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PALM Protocols - DNA Handling

Carl Zeiss MicroImaging

PALM Protocols – DNA handling

PALM Protocols

DNA handlingNon contact Laser Capture Microdissection

Carl Zeiss MicroImaging – Location Munich – Germany

5 Introduction - Some remarks on DNA

6 Preparation of slides – Samples on MembraneSlide 8 – Samples on glass slides 8 – Archived samples: removing the coverslip

9 Treatment of slides – Heat treatment 9 – UV treatment 9 – Poly-L-Lysine treatment

10 Mounting samples onto slides 10 Frozen sections 10 FormalinFixedParaffinEmbedded(FFPE)sections 11 Cytospins 11 Blood and tissue smear

12 Staining procedures 12 FormalinFixedParaffinEmbedded(FFPE)sections 12 Frozensections –Hematoxylin/Eosin(HE) 12 – Cresyl Violet 13 – Toluidine Blue 13 – Methyl Green 13 – Methylene Blue 13 – Nuclear Fast Red 13 Storage

14 Non-contactLaserCaptureMicrodissection(LCM)Procedure 14 Tips to improve morphological information 14 Diffusor CM 15 AdhesiveCap opaque 15 Liquid Cover Glass

16 Collection devices 16 AdhesiveCap 16 Other microfuge tubes 16 AmpliGrid AG480F

17 Collection procedures 17 „Dry“collection(AdhesiveCap) 18 „Wet”collection(othermicrofugetubes) 19 „Wet“collectionontoSlide48(AmpliGridAG480F) 19 Capture check – looking into the cap to see the lifted samples

20 Downstream Applications 20 DNAisolationfromFFPEsections 20 DNA isolation from frozen sections 21 QIAamp DNA Procedure 22 PCRsetup-StandardPCR(20µl)inacapillarycycler 23 -HighvolumePCR(50µl)ina96-wellblockcycler 23 -LowvolumePCR(1µl)inanEppendorf Mastercycler with In situ Adapter 24 Otherprotocols(RNA,Chromosomes,LiveCells,...)

Content

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Introduction

Some remarks on DNA

Human Genome Project has shown new insights into poorly understood biological phenomenabyprovidingvastDNAsequencingdata.Asaresultofthisexpansionofgenomicsintohumanhealthapplications,thefieldofgenomicmedicinewasborn.Geneticsisplayinganincreasinglyimportantroleinthediagnosis,monitoring,andtreatmentofdiseases.

FurtherareasthatstandtobenefitfromDNAresultsincludebiomedicalandbiological research,toxicology,drugdesign,forensics,animalandplantgenetics,andmanyothers.

Inallfieldsthemethodsformoleculartestingmustbeabletodetermineand analyzeDNAsequencesaccuratelyandrapidly.Wheneverpossibletheproceduresshouldbeeasytouse,highlyautomated,andminimizedinrequirementofmaterial.

Thereforethegenerationofdefinedsampleassourcefortheanalysisisofprimeimportance.

Non-contactLaserCaptureMicrodissection(LCM)fromCarlZeississtateoftheart forprecisesamplepreparation.

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PALM Protocols - DNA Handling PALM Protocols - DNA Handling

Preparation of slides – Samples on MembraneSlide

MembraneSlide is a glass slide covered with a membraneononeside.This membrane is easily cut together with the sample and acts as a stabilizing support duringlifting.Thereforeevenlargeareasarelifted by a single laser pulse without affecting themorphologicalintegrityofthetissue.Use of MembraneSlide is especially recommen- ded for isolating single cells or chromosomes aswellaslivecellsorsmallorganisms.CarlZeissMicroImaging(CZMI)offersslides(1mm,0.17mm)coveredwithPolyethyleneNaphthalate(PEN)-membraneorPolyethyleneTeraphthalate(PET)-membrane.

PEN-membraneishighlyabsorptiveintheUV-Arange,whichfacilitateslasercutting.The membrane can be used for all kind of applications.MembraneSlideNF(nucleasefree)iscertifiedtobefreeofDNase,RNaseandhumanDNA.

InadditiontoPEN-MembraneSlide,CZMIalsooffersPET-membranecoveredslides.Theseslidesarehelpfulforspecialprocesses,i.e.fluorescenceapplications.Evenweakfluores-cencesignalscanbedetectedwithPET-slides,duetothelowsignaltonoiseratio.

