-50pollengrains.StartingwiththeRFPfilter,manuallyadjusttheaperturesothatthedifferencebetween‘compromised’and‘intact’pollenisclear.Takeapicture.Switchtothe GFP filter – the signal will be very bright – reduce the aperture to the point where

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‘compromised’pollenarenearlyinvisible.QuicklytakeapicturebeforetheFDAsignalbleachesout.Dothisfor2-4separatefields.

9. UseboththeFDAandPIpicturestoaidincounting‘intact’vs.‘compromised’pollen.Reportas

a%ofthetotalpollen.

Eric Hamilton

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46.InVivoPollenHydrationAssay

*Measuresthehydrationofpollengrainsthatarehydratedonastigma.*Whatismeasuredisthechangeinequatorialdiameterofthepollengrainasithydratesonastigma.Emasculatedflowers(withstamensremovedpriortopollendehiscence)mustbeused.Itisconvenienttousethems1-1malesterilelineintheLandsbergbackground:halfoftheseededplantswillnotproduceanymaturepollen.*Adaptedfrom:ChapmanandGoring,2011andUpdegraffetal.,2009.PROCEDURE

1.Preparepistils a.Emasculatedormale-sterilepistilsareembeddedinagarosetopreparethemforpollination.Makeabouta1.5%agarosesolutioninwaterandprepareabedofagarosebypipettingafewhundredµlofhotagaroseontoaslide.Allowtocoolandsolidify. b.Oncetheagaroseissolid,cutthepistilatthebaseoftheflowerusingtweezers.Pushthetweezersintotheagarosetoplacethepistilwithinitsothestigmaisexposedabovetheagarosepad.2.Preparemicroscope a.Theimagingmustbedoneimmediatelyafterpollinationsothemicroscopemustbereadiedaheadoftime.Nolasersarerequired.PulluptheOlympusDPsoftwaretocontrolthe camera and just use the Fluoview software to control the microscope. At 20x theobjectivewillnottouchthesample.Setuptimelapsetocaptureanimageeveryminutefor25minutes.Grayscalehelpsreduceimagesize.3.Pollinate a.Bringthepistilsembeddedinagaroseintothemicroscoperoom.Bringtheplantsdonatingpollenaswell. b. Under the dissecting microscope, *lightly* pollinate the embedded stigma of asingle pistil with the pollen. Light pollination helps reduce obscuring tissue duringmicroscopy.Thebestlocationforpollinationistheveryedgeofthestigma(youcanimagineahalogoingaroundthestigmaatitswidestdiameter).Againthisistomakeasmanypollengrainsvisibleduringimagingaspossible.4.Image a.Placetheslideundertheobjectiveandbringituptofocus.Focusontheplanethatprovidestheclearestviewofthemaximumnumberofpollengrains.Begintimelapse. b. The focusmust bemanually adjusted throughout the timelapse, approximatelyeverycoupleofminutes.Itisusefultokeepaneyeonasinglepollengraintokeepinfocus.However,thegoalistokeepasmanygrainsaspossibleinfocusfortheentireexperiment.

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5.Analyze ImportthesequenceofimagesforasinglepollinationintoImageJ(File>Import>ImageSequence).Thisconvertsthemtoastack.Measureasinglepollengrainatatimeforthe25minutes.Drawalineacrosstheequatorialdiameterofthepollengrainforeachframeandmeasure(hitMonthekeyboard).Dothisforthe25framesandthenrepeatforasmanypollengrainsasremaininfocusfortheexperiment.

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47.PollenGermination

*Pollengerminationonsolidifiedmediumforobservingpollentubes.Canalsobeperformedinliquidmediaforotherformsofquantificationsuchasgerminationrates.PROCEDUREForgermination in liquidmedia:Pipetteabout30 µlofmediaontoanetched-ringglassslide. Take whole flowers or individual stamens and dab into media to release pollen.Generally3-5flowersprovidesagoodamountofpollen.Incubateupside-downina“humidchamber”:apipetteboxwithwateratthebottomandmoistkimwipesonthesurfacewithtipsusedasstiltsfortheslides.Canbesealedwithparafilm.Incubateascloseto22±2°Caspossible.Lightshouldnotaffectthepollen.For germination on solid media: Prepare a 1 to 1.5% agarose solution with the pollengerminationmediabyweighingout theappropriateamountofagarandboilingwith thedesiredvolume(generally1mlworkstomakeseveralagarosepads).Agarosepadsaremadeasfollows:Tapeaslideatbothends(theheightofthetapewillbetheheightofthefinalagarosepad).Pipettearound50µlofhotagarosemediaontothecenteroftheslide.Placeanother,untaped,slideontop.Thiswillevenlyspreadthemediatotheheightdictatedbythetapeseparatingthetwoslides.After20-30min,gentlypulltheslidesapart—generallythisworksbestby‘sliding’oneslideofftheother.Itmaybeusefultolettheagarosepaddryforacoupleminutes.Applypollenbytakingwholeflowersand‘brushing’themacrosstheagarosepad,usingthepetalstoevenlyspreadthepollendepositedbythestamens.Generally3-5flowersprovidesagoodamountofpollen.Incubateasforliquidmedia.SOLUTIONSPollenGerminationMedia(25ml)*fromBoavidaandMcCormick,200710%Sucrose 2.50g0.01%BoricAcid 250µl1%H3BO35mMCaCl2 125µl1MCaCl25mMKCl 125µl1MKCl1mMMgSO4 25µl1MMgSO4*pHto7.5with1MKOH(requiresverysmallvolume,soitisusefultodiluteitwithDIwaterbeforeadjustingpH).ThepHdriftsdownovertime.Canbeadjustedback,generallygoodforoneweek.Canbefiltersterilized.

