336 determining quickplant™ genetics using pcr...a pcr experimental success guidelines 28 b...
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336DeterminingQuickPlant™ GeneticsUsing PCR
Storage: See Page 3 for specific storage instructions
ExPERImEnT ObjECTIVE:
The object of this experiment is to introduce students to the concept of genetic linkage by using the
polymerase chain reaction to amplify DNA from wild type and mutant Arabidopsis plants.
This experiment is designed for DNA staining with InstaStain® Ethidium Bromide.
Determining Quick Plant™ Genetics Using PCR2
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EDVOTEK - The biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.
Experiment Components 3
Experiment Requirements 4
Background Information 5
Experiment Procedures
Experiment Overview and General Instructions 9
Laboratory Safety 10
Module I: Module I: Growing QuickPlants™ -
Arabidopsis Thaliana 11
Module II: Isolation of Genomic DNA from Arabidopsis 12
Module III: PCR of Genomic DNA from Arabidopsis 14
Module IV: Agarose Gel Electrophoresis 15
Study Questions 16
Instructor's Guidelines
Notes to the Instructor 20
Pre-Lab Preparations 23
Experiment Results and Analysis 25
Study Questions and Answers 26
Appendices
A PCR Experimental Success Guidelines 28
B Polymerase Chain Reaction Using Three Waterbaths 30
C Preparation and Handling of PCR Samples With Wax 31
D 1.0% Agarose Gel Preparation 32
E 1.0% Agarose Gels - Quantity Preparations 33
F Staining and Visualization of DNA with
InstaStain® Ethidium Bromide Cards 34
G InstaStain® Blue: One Step Staining
and Destaining 35
Material Safety Data Sheets 36
Table of Contents
Determining Quick Plant™ Genetics Using PCR
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All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor admin-istered to or consumed by humans or animals.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experi-ment components are de-rived from human sources.
Storage
A. Tubes with PCR reaction pellets™ Room Temperature
Each PCR reaction pellet™ contains
• dNTPMixture
• Taq DNA Polymerase Buffer
• Taq DNA Polymerase
• MgCl2
B. Primer mix -20°C Freezer
C. 200 base pair ladder -20°C Freezer
D. UltraPure H2O -20°C Freezer
E. Tris buffer -20°C Freezer
F. Proteinase K Room temperature
G. NaCl Room temperature
H DNA extraction buffer Room temperature
Reagents & Supplies
(Store all components below at room temperature)
• WildtypeandglabraArabidopsis seeds
• Pottingsoilpellets
• Planthomogenizationpestleswithtubes
• UltraSpec-Agarose™
• ElectrophoresisBuffer(50x)
• 10xGelLoadingSolution
• InstaStain®EthidiumBromide
• MicrocentrifugeTubes
• PCRtubes(0.2 ml - for thermal cyclers with 0.2 ml template)
• Calibratedtransferpipets
• Waxbeads(for waterbath option or thermal cyclers without heated lid)
This experiment is designed for 10 lab groups.
Sample volumes are very small. For liquid samples, it is impor-tant to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting. Spin samples for 10-20 seconds at maximum speed.
Components & Requirements
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Requirements
• Thermalcycler(EDVOTEKCat.#541highlyrecommended)
or three waterbaths*
•Horizontalgelelectrophoresisapparatus
•D.C.powersupply
• Balance
• Microcentrifuge
• Waterbath(56°C)
• Plantlights(optional)
• UVTransilluminatororUVPhotodocumentationsystem
• UVsafetygoggles
•Automaticmicropipets(5-50µl)withtips
•Microwave,hotplateorburner
•Pipetpump
•250mlflasksorbeakers
•Hotgloves
• Disposablevinylorlatexlaboratorygloves
• Icebucketsandice
•Distilledordeionizedwater
• Isopropanol
• Ethanol
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*If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three waterbaths. However, a thermal cycler assures a significantly higher rate of success.
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QuickPlants™ - Arabidopsis Thaliana
Arabidopsis thalianaisasmall,weed-likeplantfromthemustardfamily,Brassicaceae(Cruciferae).Inspiteofitshumbleappearance,Arabidopsis has become a superstar for plant geneticists and molecular biologists. There areseveralreasonsforitssuccess.First,thesmallsizeoftheplantsallowsforlargenumberstobegrowninasmallspaceinthelaboratory,growthchamberorgreenhouse.Second,Arabidopsis has a very short life cycle. Plantsfromseedsplantedtodaywillbeginfloweringinonlythreetofourweeks. This is an advantage for geneticists because they can make experi-mental crosses and raise many generations in a very short period of time. Third,Arabidopsis has a very small genome consisting of 5 chromosomes. The amount of DNA normally found in Arabidopsis cells is small compared tothatofotherplants.Someplantspeciesareknowntocontain10,000times as much DNA per cell as Arabidopsis. The small size of the Arabidopsis genome has made it possible to determine its entire nucleotide sequence. This task was completed in 2000. Annotating and identifying the genes in thesequenceandassigningfunctionstothem,willprobablytakemanymoreyears.
The same features of Arabidopsis that make it an attractive organism for re-search also make it useful in the classroom. The plant can be grown in large numbers and in a small space under classroom conditions. Genetics experi-ments can be completed in a single semester. Large numbers of interesting mutantshavebeenidentifiedandcharacterized,andseveralhavebeenselected as especially useful for education.
Examples of mutant characteristics are described below:
• gai1isagibberellicacidinsensitivedwarf.ThisArabidopsis plant is much smaller than the wild type.
• ap1-1andap3-3,arehomeoticmutants.Homeoticmutationshavetheeffect of converting one organ or body part into another; ap stands for apetala.Thenamereferstothephenotypeofthemutants,lackingpet-alsbecausetheyhavebeenconvertedintootherflowerparts.Notethatalthoughbothofthesemutationsproducesimilarphenotypes,theyaredefects in different genetic loci as indicated by their numbering.
• fus3-3,fusca,isamutantinwhichthegerminatingseedsaresplotchedwithreddishbrowncolor.Normally,seedlingsareexpectedtobeauni-form light green color.
• varmutantsarevariegated.Leavesaresplashedwithpatchesofwhite.
• yi1mutantshaveayellowinflorescence.Theflowerbudsofthismutantareaverypalegreen–yellowishcolorandtheflowerpetalsareoff-white.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2002, 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-19
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336Determining Quick Plant™ Genetics Using PCR
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QuickPlants™ - Arabidopsis Thaliana
• tt2-1mutantshaveatransparenttesta(oruncoloredseedcoat).Normally,seedcoatsarebrownandthismakestheseedsbrown.Transparenttestamutants,therefore,produceyellow,ratherthanbrownseeds.Sincetheseedcoathasnocolor,theseedsshow the color of the embryo inside.
The gl1-1 glabra are hairless mutants that are selected for inclusion in this mapping experiment. This mutant lacks the fine glandular hairs(trichomes)normallyfoundcoveringthesurfaceofanArabi-dopsis leaf.
mAPPInG STRATEGY
There are many advantages of genetic mapping vs. classical plant breeding.Withclassicalplantbreeding/genetics,manycrossesarerequired and many f1 lines must be maintained to reach a final re-sult.Withgeneticmapping,anassayfromtheDNAofasinglecrosswill yield many DNA polymorphic markers.
Traditionally,geneshavebeenlocated,ormappedtospecificlocionchro-mosomes by the technique of recombination mapping. This technique takes advantage of the fact that genes located very close together on a chromo-some are often inherited together as a package. The closer two genes are to oneanother,thelesslikelytheyaretobeseparatedbyrecombination.So,a gene is mapped by measuring the frequency of recombination between the gene of interest and other genes that have already been placed on the chromosome.
Thisstrategyformappinggenesislimitedhowever,bythenumberofgenesthat have already been mapped. Producing a very detailed map by recom-bination analysis requires many genes. Molecular biology has extended our ability to map genes by providing convenient genetic markers in numbers that literally saturate the chromosomes. Using molecular markers rather than Mendelian traits as chromosomal landmarks for mapping means that genes can be placed very precisely on the genetic map.
DnA ExTRACTIOn
Every method for extraction of DNA includes some common features: tissues aredisruptedtoreleaseDNA,cellulardebrisisremoved,andDNAisprecipi-tated to separate it from other cellular components. The method outlined inthisexperimentincludeseachofthesesteps.First,smallamountsofplantleaf tissue from Arabidopsis plants carrying the gene to be mapped and from mutant Arabidopsis plants are ground into a fine suspension in extraction
Wild Glabra
Figure 1:Wild and mutant Glabra strains. The gl1-1 glabra are hair-less mutants that lack fine glandular hairs (trichomes).