AlternativelythePET-membraneattachedtoametalframe(FrameSlidePET)isalsoavailable.ThestructureofFrameSlidePETisresistanttomicrowavetreatmentorpressurecooking.The special bonding is inert and adapted to heat treatment even in moisture or liquid so thatthemembranedoesnotruffleduringtheheatingprocess.Ifyouneedfurther informationabouttheseslides,pleasecontact:

E-Mail: [email protected]

When working with low magnifying objectives like5xor10x,bothregular1mmthickglassslidesand0.17mmglassslidescanbeused. Tokeepthisflexibilityforhighermagnifications (20x,40xor63x)CZMIrecommendsusinglongdistanceobjectives.With those the working distance can be adap-ted to the different glass slides by moving the correctioncollarontheobjective. (seepictureabove)Duetotheshortworkingdistanceonly0.17mm thin cover glass slides can be used with the 100xmagnifyingobjectives.

Regular glass slide (1 mm thick) => 1, thin slide (0.17 mm thick) => dot,

DuplexDish and FrameSlide => between dot and 0.


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MembraneSlide 1.0 PEN - Order No. 415190-9041-000 (white)MembraneSlide 1.0 PEN NF - Order No. 415190-9081-000 (white)MembraneSlide 0.17 PEN - Order No. 415190-9061-000 (uncolored)MembraneSlide 50x1.0 PEN - Order No. 415190-9091-000 (doublewidth)MembraneSlide 1.0 PET - Order No. 415190-9051-000 (blue)MembraneSlide 0.17 PET - Order No. 415190-9071-000FrameSlide PET - Order No. 415190-9101-000 (metal)

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Preparation of slides – Samples on glass slides

With PALM MicroBeam almost every kind of biological material can be microdissected andlifteddirectlyfromregularglassslides.Evenarchivalsectionscanbeusedafterremoving the coverslip and the mounting medium.To facilitate lifting additional adhesive sub-stances or “Superfrost + charged slides”should only be applied when absolutely necessary for the attachment of poorly ad- heringmaterial(e.g.somebrainsectionsorbloodvesselrings).Inthosecaseshigherlaserenergyisneededforlifting.

Archived samples: removing the coverslip

Depending on the applied mounting medium(whetheritissolubleinxyleneorwater)thewholeslideshouldbecompletely submerged in the respective solvent.

1.standingupinaglassjarfilledwith either pure xylene or warm water (30-50°C)

2. timeneededforthecoversliptoswim off may range from hours to days

3.gentlemovementofthejarmayspeed up the process

4.air-drytheslideafterremoval

Note: It is very important NOT to use any force to push off the coverslip because this might damage the section! Wait till it falls off by itself! The necessary time depends on the age of the sample and the dryness ofthemountingmedium.Freshslides(onlydaysold)canbedecover-slippedmuchfaster.

From the dry glass slide sample material can be lifted directly by “AutoLPC” function ofPALMRoboSoftware.


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Treatment of slides

Heat treatmentToensurenuclease-freeMembraneSlides,heatslidesat180°Cinadryingcabinetfor4hourstocompletelyinactivatenucleases.

UV treatmentTo overcome the hydrophobic nature of the membrane it is advisable to irradiate with UV light at 254 nm for 30 minutes (e.g.inacellculturehood).

Themembranegetsmorehydrophilic, thereforethesections(paraffin-aswell ascryosections)adherebetter. Positive side effects are sterilization and destruction of potentially contaminating nucleicacids.

Poly-L-Lysine treatmentAdditional coating of the slide with Poly-L-Lysine(0.1%w/v,e.g.SIGMA,#P8920)only will be necessary for poorly adhering materials(e.g.brainsections)andshouldbeperformedafterUVtreatment.Distribute a drop of the solution on top of theslide.Let air-dry at room temperature for 2-3 minutes.Avoidanyleakageofthemembrane,asthis might result in impairment of Laser CaptureMicrodissection.

Slides are shipped withoutanypretreatment.ToremovepotentiallycontaminatingnucleasesandDNA,MembraneSlidesandglassslidescanbetreatedinthesameway.