Grigory Maksaev

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48.XenopuslaevisOocyteRetrievalandInjection

EXPERIMENTALDESIGN

• Surgery is notperformeduntil theRNAhasbeenmade andquantified.ThispreventsunnecessarysurgeriesinthecasethatRNApreparationisnotgood.• Thesurgeryandpreparationoccuronthesamedayandinjectionsaredonethefollowingday.• Analysiscantakeplaceasearlyas24-48hoursandaslateas7-14(upto21inrarecases)daysafterinjection.TOBRINGTOANIMALFACILITY

1. Labcoat.2. Gloves.3. Eyeprotection.4. 70%ethanol.5. Timer.6. Protocol.7. IDcardforentry(PINisrequired).8. Sterilesurgicaltools,includingforcepsandscissors.9. Sutures(2types,2pieceseach).10. 10ml100XPenn/Strepsolution.11. 25mlND96bufferina50mlconical.12. 0.5LbottlefordilutingPenn/Strepsolution.13. 0.5Lbottlewith0.75gMS-222and0.42mgNaHCO3foranesthesia.14. Filter-sterilized(orautoclaved)ND96buffer.15. Wastecontainer.SURGERY

1.Makeup0.5 L of 1.5 g/LMS-222 (anesthetic) solution in treatedwater from the frogfacility,alongwith840mg/LNaHCO3asabuffer(0.75gMS-222+0.42gNaHCO3per0.5L).2.Immerse a frog in theMS-222 solution for 10-15min, until she does not respond topitchingoftheskinbetweenthetoes.13minseemstobeoptimal.3.Performthesurgerywithsterile(autoclaved)instruments,onasterilebench-top,usingsterilegloves.KeepthefrogmoistwithsterileND96solution(putthefrogonawetpapertowel).4.Makeasmallincisionthroughtheskinoftheabdomen,usingsharpscissors,followedbyanincisionofabout1cminthemusclelayer.RinsewithND96.(Whencuttingslipperyskin,graspitwithtwofingersthroughapieceofpapertoweltoincreasefriction).PlaceasheetofParafilm(withanorificeinit)aroundtheincision,sothattheoocytesextractedfromtheabdomenwill contactwith Parafilm but notwithmucous surface of the frog’s skin (notnecessary,butitlooksliketheoocytessurvivelongerinthiscase).5.Pullapprox.2-4mlofoocytesoutofthebodycavitywithdullforcepsandplacein25mlof

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ND96ina50mlconicaltube.6.Repairtheincisionwith2setsofsutures,oneforthemuscle(itwilldissolvewithtime)andone forskin (Monocrylpoliglecapron25undyed filament isused for themuscleandEthilonnylonsutureblackmonofilamentfortheskin,bothfromEthicon).Eachstitchismadewith2loopsofsingleknotsandathirdloopofdoubleknots(3stitchesperincisionof1–1.5cmarefine).Thelastknotshouldbepulledtight,andtheendstrimmedclosely.7.Afterthatwashthewholefrogwithapprox.0.5Lofcleanwatertakenfromthefacilityinordertogetridoftheleftoversoftheanestheticonitsskin.8.Then,allowthefrogtorecoverin500mlofPenn/Strepsolution(madeupintreatedwaterfromthefrogfacility)for40-60min.Tilttheplasticbathwithbuffer~450,sothatthemostofthefrog'sbodyissubmerged.Caremustbetakenthatthefrogdoesnotdrown(keephernostrilsabovethewaterlevel)butherskinstayswet(first,she'llstartbreathing,whenshestarttomoveinresponsetopitching,she'sOK).Oncesherecovers,returnhertoacleancage(withwaterreplaced).OOCYTEPREPARATION

1.Separatetheovariantissueintoclampsofabout10-20oocyteseach,usingforcepsandworking under stereomicroscope. Transfer the clamps into Ca-free ND96. Try to avoiddamagetotheoocytesasmuchaspossible.2.IncubatetheoocytesinaPetridish,withslowshaking,for40-50minatroomtemperaturein20mlofCa-freeND96with0.08–0.1gcollagenase(CollagenaseATypeIfromSigma).Ca-free medium should be used to slow down the reaction as Ca2+ strongly activatescollagenase.Stoptheprocessastheclumpsjustbegintoreleasesingleoocytes.3.Washtheoocytes5x25mlND96(Ca-free)+0.1%BSA(0.25g/250ml).4.Add25ml0.1Mpotassiumphosphate+0.01%BSA,pH6.5,incubatefor45-50minonaslowshaker.(Thesolutionismadeinadvanceandthenstoredin

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the-200Cin25mlaliquots).At15minintervals,drawtheoocytesintoawide-boreplasticpipette (madeby trimming the tip) andpipetteupanddown to facilitatebreakingupofclumps.5.Washtheoocytes5x25mlND96(Ca-free)+0.1%BSA(0.25g/250ml).6.Sortoocytes,selectingonlymatureoocyteswithclearanimal/vegetalpoledivisionsandwithoutanyspots,markingsorresidualovariantissueonthesurface.StorethoseinND96+50mg/mlGentamycinat180C.RNAPREPARATION

UsethemMessagemMachinekitfromAmbion(SP6kit,AM1340forourconstructs).Startingwith5μgofminiprepDNA,add1μglinearizedDNAtoeachRNAreaction,performedexactlyasdescribedinthekitprotocol,andre-suspendthefinalRNApelletin25μlofwater.ThisresultsinRNAatabout1μg/μl.RNAisthenstoredfrozenin3-4μlaliquots.DNAmustbecompletelylinearizedforthereactiontoproceedwell.OOCYTEINJECTION