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QuickPlants™ - Arabidopsis Thaliana
buffer.Thisbuffercontainsachelatingagent(EDTA)toprotectDNAfromthe activity of nucleases released from the tissue as the cells are disrupted. Italsoincludessaltandadetergent(SDS)whichwilldisruptcellularmem-branes.Second,theplanttissueisincubatedinahotwaterbathtofacilitatecell lysis. Cell debris is removed from the preparation by centrifugation and the pelleted material is ground a second time to maximize DNA yield. After centrifugingasecondtime,DNAisprecipitatedfromtheclarifiedsuper-natant with isopropanol. The DNA prepared by this method is sufficiently purified to work as a template in the polymerase chain reaction step that follows.
POlYmERASE ChAIn REACTIOn
Sinceitsdiscoveryinthemid1980s,thepolymerasechainreaction(PCR)hasrevolutionized biological science. The enormous utility of PCR is based on its ease of use and its ability to amplify DNA. PCR amplification uses an enzyme known as TaqDNApolymerase.Thisenzyme,originallypurifiedfromabac-teriumthatinhabitshotsprings,isstableatveryhigh(nearboiling)temper-atures.AlsoincludedinthePCRreactionmixtureareshort(15-30nucleotide)syntheticoligonucleotides,knownasprimersandtheextractedDNAthatcontainstheregiontobeamplified,knownasthe"target".
InthefirststepofthePCRreaction(Figure2),knownasdenaturation,thetargetcomplementaryDNAstrandsaremelted(separated)fromeachotherat94°C,whiletheTaqDNApolymeraseremainsstable.Inthesecondstep,knownasannealing,thesampleiscooledtoanintermediatetemperature(usuallybetween37°Cand65°C)toallowhybridizationofthetwoprimerstothetwostrandsofthetargetDNA.InthethirdPCRstep(Figure2),knownasextension,thetemperatureisraisedto72°C.Atthistemperature,theTaq DNA polymerase is maximally active and adds nucleotides to the primers to complete the synthesis of the new complementary strands to the target re-gion.Thesethreesteps-denaturation,annealing,andextension–constituteonePCR"cycle”.Thisprocessistypicallyrepeatedfor20-40cycles,amplifyingthe target sequence exponentially. PCR is performed in a thermal cycler that isprogrammedtoheat,coolandmaintainsamplesatprecisetemperaturesfor varying time intervals.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2002, 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-19
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QuickPlants™ - Arabidopsis Thaliana
Figure 2: Polymerase Chain Reaction
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9Determining Quick Plant™ Genetics Using PCR
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The Exp
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bEFORE YOU START ThE ExPERImEnT
1. Read all instructions before starting the experiment.
2. If you will be conducting PCR using a thermal cycler without a heated lid,alsoreadtheAppendixentitled"PreparationandHandlingPCRSampleswithWax".
3. IfyouwillbeusingthreewaterbathstoconductPCR,readthetwoap-pendicesentitled"PolymeraseChainReactionUsingThreeWaterbaths"and"Handlingsampleswithwaxoverlays".
4. Writeahypothesisthatreflectstheexperimentandpredictexperimen-tal outcomes.
ExPERImEnT ObjECTIVE:
The object of this experiment is to introduce students to the concept of genetic linkage by using the polymerase chain reaction to amplify DNA from wild type and mutant Arabidopsis plants.
bRIEF DESCRIPTIOn OF ExPERImEnT:
Inthisexperiment,theextractedArabidopsis(glabraandwildtype)DNAwill be amplified at two separate target sequences on chromosomes 1 and 3.Theamplifiedregion(519basepairs)onchromosome3isunlinkedtotheglabragene,whilethetargetonchromosome1(1481basepairs)islinked.Comparison of the wild type and glabra PCR products experimentally dem-onstrate the concept of genetic linkage. This experiment has three modules:
Module I: Module I: Growing QuickPlants™ - Arabidopsis Thaliana
Module II: Isolation of Genomic DNA from Arabidopsis
Module III: PCR of Genomic DNA from Arabidopsis
Module IV: Agarose Gel Electrophoresis
GEl SPECIFICATIOnS
This experiment requires a gel with the following specifications:
• Recommendedgelsize 7x14cm(longtray) • Numberofsamplewellsrequired 6 • Placementofwell-formertemplate firstsetofnotches • Gelconcentrationrequired 1.0%
Experiment Overview and General Instructions
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2002, 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-19
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laboratory Safety
1. Gloves and goggles should be worn rou-tinely as good laboratory practice.
2. Exercise extreme caution when working with equipment that is used in conjunc-tion with the heating and/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
Wear gloves and safety goggles
4. Exercise caution when using any electrical equipment in the laboratory.
• Althoughelectricalcurrentfromthepowersourceisautomaticallydisruptedwhenthecoverisremovedfromtheapparatus,firstturnoffthepower,thenunplugthepowersourcebeforedisconnectingthe leads and removing the cover.
• Turnoffpowerandunplugtheequipmentwhennotinuse.
5. EDVOTEK injection-molded electrophoresis units do not have glued junc-tionsthatcandeveloppotentialleaks.However,intheunlikelyeventthataleakdevelopsinanyelectrophoresisapparatusyouareusing,IM-MEDIATELY SHUT OFF POWER. Do not use the apparatus.
6. Always wash hands thoroughly with soap and water after handling re-agents or biological materials in the laboratory.
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The Exp
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module I: Growing QuickPlants™ - Arabidopsis Thaliana
1. Sow the seeds thinly on the surface of a moist peat-based potting mix or on moistened peat pods.
Alternatively,sprinkleseveralseedsintoatubeandadd0.5mltapwa-ter to the tube. Use a small transfer pipet to disperse the seeds evenly on the soil surface.
2. Do not cover the seeds; the seeds need light for germination.
3. Placetheseedsdirectlyunderfluorescentlightsoronthesillofabrightwindow.
4. Keep the potting medium moist to wet while the seeds germinate. This will take approximately 3-4 days.
5. Aftertheseedsgerminate,theycantoleratesomedrying,butdon’tletthemdrycompletely.Misttheplantsdailywithadilute(1/4strength)solution of balanced commercial fertilizer.
Helpful Hints and Notes:
Quick Plants™ are amazingly hardy and tolerant of abuse once they become established.
Soil and planting: Soil can be mixed from standard greenhouse components. Use light soil mixtures with ample peat moss, and sterilize before planting in order to avoid any pest contamination. Alterna-tively, use commercially prepared mixes, such as Metromix 350 or ProMix BX. The surface of the soil should be approximately 1 cm from the top of the pot. Several pots can be put together in a tub or similar container. Cover with clear plastic wrap. Perforate the wrap to maintain enough humidity for germination.
Temperature: Quick Plants™ thrive under cool conditions. Optimum temperature is 25°C. Room temperature works great.
Lighting: More than any other factor, light determines how quickly the plants will grow and develop. Fastest growth is under continuous fluorescent light (shop lights). These can be easily and inexpensively configured in a classroom or lab. These conditions also produce compact sized plants. On a bright win-dowsill, or in a cool greenhouse, the plants take one to several weeks longer to develop, but are larger in size. Slowest growth occurs under low light conditions, such as a poorly lit windowsill.
Watering: After germination, water plants as needed to avoid water stress. Avoid over-watering to prevent the potential for algal or fungal growth on the soil surface. If algae does appear, allow the pots to dry and scrape the algae from the soil surface with care.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2002, 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-19
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336Determining Quick Plant™ Genetics Using PCR
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1. Harvest4-6seedlings(~1cmtall,1-3weeksold),oraleaffromamatureplant(~1x1cm)intoamicrofugetubewithpestle.
• PlaceGlabramutantseedlingsinonetubeandwildtypeseedlingsin another.
• Keepeachtubeseparatethroughouttheentireexperiment.
2. Use the pestle to partially mash the tissue.
3. Add100µlofDNAExtractionBuffertoeachtubeandcontinuegrindingthe tissue.
module II: Isolation of Genomic DnA from Arabidopsis
WARNING!Use only screw-cap tubes when incubating in the waterbath for DNA isolation. Do not use snap-top tubes.
After the 56°C Incubation:
6. Add250µlNaClsolutiontoeachtubeandmixwellfor30seconds.
7. Centrifugethetubesat13,000rpm(microcentrifugemaximumspeed)for 15-30 minutes.
8. Re-grind the pelleted material in each tube and centrifuge the tube at 13,000rpmfor5minutes.
9. Carefully transfer the supernatant from each tube into a fresh labeled microcentrifuge tube being careful not to disturb the pellet. Discard the tubes with pellets.