MembraneSlideNF(nucleasefree)iscertifiedtobefreeofDNase,RNaseandhumanDNA. Treatments to remove nucleases and contaminating DNA are therefore not necessary using these slides.

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Mounting samples onto slides

Frozen sections

SectioningSections are mounted onto MembraneSlides the same way as routinely done using glass slides.Toallowsubsequentcuttingandlifting by the laser a coverslip and standard mountingmediummustnotbeapplied.Freezing media like OCT or similar may be used but should be kept to a minimum and havetoberemovedbeforelasercutting.

Removing the tissue freezing mediumIf OCT or another tissue freezing medium isused,itisimportanttoremoveitbeforeLaserMicrodissection,becausethesemediawillinterferewithlaserefficiency.

Removing the medium is easily done by dippingtheslide5-6timesinwater.Ifthesectionsarestainedinaqueoussolutions,the supporting substance is normally removed “automatically” by the water containingsteps.

Formalin Fixed Paraffin Embedded (FFPE) sections

SectioningFloatingthesectiononwarmwater(40°C)as well as hot plate techniques can be applied.After mounting the section let the slides dry overnight inadryingovenat56°Ctoim-prove the adhesion of the sections to the membrane.To allow laser cutting and lifting a coverslip and standard mounting medium must not beapplied.Archivalsectionswithmountingmedium and coverslip have to be processed asdescribedtoremovethecoverslip(seepage8).

DeparaffinationResidualparaffinwillreducelaserefficiency,sometimes completely inhibiting cutting andlifting.Ifyouareworkingwithunstai-ned sections it is therefore very important toremovetheparaffinbeforelasercut-tingandlifting.MembraneSlidescanbehandled like normal glass slides

Deparaffination Procedure

1.Xylene 5minutes,2times (2minutesminimum)

2.Ethanol100% 1minute




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Blood and tissue smear

Distribute a drop of blood or material of a smearovertheslide.Be careful to avoid injuries in the mem-brane,whichwouldleadtoleakageduringfixationorwashingstepsand therefore would impair the Laser CaptureMicrodissectionprocess.Letsmearsair-dryshortlyandfixthem for2upto5minutesin70%ethanol.

Cytospins

Cytospins can be prepared on glass slides oronMembraneSlides.After centrifugation in a cytocentrifuge let thecellsair-dryatroomtemperature.Thenfixfor2minutesin70%ethanolandair-dryagainbeforestaining.

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Staining procedures

ForisolationofhighqualityDNAusefreshlyprepared,autoclavedsolutions.

Formalin Fixed Paraffin Embedded (FFPE) sections

Afterdeparaffination(seepage10)continuewiththestainingprocedureofyourchoice. Most staining procedures for frozen sections canbeappliedforFFPEsections(forrecommendationssee´Frozensections`).

Frozen sections

Most standard histological stainings (e.g.HE,MethylGreen,CresylViolet, NuclearFastRed)arecompatiblewith subsequentDNAisolation.AtZEISSLabsweusuallyperformtheCresylVioletorHematoxylin/Eosin(HE)staining.

Hematoxylin/Eosin (HE)

HE-stainingisusedroutinelyinmosthistolo-gicallaboratories.Thenucleiarestainedblue,thecytoplasmpink/red.

Staining Procedure

1.afterfixation(2min,70%Ethanol)dip slide 5-6 times in distilled water

2.stain1-2minutesinMayer’sHematoxy- linsolution(e.g.SIGMA,#MHS-32)

3.rinse1-2minindistilledwateror blueingsolution(e.g.BBC,#3900)

4.stain10secondsinEosinY (e.g.SIGMA,#HT110-2-32)

5.performaquickincreasingethanol series(70%,96%,100%)

6.air-dryshortly(1-2min)

Cresyl Violet

This short staining procedure colors the nu-cleivioletandthecytoplasmweakviolet.

Staining Procedure

1.afterfixation(2min,70%Ethanol)dip slidefor30secinto1%cresylviolet acetatesolution(*)

2.removeexcessstainonabsorbentsurface

3.dipinto70%Ethanol

4.dipinto100%Ethanol


(*)Dissolvesolidcresylvioletacetate (e.g.ALDRICH#86,098-0)ataconcen- trationof1%(w/v)in50%EtOHatroom temperature with agitation/stirring for severalhourstoovernight. Filter the staining solution before use to removeunsolubilizedpowder.Sometimes Lot to Lot variations in the purchased cresyl violet powder can lead to weaker staining resultsifthedyecontentisbelow75%.