1.ThawRNAse-freewaterandRNAonice.2.Pullneedles(morethanyouthinkyouwillneed)onpullersetting#8.Trimtheendofeachneedlewithscissors~5-6mmoutsidethespotwheretheygetlessbendy(youwillneedtodothisunderthemicroscope).3.Backfilltheneedlewithabout15μlofmineraloilusingayellowtipandapipetman,thenapplytheneedletotheinjectorapparatus.Makesuretherearenobubblesintheoil,thenpull500nl(byturningtheknobcounterclockwise“50”)ofsterilewaterintothetip(fromaPetriplate),followedbyall4μlofyourRNAsample.4.PlaceoocytesintoagriddedPetridish,infreshND96.Makesureyoucaninjectwithoutbubblesbyejecting50nl(“5”onapipette)outsidethedish.Injecteachoocytewith50nlinjectionsolutioninthelightside,oratthelight/darkinterface.Returntothe6-wellplateandincubateinND96with50mg/mlGentamycinat180C.5.ChangetheND96(withantibiotic)andremovedefectiveoocytesonadailybasisuntilanalysis.ND96buffer: 1L 2L NaCl 96mM 5.61g 11.22gKCl 2mM 0.15g 0.30gCaCl2 1.8mM 1.8ml* 3.6ml*MgCl2 1mM 1.0ml* 2.0ml*Hepes 5mM 1.2g 2.4gpH adjustto7.38 *Of1Msolution,filter-sterilized,keptinafridge.

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49.PurificationofTaqPolymerase

*FromChorylab,takenfromPluthero,F.G.(1993)NAR21:4850-4851

THINGSTODOBEFORESTARTING§ RinseoutallglasswarewithddH2Opriortostartingtogetridofdetergentresidue§ NeedIPTG,bufferA,lysozyme,lysisbuffer,ammoniumsulfate,storagebufferPROCEDURE

1. Use100µlofglycerolstockIJ3(carbresistance)toinoculate1LLBa. StrainINVJaF’containingthepTqplasmid

2. Grow1Lculture11hours(O.D.~0.8)a. AddIPTGtofinalconcentrationof124mg/Lb. Incubateandshakeat37oCfor12hours

3. Centrifugeandwashwith100mlbufferAa. Resuspendin50mlbufferAb. Add4mg/mllysozymec. Incubate15minatroomtemp

4. Add50mloflysisbuffera. Incubateat75oCfor1hourb. Centrifugeat10,000rpminSS34for15minat4oCtoclearlysate

i. Don’tuseblue-captubes,theycrackathighspeedsc. Overthecourseof~30min(slowly)add30gammoniumsulfate((NH4)2SO4)

per100mlwithstirringi. Avoidbubbles,enzymesdenatureatair/liquidboundariesii. Usethesmalleststirbaronthelowsettingatroomtemp

d. Letstiranadditional1houre. Centrifugesolutionasinstep4bf. Harvestprotein

i. Somewillbeinapellet,muchwillbeonthesurfaceandwalls

5. Resuspendproteinin20mlbufferAper100mlclearedlysatea. Dialyze2x12hours(useslide-a-lyzersovernight)againststoragebuffer

6. Diluteresultingprotein1:1withstoragebuffera. RunPCRusingdilutionseriesofnewtaq(dilutewithstoragebuffer)b. Determineappropriatedilution,ifnecessary

7. Aliquotandstoreat-80oC

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SOLUTIONSBufferA LysisBuffer StorageBuffer50mMTris-HClpH7.9 10mMTris-HClpH7.9 50mMTrisHClpH7.950mMdextrose 50mMKCl 50mMKCl1mMEDTA 1mMEDTA 0.1mMEDTA 1mMPMSF* 1mMDTT 0.5%Tween-20 0.5mMPMSF* 0.5%NonidetP4050%glycerol*OrPefablocat1mMworkingconc.100mg/ml=0.4MPefabloc;25mg/ml=0.1MPefablock.Sterilization:FiltersterilizeBufferA(donotautoclave). LysisBufferdoesnotneedtobeautoclavedifusedrightaway(within24hours).DoNOTaddPMSF/PefablocorDTTuntilAFTERautoclaving.Chillbuffersbeforeuse.

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50.HypoosmoticShockAssay

(PaulBlount’sLaboratory)

Youwillneed:- Citrate-phosphateminimalmedia(CphM):normalglucose,1MNaCl+normalglucose,and0.5

MNaCl+normalglucoseversions *Thiamine-HClsolnneedstobestoredat4Cinthedark *Storeironsolnindark- SterileH2Oand0.5MNaClCphM+normalglucosepre-warmedto37Conthedayoftheassay

fordilutions(seeprotocolfordetailsonvolumes)

Day1:Transformconstructsyouaretestingintoosmoticshock-sensitiveMJF465cells.Alsotransformthefollowingcontrols: FRAG-1cells+emptyvector(positivecontrol)

*FRAG-1cellsaretheparentstrainofMJF465cellsandareresistanttoosmoticshock*

MJF465cells+emptyvector(negativecontrol)MJF465cells+WTMSionchannel(shouldhave~80%survivalofshockedvs.nonshockedcells)

Day2:- 1.Inoculate3mL0MCphMwith5-10colonies.Shakeat37CuntilOD600~0.1- 2.Remove1.5mLofculture,discard.Add1.5mLof1MNaCLCphMtoremainingculture.

ShakeuntilOD600~0.1.- 3.Add30microliters0.1MIPTG(finalconc.1mM)andshakeforanadditional30-60minto

induceconstructexpression.MeasureOD600afterinduction.- 4.Dilute50microlitersofcultureintoeither950microliterssterileH2O(shockedsamples)or

0.5MCphM(bothpre-warmedto37C)ina14mLculturetube.Vortextomixandshakeat37Cfor15min.