10. Precipitate the DNA in the supernatant by adding an equal amount of ice-cold isopropanol.
11. Incubate the tubes in the freezer for at least one hour to overnight.
After Incubation in the freezer:
12. CollecttheprecipitatedDNAbycentrifugationat13,000rpmfor10minutes.
13. Carefully remove and discard all the supernatant and leave the pelleted DNA at the bottom of the tube.
4. Addanadditional200µlofDNAExtractionBufferto each tube and grind the tissue again.
5. Incubate the tubes in a waterbath at 56°C for one hour.
Use caution not to cross-contaminate plant tissue and DNA - this will yield false positive results.
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The Exp
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module II: Isolation of Genomic DnA from Arabidopsis
OPTIOnAl STOPPInG POInT
The supernatant may be stored at -20°C until the experiment is continued.
14. Washthepelletwith1.5mlof70%ethanolorisopropanol.
15. Ifthepelletbecomesdislodged,spinatfullspeedfor2minutes.
16. Discard the supernatant and allow the DNA pellet to dry for 5 minutes.
17. Completelyresuspendthepelletin100µlofTE(10mMTris,pH8.0,0.1mMEDTA)bypipettingupanddownseveraltimesandbyvortexingortapping vigorously.
18. Proceed with the PCR preparations or store the DNA at -20°C if it will not be used immediately.
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module III: PCR of Genomic DnA from Arabidopsis
Perform one PCR reaction for each plant type.
1. TransferthePCRreactionpellet™totheappropriatesizedtube(e.g.0.5mlor0.2ml)foryourthermalcycler.
2. LabeleachPCRtubewiththeappropriatename(“glabra”or“wild”).
3. Toeachtube,addandmixthefollowing:
PCRReactionpellet™, 10µl UltraPurewater 10µl theappropriateArabidopsis DNA 5µl primermixture.
4. Ifyourthermalcyclerisequippedwithaheatedlid,proceeddirectlytopolymerase chain reaction cycling.
Ifyourthermalcyclerdoesnothaveaheatedlid,orifyouarecyclingmanuallywiththreewaterbaths,addonewaxbeadtothetubebeforeproceeding to polymerase chain reaction cycling.
5. Process the assembled reactions for polymerase chain reaction cycling in a thermal cycler as follows:
1 cycle 35 cycles 1 cycle 94°Cfor5minutes 94°Cfor1minute 72°Cfor4minutes 54°C for 30 seconds 72°Cfor90seconds
6. Afterthecompletionofthecycling,add5microlitersof10xgelloadsolution to each tube.
7. Proceedtoinstructionsforpreparinga1.0%agarosegel(7x14cm)andseparating the PCR products by electrophoresis.
OPTIOnAl STOPPInG POInT
The samples can be held in the thermal cycler at 4°C or frozen after addi-tion of 5 µl of 10x Gel Loading Solution until ready for electrophoresis.
The PCR reaction pel-let™ contains Taq DNA polymerase, the four deoxytriphosphates, Mg+2
and buffer.
Sample volumes are very small. For liquid samples, it is important to quick spin the tube contents in a microcentrifuge to ob-tain sufficient volume for pipeting. Spin samples for 10-20 seconds at maxi-mum speed.
15Determining Quick Plant™ Genetics Using PCR
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The Exp
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Agarose Gel Electrophoresis
AGAROSE GEl REQUIREmEnTS
• Recommendedgelsize: 7x14cm
7 x 14 cm gels are recommended to achieve better resolution of the PCR products. Each gel can be shared by several students or groups.
• Placementofwell-formertemplate: firstsetofnotches
• Agarosegelconcentration: 1.0%
PREPARInG ThE AGAROSE GEl
1. Closeofftheopenendsofacleananddrygelbed(castingtray)byusingrubber dams or tape.
2. Placeawell-formertemplate(comb)inthefirstsetofnotchesattheendof the bed. Make sure the comb sits firmly and evenly across the bed.
3. Toa250mlflaskorbeaker,addagarosepowderandbufferasindicatedintheReferenceTables(AppendixA)providedbyyourinstructor.Swirlthe mixture to disperse clumps of agarose powder.
4. Withamarkingpen,indicatethelevelofthesolutionvolumeontheoutsideoftheflask.
5. Heat the mixture using a microwave oven or burner to dissolve the aga-rose powder.
6. Cool the agarose solution to 60°C with careful swirling to promote even dissipationofheat.Ifdetectableevaporationhasoccurred,adddistilledwater to bring the solution up to the original volume marked in step 4.
After the gel is cooled to 60°C:
7. Placethebedonalevelsurfaceandpourthecooledagarosesolutioninto the bed.
8. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes.
9. Afterthegelissolidified,becarefulnottodamageortearthewellswhileremovingtherubberdamsortapeandcomb(s)fromthegelbed.
10. Placethegel(onitsbed)intotheelectrophoresischamber,properlyoriented,centeredandlevelontheplatform.
11. Fill the electrophoresis apparatus chamber with the appropriate amount ofdiluted(1x)electrophoresisbuffer(refertoTableBontheinstructionAppendixprovidedbyyourinstructor).
If you are unfamiliar with agarose gel preparation and electrophoresis, detailed instructions and helpful resources are available at www.edvotek.com
Important Note
Continue heating until the final solution appears clear (like water) without any un-dissolved particles. Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved.
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Agarose Gel Electrophoresis
bEFORE lOADInG ThE SAmPlES
This experiment requires a 1.0% agarose gel and is designed for staining with InstaStain® Ethidium Bromide.
lOADInG DnA SAmPlES
1. (OptionalStep)Heatthe200bpDNAladderandPCRsamplesfortwominutes at 50°C. Allow the samples to cool for a few minutes.
2. Make sure the gel is completely submerged under buffer before loading thesamples.Loadtheentirevolume(30µl)ofthesamplesinthefol-lowing sequence.
Lane 1 200 bp ladder 2 Wild type PCR DNA 3 Glabra PCR DNA
3. Record the position of your sample in the gel for easy identification after staining.
RUnnInG ThE GEl
4. AftertheDNAsamplesareloaded,properlyorientthecoverandcare-fully snap it onto the electrode terminals.
5. Insert the plugs of the black and red leads into the corresponding inputs of the power source.
6. Set the power source at the required voltage and conduct electrophore-sis for the length of time determined by your instructor.
7. Checktoseethatcurrentisflowingproperly-youshouldseebubblesforming on the two platinum electrodes.
8. Aftertheelectrophoresisiscompleted,disconnectthepowerandre-move the gel from the bed for staining.
STAInInG AnD VISUAlIzATIOn OF DnA Afterelectrophoresis,agarosegelsrequirestainingtovisualizetheseparatedDNA samples. Your instructor will provide instructions for DNA staining with InstaStain® Ethidium Bromide.
Reminder:
Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.
+-Black Red
Sample wells
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The Exp
erimen
t
Answer the following study questions in your laboratory notebook or on a separate worksheet.
1. Describe the methods involved in amplification of plant DNA from start to finish.
2. What can interfere with obtaining successful PCR results.
3. What are the advantages of using a genetic mapping strategy vs. tradi-tional plant breeding/crossing?
4. How can mapping a plant such as Arabidopsis help with other plant spe-cies or in other areas of plant breeding and genetics?
Study Questions
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Instructor’s Guide
Classsize,lengthoflaboratorysessions,andavailabilityofequipmentarefactors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. If you do not find the answers to your questionsinthissection,avarietyofresourcesarecontinuouslybeingaddedtotheEDVOTEKwebsite.Inaddition,TechnicalServiceisavailablefrom9:00amto6:00pm,Easterntimezone.Callforhelpfromourknowledge-abletechnicalstaffat1-800-EDVOTEK(1-800-338-6835).
nATIOnAl COnTEnT AnD SKIll STAnDARDS
Byperformingthisexperiment,studentswilllearntoloadsamplesandrun agarose gel electrophoresis. Analysis of the experiments will provide students the means to transform an abstract concept into a concrete expla-nation. Please visit our website for specific content and skill standards for various experiments.
EDUCATIOnAl RESOURCES
Electrophoresis hints, help and Frequently Asked Questions
EDVOTEK experiments are easy to perform and designed for maximum success in the classroom setting.However,eventhemostexperiencedstudents and teachers occasionally encounter experimental problems or difficulties. The ED-VOTEK web site provides several suggestions and remindersforconductingelectrophoresis,aswellas answers to frequently asked electrophoresis questions.