Note: In most cases this cresyl violet staining procedurewillbesufficientforcellidentifi- cation.Ifanenhancementoftheintensityis desired,areinforcementbytwoadditional stepsin50%ethanolispossible(first,beforestainingincresylviolet;second,afterthestainingincresylviolet).AmbionofferstheLCMStainingKit(#1935)whichalsocontainsacresylvioletdye.When using this kit we strongly recommend toomitthefinalxylenestepoftheAmbion instruction manual because xylene makes the tissue very brittle and reduces the ad-hesionofthesectiontothePEN-membrane.


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Toluidine Blue

Thenucleiarestaineddarkblue, thecytoplasmlighterblue.

Staining Procedure

1.afterfixation(2min,70%Ethanol) dip slide 5-6 times in distilled water

2.stain30secondsinToluidineBluesolution (0.1%inwater;SIGMA,#T-0394)

3.rinseindistilledwater

4.performaquickincreasingethanolseries (70%,96%,100%)


Methyl Green

Thenucleiarestaineddarkgreen,the cytoplasmlightgreen.

Staining Procedure


2.stain5minutesinMethylGreensolution (DAKO,#S1962)



Methylene Blue

Thenucleiarestaineddarkblue.

Staining Procedure


2.stain5-10mininMethyleneBluesolution (0.05%inwater;SIGMA,#31911-2)



Nuclear Fast Red

Thenucleiarestaineddarkred, thecytoplasmlighterred.

Staining Procedure


2.stain5to10minutesinNuclearFast Redsolution(DAKO,#S1963)



Storage

Stained slides can be used immediately orstoreddry.Iftheslidesarestored inafreezerbeforeLCM,theslidesshouldbefrozen in a tightly sealed container (e.g.twoslidesbacktobackina50mlFalcon-tube)toavoidexcesscondensationofmoistureduringthawing.For rethawing the container should not be opened before it is completely warmed up againtoambienttemperature.

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Non-contact Laser Capture Microdissection (LCM) ProceduresPlease,additionallyhavealookintothePALMMicroBeamusermanual.

Tips to improve morphological information

EmbeddingandglasscoveringofthespecimenisinapplicableforLCM.Thus,theroughopensurfaceofthesection/materialoftenresultsinimpairedviewof morphology.Effectsofdiffusor,AdhesiveCapaswellasLiquidCoverGlassarecomparabletotheusualcoverslipforenhancedvisualization.

Diffusor CM

Holders for PALM RoboMover and PALM CapMoverIIareequippedwithdiffusors.The opaque glass diffuses the incident microscopelight,whichsmoothenstheharshnessofcontrastand,dependingonmaterialandstaining,evenminutedetails asnucleiandcellboundariesshowup. Evenslightdifferencesincolorbecome visible.Formoredetailsandhandling, pleaseseeDiffusorCMproductinformation.

Diffusor CM - Order No. 415101-2100-320PALM CombiSystem


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AdhesiveCap opaque - Order No. 415190-9201-000 (500 µl) AdhesiveCap opaque - Order No. 415190-9181-000 (200 µl)

Liquid Cover Glass

The polymeric and low viscose Liquid Cover Glass completely embeds the tissue andsmoothenstheroughtissuesurface,resul-tinginenhancedmorphologyafterdrying.Formoredetailsandhandling,pleaseseeLiquidCoverGlassproductinformation.

AdhesiveCap opaque

Thewhite/opaquefillingofAdhesiveCapclearly improves visualization of morpho-logical information of the samples due toenhancedcolorbalanceandcontrast,which makes the view comparable to thoseofcoverslippedtissuesections.Two different microfuge tube sizes (200µl,500µl)withthesefilledcaps areavailablefromCZMI.Formoredetailsandhandling,please seeAdhesiveCapproductinformation.