- 5.Make1:10serialdilutionsofeachsamplebymixing20microliterssamplewith180microlitersH2Ooro.5MCphM.Repeatforatotalof6dilutions.Besuretothoroughlymixsamplesbeforeperformingeachdilution-vortextubesfrompreviousshockingstepandpipetteserialdilutionstomix.Changepipettetipsbetweeneachdilution.Themostefficientwaytoperformthisstepformultiplesamplesistoperformdilutionsina96-wellplateusingamulti-channelpipettor

- 6.Plate5microliterdropsofeachdilutionforeachsample(shockandnon-shocked)onLB+antibioticplates.Growat30CO/N.

- 7.Countcoloniesthenextday.Calculate%survival=100*(#shockedcolonies/#unsh0ckedcolonies).

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51.PharmacologicalPolarizing/DepolarizingTreatmentsand

DetectionwithaVoltageSensitiveDyeinArabidopsisRoots

Reagents:

Di-4-ANEPPS(LifeTechnologies#D-1199)-Afast-responseprobethatchangesitselectronicstructureandfluorescencepropertiesinresponsetochangesinmembranevoltage.Di-4-ANEPPSshowsanexcitationshiftdependingonitselectricenvironment.Inresponsetoincreasingmembranepotential,Di-4-ANEPPSshowsdecreasingfluorescencewhenexcitedat488nmsandincreasingfluorescencewhenexcitedat543nms.Inresponsetodecreasingmembranepotential,Di-4-ANEPPSshowsincreasingfluorescencewhenexcitedat488nmsanddecreasingfluorescencewhenexcitedat543nms.BecauseofthisaF488/F543emissionratiocanbeusedtodetectedchangesintheplasmamembranepotential.Fusicoccin(Sigma#F0537-250UG)–ActivatestheH+/ATPasecausingmembranehyperpolarization.5-Nitro-2-(3-phenylpropylamino)benzoicacid(Sigma#N4779-25MG)–BlocksCl-channelsandactsasaprotonphorecausingmembranedepolarization.StockSolutions:

2mMDi-4-ANEPPSinDimethylsulfoxide1mMFusicoccininH2O10mM5-Nitro-2-(3-phenylpropylamino)benzoicacidinDimethylsulfoxideWorkingSolutions:

20µMDi-4-ANEPPSinH2O2µMFusicoccininH2040µM5-Nitro-2-(3-phenylpropylamino)benzoicacidinH2OAllreagentsweremadefreshfromstocksolutionsforeachexperiment.WorkingsolutionsofFusicoccinandNPPBcontain0.1%(v/v)SilwetL-77,oranotherdetergent,toensuregoodcontactbetweensolutionandsample.

Protocol:

1)Carefullypullseedlingfromagarinthelaminarflowhoodandplaceina1.7mlmicrofugetubecontaining1mlof20µMDi-4-ANEPPS.Incubatefor5minutesatroomtemperature.2)Whileholdingseedlinglooselywithforcepsundertheleaves,gentlyrinseseedlingwith1mLdistilledwateroveranempty50mLbeakertoremoveanyexcessdye.Repeatasecondtime.

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3)Layseedlingoutonaglassslideandapply40 µlofNPPBorFusicoccintothewholeroot.Dothisquicklytoavoidlettingtherootdryout.4)Quicklyandgentlyplaceacoverslipovertheroot.5)Selectregionsofinterestandimagebyexcitingwitha488nmlaseranda543nmlaser,andcollectingat560-660nmsfor5minutes.Icollectdateevery12secondsoverthe5-minutetimecoursetoavoidverylargefiles.

Maggie Wilson

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52.ConfocalLaserScanningMicroscopy

*General Confocal Laser ScanningMicroscope Use and Tips for theOlympus Fluoview –FV1000.*ThisProtocoldoesnotreplacemicroscopetraining.*Adapted from the Laurier Research Instrumentation User Guidelines & StandardOperatingProcedure.http://www.wlu.ca/documents/30936/Confocal_Microscope.pdf

TURNINGONTHEMICROSCOPE

TheHaswelllabconfocalmicroscopeislocatedinroomofMcDonnellRm231.Componentsoftheconfocalmicroscopesystemarelabeled1-9.Whenturningonthesystem,eachpartshouldbeturnedoninthisorder,startingwith#1andendingwith#9.Thesystemshouldbeshutdowninthereverseorder,startingwith#9andendingwith#1,withtheexceptionof#2.#2controlsacoolingfanandthisswitchshouldremainintheonpositionuntilthefanstopsrunning.PartDescription:

1) MercuryLamp2) CoolingFan3) ArgonLasers458nm,488nm,515nm4) GreenHe-NeLaser543nm5) DiodeLasers405nm(blue)and635nm(red)6) Microscope7) Microscope8) Microscope9) Computer(Password:fluoview)

Ifyouareonlyplanningtousetheepifluorescence(mercuryburner)youdonotneedtoturnonthelasers(#2-5).Thissituationcouldoccurifyouarequicklycheckingforsignal.Ifareonlyusingthelasers,donotturnonthemercurylamp.*NOTE: Themercuryburner takesapproximately5minutes towarmup.Oncethe

lampisturnedonitshouldremainonfor30minutes.Onceturnedoff,Imustremain

offfor15-20minutestocompletelycoolbeforeturningbackon.Improperuseofthe

mercurylampwillreducethelifeoftheburner.

Formicroscopeuse(confocalornot)youneedtoopentheFV10-ASW1.7software. Login:Administrator Password:Administrator*Pleasecleanupafteryourself!

*Neverputpressureontheairtable.