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Please have the following information ready:
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Technical ServiceDepartment
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notes to the Instructor:
PCR ExPERImEnTAl SUCCESS GUIDElInES
Please refer to the Appendices section for a summary of important hints and reminders which will help maximize successful implementation of this experi-ment. This experiment has three modules:
Module I: Growing QuickPlants™ - Arabidopsis Thaliana Module II: Isolation of Genomic DNA from Arabidopsis Module III: PCR of Genomic DNA from Arabidopsis Module IV: Agarose Gel Electrophoresis
mICROPIPETTInG bASICS AnD PRACTICE GEl lOADInG
Accurate pipeting is critical for maximizing successful experiment results. EDVOTEK Series 300 experiments are designed for students who have had previous experience with agarose gel electrophoresis and micropipeting techniques.Ifyourstudentsareunfamiliarwithusingmicropipets,EDVOTEKhighlyrecommendsthatstudentsperformExperiment#S-44,MicropipettingBasics,orotherSeries100or200electrophoresisexperimentpriortocon-ducting this advanced level experiment.
APPROxImATE TImE REQUIREmEnTS
1. ThePCRstep(35cycles)willtakeabout100-120minutesorcanbepro-cessed overnight and held at 4°C.
2. The experiment can be temporarily stopped after the completion of Modules I and II and later resumed. Experimental results will not be compromised if instructions are followed as noted under the heading “OptionalStoppingPoint”attheendofModuleIandModuleII.
3. Whetheryouchoosetopreparethegel(s)inadvanceorhavethe
studentspreparetheirown,allowapproximately30-40minutesforthisprocedure.Generally,20minutesofthistimeisrequiredforgelsolidification.Seesection“OptionsforPreparingAgaroseGels”below.
4. The approximate time for electrophoresis will vary from1-5hours.Generally,thehigherthevoltageapplied,thefasterthesamplesmigrate.However,depending upon the apparatus configuration and the distancebetweenthetwoelectrodes,individualelec-trophoresis units will separate DNA at different rates. Follow manufacturer's recommendations. Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C.
Table C Time and Voltage
Recommended Time Minimum Maximum
Volts
125
70
50
55 min
2 hrs 15 min
3 hrs 25 min
1 hr 15 min
3 hrs
5 hrs
(1.0% - 7 x 14 cm gel)
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Instru
ctor’s G
uid
e
notes to the Instructor:
OPTIOnS FOR PREPARInG AGAROSE GElS
This experiment is designed for DNA staining after electrophoresis with In-staStain® Ethidium Bromide. There are several options for preparing agarose gels for the experiment.
1. Individual Gel Casting: Each student lab group can be responsible for casting their own indi-
vidual gel prior to conducting the experiment.
2. Preparing Gels in Advance: Gels may be prepared ahead and stored for later use. Solidified gels can
be stored under buffer in the refrigerator for up to 2 weeks.
Do not store gels at -20°C. Freezing will destroy the gels.
Gelsthathavebeenremovedfromtheirtraysforstorage,shouldbe"anchored"backtothetraywithafewdropsofhot,moltenagarosebefore placing the gels into the apparatus for electrophoresis. This will prevent the gels from sliding around in the trays and the chambers.
3. Batch Gel Preparation: A batch of agarose gel can be prepared for sharing by the class. To save
time,alargerquantityofUltraSpec-Agarosecanbepreparedforsharingbytheclass.Seeinstructionsfor"BatchGelPreparation".
GEl COnCEnTRATIOn AnD VOlUmE
The gel concentration required for this experiment is 1.0%. Prepare gels ac-cording to Table A.1 or A.2 in Appendix D.
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EDVO-Kit #In
stru
cto
r’s
Gu
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notes to the Instructor:
GEl STAInInG AnD DESTAInInG AFTER ElECTROPhORESIS Afterelectrophoresis,theagarosegelsrequirestaininginordertovisualizethe separated DNA samples. This experiment features a proprietary stain called InstaStain®.
InstaStain® Etbr (Appendix F)
Optimal visualization of PCR products on gels of 1.0% or higher concentra-tionisobtainedbystainingwithInstaStain®EthidiumBromide(InstaStain®EtBr)cards.ExercisecautionwhenusingEthidiumBromide,whichisalistedmutagen.DisposaloftheInstaStain®EtBrcards,whichcontainonlyafewmicrogramsofethidiumbromide,isminimalcomparedtothelargevolumeof liquid waste generated by traditional ethidium bromide staining pro-cedures. Disposal of InstaStain® cards and gels should follow institutional guidelines for chemical waste.
InstaStain® blue: One-step Staining and Destaining (Appendix G)
InstaStain® Blue can be used as an alternative for staining gels in this experi-ment.However,InstaStain®BlueislesssensitivethanInstaStain®EtBrandwill yield variable results.
Agarosegelscanbestainedanddestainedinoneeasystep,whichcanbecompletedinapproximately3hours,orcanbeleftinliquidovernight.Forthebestphotographicresults,leavethegelinliquidovernight.Thiswillallowthestainedgelto"equilibrate"inthedestainingsolution,resultingindark blue DNA bands contrasting against a uniformly light blue background.
Gels stained with InstaStain® Blue may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid. DO NOT FREEZE AGAROSE GELS! Used InstaStain® Blue cards and destained gels can be discarded in solid waste disposal. Destaining solutions can be disposed down the drain.
PhOTODOCUmEnTATIOn OF DnA (OPTIOnAl)
Therearemanydifferentphotodocumentationsystemsavailable,includingdigital systems that are interfaced directly with computers. Specific instruc-tions will vary depending upon the type of photodocumentation system you are using.
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Instru
ctor’s G
uid
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Pre-lab Preparations
mODUlE I: GROwInG QUICKPlAnTS™
Plan to have plants ready for harvest on the day of the lab. Allow 2- 3 weeks for adequate growth. See Growing EDVOTEK QuickPlants™ in the Experi-ment Procedures section.
mODUlE II: ISOlATIOn OF GEnOmIC DnA FROm ArAbidopsis
1. IfaprecipitatehasformedintheDNAextractionbuffer,warmat37°Cto redissolve.
2. Prepare Proteinase K solution:
• Add200µlofDNAExtractionBuffer(H)toeachtubeofProteinaseK and allow the pellets to hydrate for a couple of minutes.
• AddthedissolvedProteinaseKbacktothe10mlofDNAExtractionBuffer and mix.
• Aliquot1mlforeachgroupandkeeponice.
3. Aliquot1mlofNaClsolution(G)foreachgroup.
4. Aliquot1mlofTrisBuffer(E)foreachgroup.
5. Placebottlesof95%and70%Isopropylalcoholoniceorinthefreezer.Chill thoroughly.
Each student group will require: • Arabidopsis glabra plants• Arabidopsis wild type plants• 2microcentrifugetubeswithpestle• 1mlDNAExtractionBuffer(H)• Ice-coldisopropanolandethanol• 1mlTrisbuffer• 1mlNaClsolution• Additionalmicrocentrifugetubes
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Notes and Reminders:
• AccuratetemperaturesandcycletimesarecriticalforPCR.Apre-runforone cycle (approx. 3 to 5 minutes) is recommended to check that the ther-mal cycler is properly programmed.
• Forthermalcyclersthatdonothaveatopheatingplate,itisnecessarytoplace a layer of wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix entitled "Preparation and Handling PCR Samples with Wax ".
• ThreewaterbathscanbeusedforPCRifathermalcyclerisunavailable.The experiment will require great care and patience. Samples will require wax layers. See appendices entitled "Polymerase Chain Reaction Using Three Waterbaths" and "Handling samples with wax overlays".
mODUlE III: PCR OF GEnOmIC DnA FROm ArAbidopsis
mODUlE IV: AGAROSE GEl ElECTROPhORESIS Whenstudentsarereadytoperformtheelectrophoresis,thawthe200bpDNAladder(C).Aliquot30µlofthe200bpDNAladderforeachgeltoberun. Place on ice until students are ready to load the gels.
Pre-lab Preparations
Each student group will require: • 2 PCRbeads(intubes)• 50µl UltraPurewater• 15µl primermixture• 20µl 10xGelLoadsolution
• Thawtheprimermix(B)andplaceonice.Aliquot15µlforeach student group.
25Determining Quick Plant™ Genetics Using PCR
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Instru
ctor’s G
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Experiment Results and Analysis
Theamplifiedregion(519basepairs)onchromosome3isunlinkedtotheglabragene,whilethetar-getonchromosome1(1481basepairs)islinked.Comparisonofthewild type and glabra PCR products experimentally demonstrate the concept of genetic linkage.
Lane 1 - 200 bp ladderLane 2 - Wild Type PCR DNALane 3 - Glabra PCR DNA
Note: Depending on the PCR conditions used, a diffuse, small-molecular weight band, known as a "primer di-mer", may be present below the 200 bp marker. This is a PCR artifact and can be ignored. Other minor bands may also appear due to nonspecific primer binding and the subsequent amplification of these sequences.