Liquid Cover Glass - Order No. 415190-9020-000

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Collection devices

AdhesiveCap

The intention of AdhesiveCap is to allow LCM(LaserCaptureMicrodissection) without applying any capturing liquid intothecapspriortoLCM. Thisminimizestheriskofnucleaseactivity.Beside the quick relocation of the lifted samples inside the cap due to instant immobilization there is no risk of evapo-ration and crystal formation of the buffer duringextendedspecimenharvesting.Formoredetailsandhandling,pleaseseealsoAdhesiveCapproductinformation.

Other microfuge tubes

Other commercially available plasticware canbeused,too. (e.g.ABgene#AB-0350;0.5mltubes)

AmpliGrid AG480F

Using the SlideCollector 48 in conjunction with AmpliGrid technology from Advalytix enables working in a higher throughput LCM(48samplessimultaneously).The AmpliGrid technology allows DNA analysisinanextremelylowvolume(1µl)directlyonchip.

Please,seethebrochure([email protected])

On-chip Single Cell Analysis for PALM MicroBeam.AdhesiveCap opaque - Order No. 415190-9201-000 (500 µl) AdhesiveCap opaque - Order No. 415190-9181-000 (200 µl)

AdhesiveCap clear - Order No. 415190-9211-000 (500 µl) AdhesiveCap clear - Order No. 415190-9191-000 (200 µl)


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Collection procedures

PleasehavealookintothePALMMicroBeamusermanual.

„Dry collection“ procedure

Note: The time necessary for complete Proteinase K digestion depends on the kindandtheamountofcollectedmaterial.After the Proteinase K digest the regular procedure of the QIAamp® DNA Micro Kit #56304(page21,step4)canbeattached.

„Dry“ collection (AdhesiveCap)

Note:CZMIrecommendsAdhesiveCapascollectiondeviceformostexperiments.

1.puttheAdhesiveCapintothecollector and check the right position of the correctioncollar(seepage6)

2.performnon-contactLCMofselectedcells

3.afterLCMadd15µllysisbuffertothe sample inside the cap (QIAamp®DNAMicroKit#56304)

4.add10µlProteinaseK(20mg/ml)and mix by pulse-vortexing for 15 sec

5.placethetubeinan“upsidedown” positioninanincubatorat56°Cfor 2 - 18 h with occasional agitation

6.centrifugethetubeat10000rcffor 5min(TabletopMicrocentrifuge)

Ifnotgoingonimmediately,storethe samplesat-20°C.

Note: Please do not use any water bath fortheupsidedownincubation.

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Collection procedures

Note: The time necessary for complete Proteinase K digestion depends on the kindandtheamountofcollectedmaterial.After the Proteinase K digest and the in-activation step the routine downstream applicationofchoicecanbecontinued.

Ifnotgoingonimmediately,storethesamplesat-20°C.

Collection Procedure

1.takeanautoclavedmicrofugetube

2.pipette3-15µlofCapturingBuffer without Proteinase K or DNase-free water in the middle of the cap

3.putthecap/tubeintothecollector check the right position of the correction collar(seepage6)

4.performnon-contactLCMofselectedcells

5.centrifugethetubeat10000rcffor5min (TabletopMicrocentrifuge)

6.add10-15µlCapturingBuffercontaining Proteinase K and mix by pulse-vortexing for 15 sec

7.incubatethetubeat56°Cfor2-18hwith occasional agitation


9.finalheatingstepat90°Cfor10minto inactivate Proteinase K


„Wet“ collection (other microfuge tubes)

Whenusing“unfilled”routinemicrofugetubes it is necessary to add a liquid into the cap to facilitate the adhesion of the cap-turedcells. The detergent Igepal CA-630 in the capturing buffer allows to smear out a small amount of liquidinthewholecaparea.Note: All kinds of aqueous solutions will run drywithextendedworkingtime.

Prearrangements - Capturing Buffer

0.05MEDTApH8.0 20µl

1MTrispH8.0 200µl

IgepalCA-630(SIGMA#I-3021) 50µl

(ProteinaseK)* (100µl)ddH

2O fillupto10ml

*ProteinaseK20mg/ml(Qiagen#19131)

FinalConcentration:20mMTris,0.1mMEDTA,0.5%Igepal,1%ProteinaseK

Always prepare a fresh mixture of CapturingBufferandProteinaseK.