*Doublechecksignupcalendar(bythedoorinMCDRm250)beforeturningoff.

*Alwaysrecovermicroscopewhenfinished!Dustisbad.

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VIEWINGSAMPLEWITHTRANSMITTEDLIGHT

Before viewing specimenwith transmitted light (bright-field) themicroscope condensershould be properly aligned to provide even and bright illumination (commonly calledKolher).

1. In theAcquisitionSettingwindowor thecontrolpanel thatsits to therightof themicroscope,selectthe10xobjective(dropdownlist,#6inFigure1).

2. Manuallymovetheobjectivedownbyturningtheknobclockwise.3. Mountyourslideonthemicroscope,coverslipup.4. Select“DICT”fromtheMicroscopeControllerwindow(orcontrolbox)andtheDIC

cubewillberotatedintoplaceautomatically(#15inFigure1).5. UsethesoftwaretoselectTransmittedLightmode(turnonTransLamp,upperbutton

by#8inFigure1).6. Focusonthespecimenusingthemicroscopeoculars.

a. Adjustthebrightnessusingthebuttonsonthefrontofthemicroscope.b. ThefocusmechanismcanbesettofineusingeithertheF/Cbuttonbelowtheright

focusknob,orintheMicroscopeControllerwindow.i. Closeyourlefteyeandfocusonthespecimenusingthefinefocusknob.ii. Closeyourrighteyeandfocusonthespecimenusingthediopterringonthe

leftocular.iii. Openbotheyesandconfirmthatthefocusiscomfortable.c. TurntheshearknobontheDICprism(justabovetheobjectives) toadjust the

contrast.d. AdjusttheDICprismfocalplanebypullingoutorpushinginthesmallL-shaped

knobjustbesidetheshearknob.(Pushitinorpullitoutuntilthebestimageisobserved).

7. Completelyopenthecondenseraperturediaphram(justunderthestage)andclosethefielddiaphragmenoughsothatyoucanseethediaphragmedgesintheeyepieces.

8. FocusthecondenserknobsothattheedgesofthediaphragmANDthespecimenappearverysharp(donotusethefine/coursefocusfortheobjectives).

9. Ifnecessary,centerthecondenserusingthecenteringscrews(ithelpstoopenthefielddiaphragmsothattheedgesofthediaphragmalmostfillthefieldofview).

10. When the condenser is focused and centered, open the field diaphragm so that itcompletelyfillstheeyepieces.

11. Enjoytheview!

EPIFLUORESCENCEIMAGING

Examiningyourspecimenusingepifluorescencebeforecollectingaconfocal imageallowsfocusingandobjective/magnificationselectionwithoutthephotobleachingrisksassociatedwithhighintensitylaserlight.Useepifluoresencetofindtheregionofinterestandfocusatthedesiredmagnificationasfollowsbeforeproceedingtolaserscanning.

1. Closethemanualshutteronthemercurylamp.

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2. ClickontheEpiLampbuttonatthetopleftcorneroftheAcquisitionSettingwindow(#8inFigure1).

3. Choose the appropriate filter cube (mirror) for your specimen in theMicroscopeControllerwindow(GFP,YFP,orDSRED/RFP)(#15inFigure1).

4. Opentheshutteronthemercurylamptoilluminateyoursample,andusethejoystickandfinefocustolocateyourregionofinterest.

a. Note: Ifyoudon’t seeany light, check tomakesure that theshutteron themercurylampisopen.Themercurylampbrightnesscanalsobeadjustedusingthisshutter.

5. Whenyouhavefoundtheregionofinterestandfocusedwiththe10xobjective,movesequentiallyuptothehighpoweredobjectives:switchtothe20x,focus,thenswitchto60xwaterobjectiveandfocusagain.(Ifyouchosetousethe100xoilobjective,youcannotreturntolowerobjectivesusingthesameslide.Oilandwaterdonotmix!)

6. Whenyouhavefoundtheregionofinterestandfocusedwiththedesiredobjective,closetheshutteronthemercurylamptoavoidbleachingyoursample.

CONFOCALIMAGING

1. Turnoffbright-fieldorepifluorescenceilluminationbyclickingtherelevantbuttonatthetopleftoftheAcquisitionSettingwindow(#8inFigure1).Themicroscopethenautomaticallysetsupforlaserscanning(LSM).

2. Changethefocusmechanismtofine(usingthegreenbuttonsbesidethefocusknoborusingthedropdownlist-#16inFigure1).

3. ClickontheDyeListbuttonontheleftintheImageAcquisitionControlwindow(#9,Figure1).

a. Double click on the dyes that you have in your sample to add them to theSelectedDyesbox.Uptothreedyescanbeselectedinadditiontotransmittedlight.

b. Ifthedyeyouareusingisnotinthelist,itcanbeaddedbyclickingonToolsàMaintenanceàUserSettings.ClickontheDyetab,andthenclickthe“add”buttononthefarrightsideofthewindow.Typeinthenameofthedyeandthen enter the excitation and emission wavelengths near the bottom leftcorner of the screen. Select the laser thatmost closely corresponds to theexcitationwavelength.Press“saveandclose”.

c. ClickApply.Theappropriatefilterswillautomaticallymoveintoplace.d. ClosetheDyeListwindow.

4. Thefourthdetectorchannel,TD1,isforDICorbright-fieldimagecollectiononly.IfyouplantocollectaDICimageaswell,thenclicktheboxbesideTD1intheAcquisitionControlwindow.YoucanselectanylasertocorrespondwiththeTD1channelexceptthe405.