200bp
400bp
600bp
800bp1000bp1200bp1400bp
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336EDVO-Kit #
A PCR Experimental Success Guidelines
B Polymerase Chain Reaction Using Three Waterbaths
C Preparation and Handling of PCR Samples With Wax
D 1.0% Agarose Gel Preparation
E 1.0% Agarose Gels - Quantity Preparations
F Staining and Visualization of DNA with
InstaStain® Ethidium Bromide Cards
G InstaStain® Blue: One Step Staining
and Destaining
Material Safety Data Sheets
Appendices
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PCR Experimental Success Guidelines
EDVOTEKexperimentswhichinvolvetheextractionandamplificationofDNAareextremelyrelevant,excitingand stimulating classroom laboratory activities. These experiments have been performed successfully in many classroomsacrossthecountry,butdorequirecarefulexecutionbecauseofthesmallvolumesused.Thefollow-ingguidelinesoffersomeimportantsuggestions,remindersandhintsformaximizingsuccess.
DnA ExTRACTIOn AnD SAmPlE PREPARATIOn:
1. Sufficient Cells: It is critical that there are sufficient cells to obtain enough DNAthatwillyieldpositiveresults.Cellsourcesincludehuman,plant,dro-sophilaandbacterialcells.Withoutenoughcells,therewillnotbeenoughDNA template for the PCR reaction.
2. Centrifugation: Centrifuge the cell suspension carefully. If the pellet loos-ens,repeatthestep.Thesupernatantshouldbeclear,notcloudy,andthepellet should be solid at the bottom of the tube. Repeat centrifugation for a longerperiodoftime,ifnecessary.
ThE PCR REACTIOn
3. Add Primers and DnA to the PCR Reaction bead: Add the primer mixture (forwardandreverseprimers)andthecellDNA(supernatant)asspecifiedin the experimental procedures to the microcentrifuge tube containing the PCRreactionbead.Makesurethatthebead(whichcontainstheTaq DNA polymerase,the4XdTPs,MgandthePCRreactionbuffer)iscompletelydis-solved. Do a quick spin in a microcentrifuge to bring the entire sample to the bottom of the tube. Prepare the control reaction similarly.
4. The Thermal cycler: The thermal cycler must be programmed for the correct cycle sequence. It is critical that the temperatures and the time for each of the cycles are accurate.
5. Oil or wax: Forthermalcyclersthatdonothaveatopheatingplate,there-action in the tubes must be overlaid with oil or wax to prevent evaporation.
6. manual water bath PCR: Three water baths can be used as an alternative to athermalcyclerforPCR,butresultsaremorevariable.Samplesrequireoilorwax layers. This method requires extra care and patience.
Appendix A
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336EDVO-Kit #
PCR Experimental Success Guidelines(continued)
Appendix A
GEl PREPARATIOn AnD STAInInG
7. Concentrated agarose:Gelsofhigherconcentration(>0.8%)requirespecialattention when dissolving or re-melting. Make sure that the solution is com-pletelyclearof“clumps”orglassygranules. Distorted electrophoresis DNA band patterns will result if the gel is not properly prepared.
8. Electrophoretic separation: The tracking dye should travel at least 6 cm from the wells for adequate separation before staining.
9. Staining:Stainingofhigherconcentrationgels(>0.8%)requireadditionalcaretoobtainclear,visibleresults.
• Afterstaining(15to30min.)withInstaStain®EthidiumBromideorliq-uidethidiumbromide,examinetheresultsusingaUV(300nm)transil-luminator. Repeat the staining as required.
• GelsstainedwithInstaStain®Blueorotherliquidbluestainmayfadewith time. Re-stain the gel to visualize the DNA bands.
10. DnA 200 bp ladder: Afterstainingtheagarosegel,theDNA200bpladder(markers)shouldbevisible.Ifbandsarevisibleinthemarkersandcontrollanes,butbandsinthesamplelanesarefaintorabsent,itispossiblethatDNAwasnotsuccessfullyextractedfromthecells.Iftheladder,controlandDNAbandsareallfaintorabsent,potentialproblemscouldincludeimprop-ergelpreparation,absenceofbufferinthegel,impropergelstainingoradysfunctional electrophoresis unit or power source.
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336EDVO-Kit # Appendix B
PREPARATIOn OF ThE PCR REACTIOn:
1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains three critical components:
•PCRReactionpellet™ •Primermix •DNAforamplification
2. AfteraddingthecomponentsofthePCRreactionsample,usecleanforcepstotransferonewaxbeadtothePCRtube.AtthestartofthePCRreaction,the wax will melt and overlay the samples to prevent evaporation during heating.
POlYmERASE ChAIn REACTIOn CYClInG
3. Inthethree-waterbathPCRmethod,thePCRreactionsampleissequentiallycycledbetweenthreeseparatewaterbaths,eachsetatdifferenttempera-tures,foraspecifiedperiodoftime.Thesequentialplacementofthereac-tion sample in the waterbaths maintained at three different temperatures constitutes one PCR cycle. One example of a PCR cycle might be as follows:
94°C for 1 minute 50°C for 1 minute 72°Cfor1minute
See experiment instructions for specific program requirements.
4. The PCR tube must be handled carefully when sequentially cycled between the three waterbaths. For each cycle:
• CarefullyplacethePCRtubeinawaterbathfloat.Makesurethatthesample volume is at the bottom of the tube and remains undisturbed. If necessary,pulsespinthetubeinabalancedmicrocentrifuge,orshakethe tube to get all of the sample to the bottom of the tube.
• Useforcepstocarefullylowerthewaterbathfloat(withtubes)sequen-tially into the waterbaths.
5. Process the PCR reaction sample for the total number of cycles specified in theexperimentinstructions.Onthefinalcyclethe72°Cincubationcanbe extended to 5 minutes.
6. Afterallthecyclesarecompleted,thePCRsampleispreparedforelectro-phoresis.
Polymerase Chain Reaction Using Three waterbaths
SuperiorPCRresultsareobtainedusinganautomatedthermalcycler.However,ifyoudonothaveathermalcycler,thisexperimentcanbeadaptedtousethreewaterbaths(Cat.#544).Muchmorecareneedstobetakenwhen using the three-waterbath PCR method. The PCR incubation sample is small and can easily be evapo-rated. Results using three waterbaths are often variable. Please refer to the Appendix entitled "PCR Samples with wax Overlays" for sample handling and preparation tips.
Each PCR Reaction pellet contains Taq DNA polymerase, four deoxytriphosphates, Mg+2
and buffer.
It is imperative that the temperatures are accurately maintained throughout the experiment.
Important Note
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336EDVO-Kit #
Preparation and handling of PCR Samples with wax
For Thermal Cyclers without heated lids, or PCR Using Three waterbaths
Automated thermal cyclers with heated lids are designed to surround the entire sample tube at the appropri-ate temperature during PCR cycles. Heating the top of the tubes during these cycles prevents the very small samplevolumesfromevaporating.Forthermalcyclerswithoutheatedlids,orwhenconductingPCRbythethree-waterbathmethod,itisnecessarytoaddawaxbeadtothereactionsample.DuringthePCRprocess,thewax will melt and overlay the samples to prevent evaporation during heating.
PREPARInG ThE PCR REACTIOn:
1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains the following three critical components:
• PCRReactionpellet™ • Primermix • DNAforamplification
2. AfteraddingthecomponentsofthePCRreactionsample,usecleanforcepsto transfer one wax bead to the PCR tube.
3. Process the PCR reaction sample for the total number of cycles specified in the experiment instructions.
PREPARInG ThE PCR REACTIOn FOR ElECTROPhPORESIS:
4. Afterthecyclesarecompleted,transferthePCRtubetoarackandpreparethe PCR sample for electrophoresis.
• PlacethePCRtubeina94°Cwaterbathlongenoughtomeltthewaxoverlay. Use a clean pipet to remove most of the melted wax overlay.
• Allowathinlayerofthewaxtosolidify.
• Useacleanpipettiptogentlypokeaholethroughthesolidifiedwax.Remove the tip.
• Useanothercleanpipettiptoentertheholetoremovethevolumeofmixture specified in the experiment instructions. Transfer this volume to a clean tube.
• Addotherreagentsaccordingtoexperimentinstructions,ifapplicable,.
• Add5µlof10xGelLoadingsolutiontothesampleandstoreonice.
5. Proceed to delivery of the sample onto an agarose gel for electrophoresis as specified in the experiment instructions.
Appendix C
Each PCR Reaction pellet contains Taq DNA polymerase, four deoxytriphosphates, Mg+2
and buffer.
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ForDNAanalysis,therecom-mended electrophoresis buffer is Tris-acetate-EDTA,pH7.8.Theformula for diluting EDVOTEK (50x)concentratedbufferisonevolume of buffer concentrate to every 49 volumes of distilled or deionized water. Prepare buffer as required for your electropho-resis unit.
If preparing the gel with concentrated(50x)buffer,use Table A.1.