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„Wet“ collection onto Slide48 (AmpliGrid AG480F)

UsingSlide48technology,DNAamp-lificationdoesn’trequireanytemplatetransferandpreparation.Analysis(PCR,cyclesequencing)canbeperformedonthe same reaction site of the AmpliGrid AG480F(seepage23):LowvolumePCR(1µl)inanEppendorfMasterCycler.

A preloading of 48 ReactionSites of the AmpliGridwith1µlliquid(e.g.1%Glycerol inwater)enableselongationoftheworking time and is necessary for adhesion of the capturedsamples.TheLCMprocessonto48 reaction sites can be operated automa-tically and is controlled by PALM RoboSoft-ware.

SlideCollector 48 with AmpliGrid AG480F

Capture check – looking into the cap to see the lifted samples

Tocontrolanddocumenttheefficiencyoflifting it is possible to have a look into the collectiondevice(e.g.microfugecap)withthe5x,10x,20x,40xand63xobjectives.

By using the software function “go to checkpoint” the slide is moved out of the light path and the objective lifted for lookinginside.

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Downstream Applications

DNA isolation from frozen sections

To capture microdissected samples we recommendtheuseofAdhesiveCap.For DNA isolation any procedure of choice canbeused.

In our hands the QIAamp® DNA Micro Kit(#56304)combinedwithAdhesiveCapresultsingoodyieldandqualityofDNA.This QIAamp® DNA Micro Kit is designed foruseofsmallamountsoftissue.The subsequently described protocol is suitableevenforsinglecells.

Note: For DNA elution incubating the QIAampMinEluteColumnloadedwithwater for 5 min at room temperature before centrifugation generally increases thefinalDNAyield.

Dilutedsolutionsofnucleicacids(e.g. dilutionseriesusedasstandards)should be stored in aliquots and thawed once only.Werecommendstorageofaliquots insiliconizedtubesifpossible. This avoids adsorption of nucleic acids to thetubewalls,whichwouldreducetheconcentrationofnucleicacidsinsolution.

DNA isolationfrom FFPE sections

Deparaffinationandstainingaredone according to standard procedures for slides(pleaseseepage10-13).

Note: Proteinase K digestion step is essential forformalinfixedsamples.The time necessary for optimal digestion dependsonmanyfactorsliketissuetype,fixationprocedureorthicknessofliftedmaterial.Anovernightdigestion(12-18hours)is a good starting point for optimization but shorter digestion times may be tested as well.Toourexperienceatleast3hoursdi-gestion should be applied with any extraction procedureandmaterial.

ForsubsequentDNAextractionfromFFPEsectionsZEISSLabsprefertheQIAampDNAMicroKit(#56304),pleaseseeDNAisolationfromfrozensections.


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Applying the components of the QIAamp® DNA Micro Kit for isolation of genomic DNA from Laser Microdissected samples

1. Add15µlATLtothemicrodissectedsampleintheAdhesiveCap.

2. Add10µlProteinaseKandmixbypulse-vortexingfor15sec.

3. Placethe0.2mltubeinan“upsidedown”positionat56°Cinanincubatorfor3-18h withoccasionalagitation.

Note: The time necessary for complete Proteinase K digestion depends on the kind ofcollectedmaterial.EspeciallyFFPEsamplesmustbedigestedlonger.

4. Add25µlBufferATLand50µlBufferAL,closethelidandmixbypulse-vortexingfor15sec. Toensureefficientlysis,itisessentialthatthesampleandBufferALarethoroughlymixedto yieldahomogeneoussolution.

5. Add50µlethanol(96-100%),closethelid,andmixthoroughlybypulse-vortexingfor15sec. Incubatefor5minatroomtemperature(15-25°C). Ifroomtemperatureexceeds25°C,cooltheethanolonicebeforeaddingtothetube.

6. Brieflycentrifugethe0.2mltubetoremovedropsfromthelid.

7. CarefullytransfertheentirelysatetotheQIAampMinElutecolumnwithoutwettingtherim, closethelid,andcentrifugeat6000xg(e.g.Eppendorf5415D:8000rpm)for1min. PlacetheQIAampMinEluteColumninaclean2mlcollectiontube,anddiscardthecollection tubecontainingtheflow-through. Ifthelysatehasnotcompletelypassedthroughthecolumnaftercentrifugation,centrifuge againatahigherspeeduntiltheQIAampMinEluteColumnisempty.