5. SetupyourscanningparametersintheAcquisitionSettingwindow:a. Mode (#1, Figure 6-2): one way raster scanning (normal) or bidirectional

(high speed. Bidirecitonal is best for live cell imaging but can cause slightimageaberration.ThisimagecanbecorrectedusingtheImageAdjustbutton,nexttothescanningspeedslider).

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b. Thescanningareamustbenormal(rectangle)tostart.i. Afteranimagehasbeencollected,differentregionsofinterest(ROIs)canbeselectedusing thebuttons in thissection:clipscan(diamondbutton;selectrectangle,ellipse,orpolygon),line(linebutton;straightor curved line), or point (point button).More than one area can bescannedatatimeusingtheROImanagerbuttoninthe2DControlPanelwindow,andlaserparameterscanbesetindividuallyforeacharea.

c. Imageacquisitionspeed(#2,Figure6-2):Selectfastscanningspeedforimageacquisitioninitially(aslowerspeedwillgiveabettersignal,butalsocauseshigherphotobleaching).Makesure“AutoHV”isnotselectedatthispoint.

d. Size (#3,Figure6-2): initially setat512x512pixels, andadjusted later tooptimizeresolution.1024x1024pixelsisgoodforpublicationqualityimages.

e. Area(#4,Figure6-2):leaveat1:1initially(nozoom).f. Laser (#5, Figure 6-2): lasers are selected automatically for fluorescence

according to thedye thatyouselect.Donotadjust the laserpowersat thispoint.

g. Microscope(#6,Figure6-2):makesurethecorrectobjectiveisselectedfromthedropdownmenu.

6. IntheImageAcquisitionControlwindow:a. If you are usingmore than one dye, check the Sequential box tominimize

bleedthrough.i. Linesequentialscanminimizesthetimedifferencebetweenimagesforeachchannelbyscanningonlyalineineachchannelatatime.Thisistypicallythebestchoice.

ii. Framesequentialscantakesonefullimageonagivenchannelbeforescanningthenextchannel.

b. ClickonXYrepeattoinitiatesamplescanning(#10,Figure6-2).Alternatively,theFocusx2orFocusx4buttonscanbeusedtoobtainaveryroughscanthatskips lines, doubles or quadrupoles the scanning speed, and minimizesphotobleaching.Remember that while the image is being scanned, thespecimenispronetophotobleaching–avoidscanningforanylongerthannecessary.

c. Thefocusbetweenwhatisseenwithepi(thoughtheoculars)andwhatisseenonthescreenisslightlydifferent.YouwilneedtomovetheobjectiveupVERYCAREFULLY (while in FINE FOCUS, turn the knob very slowely counterclockwise) to bring the image into focus. Only a very small amount ofadjustmentshouldberequired.Iftheimagedoesnotcomeintofocuseasily,switchbacktoalowermagnificationand/orswitchbacktoepi-flourescencemodetofocusagain.

d. Adjustthebrightnessoftheimage:i. If the signal is low in all channels, slow the scan speed downwhilescanninguntilsignalcanbeobservedinallchannels.

ii. PressCtrl+Htoshowthehighandlowlimitsofdetection.Ifthesignalistoohighandsaturatingthedetector,itwillshowupasred.Anyblueontheimageindicateszerosignaldetection.

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iii. AdjustHVoneachofthechannels(#12,Figure1)independentlyuntiljustafewpixelsofredcanbeseen(avoidsettingHVhigherthan700asthiswillincreasebackgroundinstrumentnoise).(Note:TheHVforDICintheTD1channelisusuallymuchlower–trystartingaround200).IftheHVisquitehighandthesignalisstilltoodim,tryadjustingtheC.A.(confocalaperture);anincreasedCAwillincreasebrightness,butalsodecreaseresolution,andincreasecross-sectionthickness.Thescan rate can also be decreased further, or the laser power can beadjustedasa last resort.Typical starting laserpowersasas follows:405,458,488,and635:0-5%;515:10-20%;543:45-55%.

iv. Avoidadjustingthegainoroffsetifpossible(#13and#14,Figure1),however; ifHVishighandtheimageisstill toodim,thegaincanbeincreased.Increasingtheoffsetwillturnthebackgrounddarker.

v. Checkforbleedthroughbyswitchingoff(unchecking)allbutonelaserthenmakesurethatthereisonlysignalintherelevantchannel.Dothisforeachlaserinturn.(Ifyouarealreadydoingsequentialscanning,thisisunnecessary).

vi. Kalmanintegrationcanbeusedtocollectseveralimagesandaveragethem to decrease S/N. This slows scanning, can dim an image, andincreasesphotobleaching.

vii. Note:forveryweaksignals,thephoton-countingmodeprovidesbettersensitivity.

e. PressCtrl+Htoreturntoregularviewingmode.f. If desired, optimize the resolution by pressing “i” in the ImageAcquisition

Control window. To achieve optimal resolution the pixel size should beapproximately ½ of the optical resolution. Adjust the size of the image(numberofpixels)andthezoomorchangeobjectivestooptimizeresolution.

i. When setting the pixel size of your image, keep in mind your finalformat;journal,poster,etc.Animagethatwillbeblownupforaposterwillneedahighpixelcounttoavoidbecomingpixilated.Animageforajournalshouldhavethedots-per-inchrequiredbythatjournal,whichcan be determined based on image size and pixel number (i.e. theJournalofMicroscopyrequires300dpi).

g. Thefinefocuscanbeadjustedagainslightlytoobtainthebrightestimage.7. When adjustments for brightness and sensitivity are complete, press the Stop

button.8. Theimageisnowoptimized.