1.0% Agarose Gel Preparation
If preparing the gel with diluted(1x)buffer,useTable A.2.
Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C. The approxi-mate time for electrophoresis will vary from ap-proximately 1 - 5 hours depending upon various factors. Conduct electrophoresis for the length of time determined by your instructor.
50x Conc.Buffer (ml)
DistilledWater (ml)
6
8
10
20
294
392
490
980
+EDVOTEKModel #
Total Volume Required (ml)
Electrophoresis (Chamber) Buffer
M6+
M12
M36 (blue)
M36 (clear)
300
400
500
1000
Dilution
Table
B
Appendix D
Amt ofAgarose
(g)
ConcentratedBuffer (50X)
(ml)
Size of Gel(cm)
7 x 7
7 x 14
0.25
0.5
0.5
1.0
+
Table
A.1 Individual 1.0% UltraSpec-Agarose™ Gel
DistilledWater(ml)
TotalVolume
(ml)
24.5
49.0
25
50
=+
Amt ofAgarose
(g)
DilutedBuffer (1x)
(ml)
Size of Gel(cm)
7 x 7
7 x 14
0.25
0.5
25
50
+
Individual 1.0% UltraSpec-Agarose™ Gel
Table
A.2
Table C Time and Voltage
Recommended Time Minimum Maximum
Volts
125
70
50
55 min
2 hrs 15 min
3 hrs 25 min
1 hr 15 min
3 hrs
5 hrs
(1.0% - 7 x 14 cm gel)
The biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2002, 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-19
33
336EDVO-Kit #
Tosavetime,electrophoresisbufferandagarosegelsolutioncanbepreparedinlargerquantitiesforsharingby the class. Unused diluted buffer can be used at a later time and solidified agarose gel can be remelted.
1.0% Agarose Gels - Quantity Preparations
bUlK ElECTROPhORESIS bUFFER
Quantity(bulk)preparationfor3litersof1xelectro-phoresis buffer is outlined in Table D.
bATCh AGAROSE GElS (1.0%)
Forquantity(batch)preparationof1.0%agarosegels,seeTableE.
1. Usea500mlflasktopreparethedilutedgelbuf-fer
2. Pour appropriate amount of UltraSpec-Aga-rose™ into the prepared buffer. Swirl to disperse clumps.
3. Withamarkingpen,indicatethelevelofsolutionvolumeontheoutsideoftheflask.
4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution.
5. Cool the agarose solution to 60°C with swirling to promote even dissipation of heat. If evaporation hasoccurred,adddistilledwaterto bring the solution up to the original volume as marked on the flaskinstep3.
6. Dispense the required volume of cooled agarose solution for casting each gel. The volume re-quired is dependent upon the size of the gel bed.
7. Allowthegeltocompletelysolidify.Itwillbecome firm and cool to the touch after approxi-mately 20 minutes. Then proceed with preparing the gel for electrophoresis.
60˚C
Note: The UltraSpec-Agarose™ kit component is often labeled with the amount it contains. Please read the label carefully. If the amount of agarose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount.
Appendix E
Table
D
ConcentratedBuffer (50x)
(ml)
DistilledWater(ml)
TotalVolume
(ml)
60 2,940 3000 (3 L)
=+
Bulk Preparation of Electrophoresis Buffer
Table
E
Amt ofAgarose
(g)
ConcentratedBuffer (50x)
(ml)
DistilledWater(ml)
TotalVolume
(ml)
3.0
4.0
6.0
8.0
294
392
300
400
+ =+
Batch Preparation of 1.0% UltraSpec-Agarose™
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2002, 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-19
34
The biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
336EDVO-Kit #
DNA InstaStain™
Patents Pending
DNA InstaStain™
Patents Pending
- - - - -
- - - - -
1
2
3
4
5
Press firmly.
Moisten the gel.
Place the InstaStain® card on the gel.
Place a small weight to ensure good contact.
View on U.V. (300 nm) transilluminator
Wear gloves and safety goggles
Do not stain gel(s) in the electrophoresis apparatus.
1. Afterelectrophoresis,placethegelonapieceofplasticwraponaflatsurface.Moistenthegel with a few drops of electrophoresis buffer.
2. Wearinggloves,removetheclearplasticpro-tectivesheet,andplacetheunprintedsideofthe InstaStain® EtBr card on the gel.
3. Firmly run your fingers over the entire surface of the InstaStain® EtBr. Do this several times.
Visit our web site for an animated demonstration of InstaStain® EtBr.
Disposal of InstaStain
Disposal of InstaStain® cards and gels should follow institutional guidelines for chemical waste.
Additional notes About Staining
• Ifbandsappearfaint,orifyouarenotusingEDVOTEKUltraSpec-Agarose™,gels may take longer to stain with InstaStain® EtBr. Repeat staining and increase the staining time an additional 10-15 minutes.
Caution: Ethidium Bromide is a listed mutagen.
Staining and Visualization of DnA
InSTASTAIn® EThIDIUm bROmIDE CARDS
• DNA200bpmarkersshouldbevisibleafterstainingeveniftheamplifiedDNAsamplesarefaintorabsent.Ifmarkers are not visible, troubleshoot for problems with the electrophoretic separation.
4. Place the gel casting tray and a small empty beaker on top to ensure that the InstaStain® card maintains direct contact with the gel surface.
Allow the InstaStain® EtBr card to stain the gel for 10-15 minutes.
5. After10-15minutes,removetheInstaStain®EtBrcard.Transferthegeltoaultraviolet(300nm)transilluminatorforviewing.BesuretowearUVprotec-tive goggles.
Appendix F
35
336EDVO-Kit #material Safety Data Sheets
Fullsize(8.5x11”)pdfcopyofMSDSavailableatwww.edvotek.comorbyrequest.
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
Ag
aro
se
10/0
5/06
This
pro
du
ct c
on
tain
s n
o h
azar
do
us
mat
eria
ls a
s d
efin
ed b
y th
e O
SHA
Haz
ard
Co
mm
un
icat
ion
Stan
dar
d.
CA
S #9
012-
36-6
For
1% s
olu
tio
n 1
94 F
N
o d
ata
N
o d
ata
No
dat
a
No
dat
a
No
dat
a
Inso
lub
le -
co
ld
W
hit
e p
ow
der
, no
od
or
N.D
. = N
o d
ata
No
dat
a
N
.D.
N.D
.
Wat
er s
pra
y, d
ry c
hem
ical
, car
bo
n d
ioxi
de,
hal
on
or
stan
dar
d f
oam
Poss
ible
fir
e h
azar
d w
hen
exp
ose
d t
o h
eat
or
flam
e
No
ne
ED
VO
TE
K®
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
G
en. d
iluti
on
ven
tila
tio
n
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
Yes
Sp
lash
pro
of
go
gg
les
Imp
ervi
ou
s cl
oth
ing
to
pre
ven
t sk
in c
on
tact
No
neX
N
on
e
No
dat
a av
aila
ble
X
No
ne
Yes
Y
es
Yes
Inh
alat
ion
: N
o d
ata
avai
lab
le
In
ges
tio
n:
Larg
e am
ou
nts
may
cau
se d
iarr
hea
No
dat
a av
aila
ble
No
dat
a av
aila
ble
Trea
t sy
mp
tom
atic
ally
an
d s
up
po
rtiv
ely
Swee
p u
p a
nd
pla
ce in
su
itab
le c
on
tain
er f
or
dis
po
sal
No
rmal
so
lid w
aste
dis
po
sal
No
ne
No
ne
Ch
emic
al c
artr
idg
e re
spir
ato
r w
ith
fu
ll fa
cep
iece
.
ED
VO
TE
K®
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
50x
Elec
tro
ph
ore
sis
Bu
ffer
This
pro
du
ct c
on
tain
s n
o h
azar
do
us
mat
eria
ls a
s d
efin
ed b
y th
e O
SHA
Haz
ard
Co
mm
un
icat
ion
Sta
nd
ard
.
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
Ap
pre
ciab
le, (
gre
ater
th
an 1
0%)
Cle
ar, l
iqu
id, s
ligh
t vi
neg
ar o
do
r
No
dat
a
N.D
. = N
o d
ata N.D
.
N.D
.
Use
ext
ing
uis
hin
g m
edia
ap
pro
pri
ate
for
surr
ou
nd
ing
fir
e.
Wea
r p
rote
ctiv
e eq
uip
men
t an
d S
CB
A w
ith
fu
ll fa
cep
iece
op
erat
ed in
po
siti
ve p
ress
ure
mo
de.