8. CarefullyopentheQIAampMinEluteColumnandadd500µlBufferAW1withoutwetting therim.Closethelidandcentrifugeat6000xg(8000rpm)for1min. PlacetheQIAampMinEluteColumninaclean2mlcollectiontube,anddiscardthecollection tubecontainingtheflow-through.

9. Repeatprocedureofstep8with500µlBufferAW2thistime. Note: ContactbetweentheQIAampMinElutecolumnandtheflow-throughshouldbe avoided.Somecentrifugerotorsmayvibrateupondeceleration,resultinginthe flow-through,whichcontainsethanol-comingintocontactwiththeQIAamp MinEluteColumn.TakecarewhenremovingtheQIAampMinEluteColumnand collectiontubefromtherotor,sothatflow-throughdoesnotcomeintocontact withtheQIAampMinEluteColumn.

10. Centrifugeatfullspeed(20,000xg;14,000rpm)for3mintodrythemembrane completely.Thisstepisnecessary,sinceethanolcarryoverintotheeluatemayinterfere withsomedownstreamapplications.

11. PlacetheQIAampMinEluteColumninaclean1.5mlmicrocentrifugetube(notprovided) anddiscardthecollectiontubecontainingtheflow-through.Carefullyopenthelidofthe QIAampMinEluteColumnandapply20µldistilledwatertothecenterofthemembrane. Ensurethatdistilledwaterisequilibratedtoroomtemperature(15-25°C).Dispensedistilled waterontothecenterofthemembranetoensurecompleteelutionofboundDNA.

Note: QIAampMinEluteColumnsprovideflexibilityinthechoiceofelutionvolume. Chooseavolumeaccordingtotherequirementsofthedownstreamapplication. Rememberthatthevolumeofeluatewillbeupto5µllessthanthevolumeof elutionsolutionappliedtothecolumn.

12. Closethelidandincubateatroomtemperature(15-25°)for1-5min.Centrifugeatfull speed(20,000xg;14,000rpm)for1min.

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Downstream Applications

PCR setup

Depending on the concentration of the isolated DNA the suitable setup for the amplificationhastobeselected:

The standard volume PCR (20 µl) in a capillary cycler is useful for highly concen-tratedDNAeluates,becausethemaximal input of target DNA in the reaction setup islimited.Only30-50%oftheeluatecan beanalysed.

ForlowconcentratedDNAeluates,e.g.fromasinglemicrodissectedcell,thehigh volume PCR (50 µl) in a 96-well block cyclerisrecommendable,as100%oftheeluatecanbeusedforthereactionsetup.

The low volume PCR (1 µl) in an Eppen-dorf Mastercycler allows a direct analysis without separate DNA isolation and trans- ferstep.Thismethodofferstheadvantageof the combination of LCM and low volume PCRonthesameslide.

Standard PCR (20 µl) in a capillary cycler

QuantiFastSYBRGreenPCR(QIAGEN#204052)in ourhandsresultsinexactamplificationproducts.

PCR Procedure

1.Thaw2xQuantiFastSYBRGreenPCRMaster Mix,templateDNA,primers,andwater. Mixtheindividualsolutions.

2.Prepareareactionmixaccordingtosetup. Duetothehotstart,itisnotnecessaryto keep samples on ice during reaction setup or whileprogrammingthereal-timecycler.

Note: We recommend starting with the Mg2+ concentration as provided in 2x QuantiFast SYBR GreenPCRMasterMix.

Reaction Setup2x QuantiFast SYBRGreenPCRMasterMix 10µlPrimerA(10µM) 0.5µlPrimerB(10µM) 0.5µlTemplateDNA ≤100ng/reactiondistilledwater(PCRclean) variableTotalreactionvolume 20µl

3.Mixthereactionmixthoroughlyanddispense appropriatevolumesintoPCRcapillaries.

4.AddtemplateDNA(≤100ng/reaction)tothe individualcapillariescontainingthereactionmix.