XYIMAGEACQUISITION

1. Ifyouaresatisfiedwiththeimage,clicktheXYbuttontodoasingleXYscan(#11,Figure1),andthenpressStop.

2. Whenimageacquisitioniscomplete,theimagewillappearinthe2DViewwindow.Thiswindowcanbeusedtomanipulatethe2Dimageinanumberofways;seesectionbelowforinstructionsonimageanalysis,manipulation,andsavingyourfiles.

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Z-SERIESOR3-DSTACKACQUISITION

1. Optimizeyourimageasdescribedabove.2. RightbelowtheXYbutton(#11,Figure1)intheImageAcquisitionControlwindow,

selecttheDepthbutton.XYZshouldnowbeboldedinthebutton.3. ScanrapidlyusingXYRepeatorFocusx2orFocusx4.4. theImageAcquisitionwindow(z-stackcontrolsindicatedby#7onFigure1):

a. Usethedownbutton(orfinefocusknob)tomovethefocustothebottom(orjust past the bottom) of your sample. Press the Set button below Start toindicatethestartposition.

b. Usetheupbutton(orfinefocusknob)tomovetothetopofthesampleortothelastXYimagethatyouwanttotake.PresstheSetbuttonbelowEnd.

c. Clickthe“go”buttonnexttoCentertogothemiddleofyourZ-series.AdjustthebrightnessasneededbychangingtheHVorlaserpowers.

d. StoptheXYScanning.e. Setyourstepsizeasdesired,orpress“Op”totherightoftheStepSizebox.

ThiswillautomaticallysettheslicethicknesstoNyquist/2,whichguaranteesthatyouwilloversampletheZ-seriesandcaptureallpossibledetail.Keepinmind that more sampling leads to longer scanning and increasedphotobleaching. The “S” at the top of the Acquisition Setting window willindicatethetotaltimetoacquirethestack.Ifyourequireaverysmallstepsizeyou can also increase the scan speedordecrease thedepthof the stack todecreasescanningtime.

5. StartthescanusingtheXYZscanbutton.6. ClickonSeriesDoneifyouaresatisfiedwiththestackcollected,orAppendImagesto

addadditionalframes.ADDINGASCALEBAR

1. In 2D viewwindow, click on the Pencil button, and then click on the ruler at thebottom.

2. Intheareaofinterest,clickanddragtherulertoshowascalebar.

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Figure1.

IMAGEANALYSIS

When imageacquisition is complete, the imagewill appear in the2DViewwindow.Thiswindow, alongwith the 2-D control panel, can be used tomanipulate the 2D image in anumberofways,andthevarioustoolsaredescribedinTable1and2.Theimagecanalsobesavedandviewedindifferentways(Table3),andprocessed(Table4).Table1:2D-ViewWindowToolbutton FunctionLUT(lookuptable) Use the LUT to change the gamma,

intensity, contrast, and pseudocolour ofany channel to enhance dim images orhighlightcertainfeatureswithadifferentcolour.

Single/Panel/Tile Changetheviewofthesample,orlookatmore than one image at once. Channelscan be viewed separately or overlaidusingtheTilebutton.

Zoom,1:1,FittoWindow Thesebuttonsadjustthesizeoftheimageonthescreen.

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Slider Usetoscanthroughtheframescollectedfor thisspeciman(primarily forZ-orT-seriesimages).

ActiveOverlay Add text to the image describing Zposition,time,orwavelength.

Numberedbuttons down the side of thewindow(1,2,etc)

Use to select the desired channelsdisplayedintheimage.

Pencilbutton Allowsaccesstoregionofinterest(ROI)buttonsandotherdrawingtools(i.e.text,scale bar, grid). After a ROI is selected,imaging can be conducted on that areaspecifically to avoid unnecessarybleachingofentirespecimaninlightpath.

Animationarrows Used to play an animation or scrollthroughZ-orT-series.

3-Dbutton Allows viewing of a Z-series from anyangle,selectionofcrosssections,rotationalong a given axis and creation ofanimation(usingtheMorearrow).

Intensityprofilebutton Displays the intensity profile for aselectedROI.Thisprofilecanbeusedtodetermine the size of a given structure,forexample.

Measurementbutton Measure the size, area, etc., of selectedROI. Also accessible from the AnalysisMenu.

Histogrambutton Displaysanintensityprofile(intensityvs.frequency) for the selected ROI. AlsoaccessiblefromtheAnalysisMenu.

SeriesAnalysis Displays intensity vales for a given ROIand each channel for each frame in aseries(i.e.canuseittodeterminewhichframebestdisplaysagivenfeature/ROI).AlsoaccessiblefromtheAnalysisMenu.

LineSeries Illustratesthechangeinintensityofalineasaseries(ZorT)progresses.

Colocalization Graph displays extent of colocalization,andstatisticspagecalculationsPearson'scoefficient, overlap and colocalizationindexes.

Table2:2DControlPanelWindowToolbutton FunctionDigitalZoom Adjustdigitalzoom.

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ROIFormatandManagerbuttons Change the text and line formatting forlabeling ROIs, and view information onROIsselected.

LoadScandatabutton If accessible, the acquisition conditionsfortheimagecanberead.

Steppingbox Allows stepping through Z, time, oranimationseriesframes.

Tilebox Determines the organization of the tilesfromasetofimages.

Intensityprofiles Clicktodisplaytheverticalorhorizontalintensitydistributionalongaline.

Table3:MainFluoviewWindowToolbutton FunctionProperties(“i”) Display image information in Data

ManagerWindow.Report, Thumbnail, Thumbnail +property

Change how files are displayed in theExplorerwindow.

Various buttons to organize howwindowsaredisplayed

Assists in organizing the screen-viewwhenmanywindowsareopen.

Darkroomcolorbutton Dims the monitor display to minimizestraylight.