No
ne
iden
tifi
ed
10/0
5/06
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X
N
on
e
Stro
ng
oxi
diz
ing
ag
ents
Car
bo
n m
on
oxi
de,
Car
bo
n d
ioxi
de
X
N
on
e
Yes
Y
es
Y
es
No
ne
No
ne
iden
tifi
ed
Irri
tati
on
to
up
per
res
pir
ato
ry t
ract
, ski
n, e
yes
No
ne
Ing
esti
on
: If
co
nsc
iou
s, g
ive
larg
e am
ou
nts
of
wat
er
Eyes
: Fl
ush
wit
h w
ater
In
hal
atio
n:
Mo
ve t
o f
resh
air
Sk
in:
Was
h w
ith
so
ap a
nd
wat
er
Wea
r su
itab
le p
rote
ctiv
e cl
oth
ing
. M
op
up
sp
ill
and
rin
se w
ith
wat
er, o
r co
llect
in a
bso
rpti
ve m
ater
ial a
nd
dis
po
se o
f th
e ab
sorp
tive
mat
eria
l.
Dis
po
se in
acc
ord
ance
wit
h a
ll ap
plic
able
fed
eral
, sta
te, a
nd
loca
l en
viro
men
tal r
egu
lati
on
s.
Avo
id e
ye a
nd
ski
n c
on
tact
.
No
ne
Yes
N
on
e
Yes
N
on
e
Yes
_Saf
ety
go
gg
les
No
ne
No
ne
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X X
Trea
t sy
mp
tom
atic
ally
an
d s
up
po
rtiv
ely
Rin
se c
on
tact
ed a
rea
wit
h c
op
iou
s am
ou
nts
of
wat
er.
No
ne
req
uir
ed
No
ne
Yes
Y
es
Y
es
May
cau
se s
kin
or
eye
irri
tati
on
No
ne
rep
ort
ed
Rin
se c
on
tact
ed a
rea
wit
h c
op
iou
s am
ou
nts
of
wat
er.
Ob
serv
e al
l fed
eral
, sta
te, a
nd
loca
l reg
ula
tio
ns.
Avo
id e
ye a
nd
ski
n c
on
tact
.
No
ne
Ch
emic
al c
artr
idg
e re
spir
ato
r w
ith
org
anic
vap
or
cart
rid
ge.
Yes
Yes
Yes
No
ne
yes
Sp
lash
pro
of
go
gg
les
Do
no
t in
ges
t. A
void
co
nta
ct w
ith
ski
n, e
yes
and
clo
thin
g.
Was
h t
ho
rou
gh
ly a
fter
han
dlin
g.
No
ne
No
ne
kno
wn
Sulf
ur
oxi
des
an
d b
rom
ides
Acu
te e
ye c
on
tact
: M
ay c
ause
irri
tati
on
N
o d
ata
avai
lab
le f
or
oth
er r
ou
tes
No
ne
No
dat
a
No
dat
a
N
o d
ata
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
Gel
load
ing
so
luti
on
co
nce
ntr
ate,
10x
10/1
0/06
This
pro
du
ct c
on
tain
s n
o h
azar
do
us
mat
eria
ls a
s d
efin
ed b
y th
e O
SHA
Haz
ard
Co
mm
un
icat
ion
Sta
nd
ard
.
No
dat
a
No
dat
a
No
dat
a
No
dat
a
N/A
No
dat
a
solu
ble
Blu
e liq
uid
, no
od
or
Un
kno
wn
No
dat
a
No
dat
a
No
dat
a
Dry
ch
emic
al, c
arb
on
dio
xid
e, w
ater
sp
ray
or
foam
Use
ag
ents
su
itab
le f
or
typ
e o
f su
rro
un
din
g f
ire.
Kee
p u
pw
ind
, avo
id
bre
ath
ing
haz
ard
ou
s su
lfu
r o
xid
es a
nd
bro
mid
es.
Wea
r SC
BA
.
ED
VO
TE
K®
36
336EDVO-Kit # material Safety Data Sheets
Fullsize(8.5x11”)pdfcopyofMSDSavailableatwww.edvotek.comorbyrequest.
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Iden
tify
Info
rmat
ion
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
ED
VO
TE
K®
Inst
aSta
in®
Met
hyl
ene
Blu
e, M
eth
ylen
e B
lue
Plu
s™
10/0
5/06
Met
hyl
ene
Blu
e
3.7
Bis
(D
imet
hyl
amin
o)
Phen
oth
iazi
n 5
IUM
C
hlo
rid
e
No
dat
a av
aila
ble
CA
S #
61-7
3-4
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
Solu
ble
- c
old
Ch
emic
al b
ou
nd
to
pap
er, n
o o
do
r
No
dat
a av
aila
ble
No
dat
a
N
o d
ata
Wat
er s
pra
y, c
arb
on
dio
xid
e, d
ry c
hem
ical
po
wd
er, a
lco
ho
l or
po
lym
er f
oam
Self
co
nta
ined
bre
ath
ing
ap
par
atu
s an
d p
rote
ctiv
e cl
oth
ing
to
pre
ven
t co
nta
ct
wit
h s
kin
an
d e
yes
Emit
s to
xid
fu
mes
un
der
fir
e co
nd
itio
ns
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X
No
ne
Stro
ng
oxi
diz
ing
ag
ents
Toxi
c fu
mes
of
Car
bo
n m
on
oxi
de,
Car
bo
n d
ioxi
de,
n
itro
gen
oxi
des
, su
lfu
r o
xid
es, h
ydro
gen
, ch
lori
de
gas
X
N
on
e
Yes
Y
es
Yes
Skin
: M
ay c
ause
ski
n ir
rita
tio
n
Eyes
: M
ay c
ause
eye
irri
tati
on
In
hal
atio
n:
Cya
no
sis
Mee
ts c
rite
ria
for
pro
po
sed
OSH
A m
edic
al r
eco
rds
rule
PER
EAC
47.
3042
0.82
No
dat
a av
aila
ble
No
dat
a av
aila
ble
Trea
t sy
mp
tom
atic
ally
Ven
tila
te a
rea
and
was
h s
pill
sit
e
Mix
mat
eria
l wit
h a
co
mb
ust
ible
so
lven
t an
d b
urn
in c
hem
ical
inci
ner
ato
r eq
uip
ped
wit
h a
fter
bu
rner
an
d s
cru
bb
er.
Ch
eck
loca
l an
d s
tate
reg
ula
tio
ns.
Kee
p t
igh
tly
clo
sed
. St
ore
in c
oo
l, d
ry p
lace
No
ne
MIO
SH/O
SHA
ap
pro
ved
, SC
BA
Req
uir
ed
Ru
bb
erC
hem
. saf
ety
go
gg
les
Ru
bb
er b
oo
ts
Mat
eria
l Saf
ety
Dat
a S
hee
tM
ay b
e us
ed to
com
ply
with
OSH
A's
Haz
ard
Com
mun
icat
ion
Stan
dard
. 29
CFR
191
0.12
00 S
tand
ard
mus
t be
cons
ulte
d fo
rsp
ecifi
c re
quire
men
ts.
IDEN
TITY
(As
Use
d on
Lab
el a
nd L
ist)
Not
e: B
lank
spa
ces
are
not p
erm
itted
. If
any
item
is n
ot
appl
icab
le, o
r no
info
rmat
ion
is a
vaila
ble,
the
spac
e m
ust
be m
arke
d to
indi
cate
that
.
Sec
tio
n I
Man
ufac
ture
r's N
ame
Sec
tio
n II
- H
azar
do
us
Ing
red
ien
ts/Id
enti
fy In
form
atio
n
Emer
genc
y Te
leph
one
Num
ber
Tele
phon
e N
umbe
r for
info
rmat
ion
Dat
e Pr
epar
ed
Sign
atur
e of
Pre
pare
r (op
tiona
l)
Addr
ess
(Num
ber,
Stre
et, C
ity, S
tate
, Zi
p Co
de)
ED
VO
TE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ardo
us C
ompo
nent
s [S
peci
fic
Chem
ical
Iden
tity;
Com
mon
Nam
e(s)
]
OSH
A P
ELAC
GIH
TLV
Oth
er L
imits
Re
com
men
ded
% (O
ptio
nal)
(301
) 25
1-59
90
(301
) 25
1-59
90
Boili
ng P
oint
Sec
tio
n II
I - P
hys
ical
/Ch
emic
al C
har
acte
rist
ics
Unu
sual
Fire
and
Exp
losi
on H
azar
ds
Spec
ial F
ire F
ight
ing
Proc
edur
es
Vapo
r Pre
ssur
e (m
m H
g.)