5.Programthecycleraccordingtoconditions

Capillary Cycler conditions (exemplary)Step Time Temp. Ramp ratePCRinitialactivation 5min 95°C fastmodeTwo-step cyclingdenaturation 10sec 95°C fastmodecombined annealing/extension 30sec 60°C fastmodenumber of cycles 40

6.PlacethePCRcapillariesinthecyclerandstart thecyclingprogram.

7.Optional:CheckthespecificityofthePCR product(s)byagarosegelelectrophoresis


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High volume PCR (50 µl) in a 96-well block cycler

Theinputofthewholeeluate(20µl)tothe PCR reaction mix requires an increased total reactionvolumeof50µl.

PCR Procedure

1.ThawPCRbuffer,dNTPs,templateDNA, primers,andwater.Mixtheindividualsolu- tions.Keepsamplesoniceduringreaction setuporwhileprogrammingthecycler.

2.Prepareareactionmixaccordingtosetup:

Reaction Setup10xBuffer 5µldNTP-Mix(2mMeach) 5µlPrimerA(10µM) 1µlPrimerB(10µM) 1µltemplate DNA variableQiagenHotStarTaqPolymerase 0.5µldistilledwater(PCRclean) variableTotalreactionvolume 50µl

3.Mixthereactionmixthoroughlyanddispense appropriatevolumesintoPCRtubes.

4.AddtemplateDNA(≤100ng/reaction)to thePCRtubescontainingthereactionmix.

5.Programthecycleraccordingtoconditions.

Block Cycler conditions (exemplary)Step Time Temp.activationstep 15min95°Cdenaturation 30sec 95°Cannealing 30sec 50°Cextension 30sec 72°Cfinalextension 5min72°Cnumber of cycles 40

6.PlacethePCRtubesinthecyclerandstart thecyclingprogram.

7.Optional:CheckthespecificityofthePCR product(s)byagarosegelelectrophoresis

Depending on the experiment a subsequent nestedPCRbasedonthefirstPCRproductandinternalprimerscanbeattached.

Low volume PCR (1 µl) in an Eppendorf Mastercycler

DNAamplificationandcyclesequencing,forexampleofasinglecell,arepossibleinan extremelylowvolumereactionformat(1µl)withtheSlide48/AmpliGridtechnology.After lifting the cell onto the chip analysis can be performed directly on-chip without any templatepreparation.

PCR Procedure

1.ThawPCRbuffer,dNTPs,templateDNA, primers,andwater.Mixtheindividualso- lutions.

2.Prepareareactionmixaccordingtosetup:

Reaction Setup (see Advalytix protocols *1)AmpliTaqGold 0.1µl10x GeneAmp Buffer I with 15mM MgCl

2 0.1µl

Primer(5pmol/µleach) 0.1µldNTP-Mix(2.5µMeach) 0.1µldistilledwater(PCRclean) 0.6µlTotalreactionvolume 1.0µl

3.Mixthereactionmixanddispense1µlto eachreactionsiteoftheAmpliGridslide.

4.CoverthePCRdropletwith5µlofsealing solution.

5.PlacetheloadedAmpliGridontheEppen- dorfMastercycler.

6.Programthecycleraccordingtoconditions.

Eppendorf Mastercycler conditions (example)Step Time Temp.PCRinitialstep 10min95°Cdenaturation 40sec 94°Cannealing 30sec 56°Cextension 30sec 72°Cfinalextension 5min72°Cnumberofcycles40 10min72°C

Depending on the experiment a sequencing reaction with subsequent capillary electrophoresis analysis or a check of the specificityofthePCRproduct(s)bygelelectro-phoresiscanbeattached.

*1 Advalytix protocols: www.advalytix.com/images/download

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Brochures and protocols

Live cells Chromosomes On-chip Single Cell Analysis

FISH Immunofluorescence RNA

Forquestions,commentsorprotocolrequestspleasecontact:

ZEISS Labs

E-Mail: [email protected] Hotline: +49 8990 9000 900


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Carl Zeiss MicroImaging GmbH07740 Jena, Germany

BioSciences I Location MunichPhone : +49 8990 9000 800Telefax: +49 8990 9000 820E-Mail : [email protected]

www.zeiss.de/microdissection

ForscientificquestionspleasecontactE-Mail:[email protected]:+4989909000900www.zeiss.de/labs

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