Table4:ProcessingMenuMenuItem FunctionFilterSetting…andFilter

Mathematical filters can be used toimprove image clarity or emphasizecertain characteristics. The filtersavailable are: sharpen, average, DIC,sobel,median,shading,lapacian.Sharpen:highlights theedgesof images,improves blurred images, but alsoincreasesnoise.Sobel:Emphasizescontours.Lapacian:emphasizesintensitychanges.Press “Preview” at bottom of Filterwindowfirst,thenselectvariousfilterstoobserveresult.SelecttheSingleorSeriesbutton,ifdoingasingleimage,enterthedesiredframeintheImagePositionbox.If you want to save the filtered image,pressNewImage.

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Threshold This feature is used tomake the imagebinary(twocolor).TheCandZ indicatethe channel and frame to display. Thethresholdscanbeadjustedbyclickingonthechannelinthetablebelowthreshold,andmanuallyenteringnewvalues,orbymovingthebarsonthegraph

ImageCalculation Used to subtract/add/divide images orconstants.

CorrectingPixelGaps Thiswindow canbe used to correct forimage shift between channels. Imagepositioncanbeusedtoselectthedesireframe.ThewhiteboxinthePreviewareacanbedraggedtoviewotherareasoftheimage.

CorrectingZGaps To correct for Z position shift betweenchannels.MovingtheyellowbarchangestheZ-sliceimage.

Ratio/Concentration TheRatiotabisusedtolookatchangesinintensitybetweentwochannels.Usethedrop down menu to select the desiredoperation, and the equation for thatoperation will be displayed in theEquation box. Equations includebaseline/background subtraction, ratioof channels, FRET image comparison(photobleachedimagerequired).TheConcentrationtabisusedtoanalyzeachangeinionintensityovertime.Thisfeature requires knowledge of intensityvalues with and without ion, and thedissociation constant (in nM). See theHelpmenuforanexplanationofFvalues.

EditExperiment Combine single channels, append orextractseries,fromtwodifferentimagestocreateanewimageorseries

SAVINGANDVIEWINGFINALIMAGES

1. Whenyouarefinishedcollectingandanalyzingyourimage,selectFileàSaveAsandsave it to your folder as a .oif or .oib file (automatically saved in D:\FV10-ASW\users\yourusername\Image). Make sure “Include all ROI” (or selectedROIs) are checked before you save the image so that scale bars and other

additionstotheimageareincluded.a. YoucanalsorightclickontheimagetoexportasaTIFF,JPEG,orotherimage

format.

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b. AnOIFformatsavestheimagesasTIFFsinafolder,andcreatesafilewiththesampleinformation.BoththefileandtheTIFFsarerequiredtoopentheimage.IfyousavethisdatatoamemorykeyorCD,youneedtocopyboththe

.oiffilesandtheassociatedsub-foldercontainingtheimages.c. AnOIBformatsavesalloftheimagesinasetoffiles.

2. Manipulationscanbedoneusingthefluoviewsoftwareontheconfocalcomputer,oranotherprogramorfluoviewsoftwareonadifferentcomputer(recommended).

a. A free version of the Fluoview software can be downloaded from:.https://support.olympus.co.jp/cf_secure/en/lisg/bio/download/ga/fv10_asw/

b. ImageJ (http://rsb.info.nih.gov/ij/) and VOXX(http://www.nephrology.iupui.edu/imaging/voxx/) software can also beusedtoview.oiffiles.

CONFOCALMICROSCOPEQUICKREFERENCEGUIDE

1. Removethecoverfromthemicroscope.2. Turnonthemicroscopecomponentsaccordingtothenumbersonlabels3. Logontothecomputerandthesoftware(FV10-ASW1.7)4. Waitforthesoftwareandmicroscopetoinitialize.5. Carefullyplaceyourslideonthestage.6. Viewtheimageusingepifluorescenceandtheappropriatefiltercubetofine-tunethe

focus.7. Whenyouhavefoundtheregionofinterestandfocusedwiththedesiredobjective,

closetheshutteronthemercurylamptoavoidbleachingyoursample.8. Turn off bright-field or epifluorescence illumination. The microscope then

automaticallysetsupforlaserscanning(LSM).9. IntheImageAcquisitionControlwindow,selectdesireddyes.10. SetupyourimageparametersintheAcquisitionSettingwindow.

a. Onewayscanning(normal)orbidirectional.b. Selectfastscanningspeedforinitialimageacquisitioninitially.c. Setthesizeto512x512pixelsd. Leavezoomat1:1initially.e. Setthelaserpowersshouldbesetaslowaspossibletominimizebleaching

(exceptthe543laser,whichshouldbeatleast50%).f. Selectthecorrectobjectivefromthedropdownmenu.

11. IntheImageAcquisitionControlwindow:a. If you are usingmore than one dye, check the Sequential box tominimize

bleedthrough.b. ClickonXYrepeatorFocusx2orFocusx4toinitiatescanning.

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c. UsethefinefocusortheupanddownbuttonintheAcquisitionSettingwindowtoalignthemicroscopewiththecrosssectiontobeobserved.

d. AdjustHVontheactivechannelsuntiltheimagecanbeobserved(donotsethigherthan700).

e. AdjustthebrightnessoftheimageusingHV,CA,scanspeed,orlaserpowers,andcheckforsaturation(Ctrl+H).

f. If desired, optimize the resolution (Nyquist) by pressing “i” in the ImageAcquisitionControlwindow.

12. When adjustments for brightness and sensitivity are complete, press the Stopbutton.

13. Theimageisnowoptimized.14. CollectasingleXYscan,orcreateaZ-orT-series.15. Whenimageacquisitioniscomplete,theimagewillappearinthe2DViewwindow.16. AddScaleBarandsaveimagetoyourfolder.