Vapo
r Den
sity
(AIR
= 1
)
Solu
bilit
y in
Wat
er
App
eara
nce
and
Odo
r
Sec
tio
n IV
- P
hys
ical
/Ch
emic
al C
har
acte
rist
ics
Flas
h Po
int (
Met
hod
Use
d)
Extin
guis
hing
Med
ia
Flam
mab
le L
imits
UEL
LEL
Mel
ting
Poin
t
Evap
orat
ion
Rate
(But
yl A
ceta
te =
1)
Spec
ific
Gra
vity
(H 0
= 1
) 2
EDV
OTE
K®
Tris
-ED
TA B
uffer
(TE
)
10/1
0/06
CAS
# 13
9-33
-3
----
----
----
----
--- N
o da
ta --
----
----
----
--
No
data
No
data
No
data
No
data
No
data
No
data
Solu
ble
Clea
r, no
odo
r
No
data
Dry
che
mic
al, c
arbo
n di
oxid
e, h
alon
, wat
er s
pray
or s
tand
ard
foam
Ther
mal
dec
ompo
sitio
n pr
oduc
ts m
ay in
clud
e to
xic
and
haza
rdou
s oxi
des
of c
arbo
n, n
itrog
en,
and
sodi
um.
Mov
e co
ntai
ner f
rom
fire
are
a if
poss
ible
Stab
ility
Sec
tio
n V
- R
eact
ivit
y D
ata
Uns
tabl
e
Sec
tio
n V
I - H
ealt
h H
azar
d D
ata
Inco
mpa
tibili
ty
Cond
ition
s to
Avo
id
Rout
e(s)
of E
ntry
:In
hala
tion?
Inge
stio
n?Sk
in?
Oth
er
Stab
le
Haz
ardo
us
Poly
mer
izat
ion
May
Occ
urCo
nditi
ons
to A
void
Will
Not
Occ
ur
Hea
lth H
azar
ds (A
cute
and
Chr
onic
)
Carc
inog
enic
ity:
NTP
?O
SHA
Reg
ulat
ion?
IARC
Mon
ogra
phs?
Sign
s an
d Sy
mpt
oms
of E
xpos
ure
Med
ical
Con
ditio
ns G
ener
ally
Agg
rava
ted
by E
xpos
ure
Emer
genc
y Fi
rst A
id P
roce
dure
s
Sec
tio
n V
II -
Pre
cau
tio
ns
for
Saf
e H
and
ling
an
d U
seSt
eps
to b
e Ta
ken
in c
ase
Mat
eria
l is
Rele
ased
for S
pille
d
Was
te D
ispo
sal M
etho
d
Prec
autio
ns to
be
Take
n in
Han
dlin
g an
d St
orin
g
Oth
er P
reca
utio
ns
Sec
tio
n V
III -
Co
ntr
ol M
easu
res
Vent
ilatio
nLo
cal E
xhau
stSp
ecia
l
Mec
hani
cal (
Gen
eral
)
Resp
irato
ry P
rote
ctio
n (S
peci
fy T
ype)
Prot
ectiv
e G
love
s
Oth
er P
rote
ctiv
e Cl
othi
ng o
r Equ
ipm
ent
Wor
k/H
ygie
nic
Prac
tices
Eye
Prot
ectio
n
Haz
ardo
us D
ecom
posi
tion
or B
ypro
duct
s
Rena
l or h
eart
dis
ease
, pot
assi
um d
efici
ency
, in
sulin
dep
ende
nt, d
iabe
tes,
seiz
ures
or i
ntra
cran
ial l
esio
ns.
X
Exce
ssiv
e he
at, s
park
s or
ope
n fla
me
X
Impe
rvio
us c
loth
ing
to p
reve
nt s
kin
cont
act
Emer
genc
y ey
e w
ash
shou
ld b
e av
aila
ble
Acid
s, al
umin
um, m
etal
s, ox
idiz
ers
(str
ong)
Yes
Ye
s
Y
es
Muc
ous
mem
bran
e irr
itatio
n, e
ye/s
kin
irrita
tion,
irrit
atin
g to
gas
troi
ntes
tinal
sys
tem
.
Trea
t sym
ptom
atic
ally
and
sup
port
ivel
y
Mop
up
with
abs
orpt
ive
mat
eria
l. C
onta
iner
ize
to d
ispo
se o
r pro
perly
Obs
erve
fede
ral,
stat
e, a
nd lo
cal l
aws.
Stor
es a
way
from
str
ong
oxid
izer
s or
hea
t. A
void
ski
n/ey
e co
ntac
t.
Non
e
Yes
N
one
V
ent.
Sys.
N
one
Yes
Sp
lash
pro
of g
oggl
es
Ther
mal
dec
ompo
sitio
n pr
oduc
ts o
f tox
ic a
nd h
azar
dous
oxi
des
of C
, N, &
Na
Non
e
Mod
erat
ely
toxi
c by
inge
stio
n. S
yste
mic
toxi
city
may
resu
lt.
Non
e
No
data
N
o da
ta
N
o da
ta
Chem
ical
car
trid
ge re
spira
tor w
ith fu
ll fa
cepi
ece
and
orga
nic
vapo
r car
trid
ge
May
che
late
lead
mag
nesi
um,
zinc
, tra
ce m
etal
s if
pres
ent i
n in
test
ine
poss
. cau
sing
incr
.abs
orpt
ion.
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X
N
on
e
Stro
ng
oxi
diz
ing
ag
ents
Car
bo
n m
on
oxi
de,
Car
bo
n d
ioxi
de,
nit
rog
en o
xid
es, h
ydro
gen
bro
mid
e g
as
X
N
on
e
Yes
Y
es
Yes
No
dat
a av
aila
ble
Irri
tati
on
to
mu
cou
s m
emb
ran
es a
nd
up
per
res
pir
ato
ry t
ract
No
dat
a
Trea
t sy
mp
tom
atic
ally
an
d s
up
po
rtiv
ely
Wea
r SC
BA
, ru
bb
er b
oo
ts, r
ub
ber
glo
ves
Mix
mat
eria
l wit
h c
om
bu
stib
le s
olv
ent
and
bu
rn in
a c
hem
ical
inci
ner
ato
r eq
uip
ped
aft
erb
urn
er a
nd
scr
ub
ber
Use
in c
hem
ical
fu
me
ho
od
wit
h p
rop
er p
rote
ctiv
e la
b g
ear.
Mu
tag
en
Yes
Ch
em. f
um
e h
oo
d
No
N
on
e
Ru
bb
er
C
hem
. saf
ety
go
gg
les
R
ub
ber
bo
ots
Use
in c
hem
ical
fu
me
ho
od
wit
h p
rop
er p
rote
ctiv
e la
b g
ear.
Acu
te: M
ater
ial i
rrit
atin
g t
o m
uco
us
mem
bra
nes
, up
per
res
pir
ato
ry t
ract
, eye
s, s
kin
Ch
ron
ic:
May
alt
er g
enet
ic m
ater
ial
SCB
A
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Inst
aSta
in, I
nc.
P.O
. Bo
x 12
32W
est
Bet
hes
da,
MD
208
27
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
Inst
aSta
in®
Eth
idiu
m B
rom
ide
Eth
idiu
m B
rom
ide
D
ata
no
t av
aila
ble
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
Solu
ble
Ch
emic
al b
ou
nd
to
pap
er, n
o o
do
r
No
dat
a
N.D
. = N
o d
ata N.D
. N
.D.
Wat
er s
pra
y, c
arb
on
dio
xid
e, d
ry c
hem
ical
po
wd
er, a
lco
ho
l or
po
lym
er f
oam
Wea
r p
rote
ctiv
e cl
oth
ing
an
d S
CB
A t
o p
reve
nt
con
tact
wit
h s
kin
& e
yes
Emit
s to
xic
fum
es
10/0
5/06
CA
S# 1
39-3
3-3
(2,7
-Dia
min
o-1
0-Et
hyl
-9-P
hen
ylp
hen
anth
rid
iniu
m B
rom
ide)
ED
VO
TE
K®
37
336EDVO-Kit #material Safety Data Sheets
Fullsize(8.5x11”)pdfcopyofMSDSavailableatwww.edvotek.comorbyrequest.
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X X
Wea
r N
IOSH
/MSH
A r
esp
irat
or
Do
no
t in
ges
t. A
void
co
nta
ct w
ith
ski
n, e
yes
and
clo
thin
g.
Yes
Y
es
Y
es
----
----
----
----
----
----
-- N
o d
ata
----
----
----
----
----
----
---
Inh
aled
: M
ove
to
fre
sh a
ir E
yes/
Skin
: Fl
ush
wit
h w
ater
fo
r 15
min
ute
sIn
ges
tio
n:
Was
h m
ou
th w
ith
wat
er a
nd
cal
l ph
ysic
ian
Plac
e in
bag
. Ven
tila
te a
rea
and
was
h s
pill
sit
e
Ob
serv
e fe
der
al, s
tate
, an
d lo
cal r
egu
lati
on
s
Avo
id s
kin
co
nta
ct.
No
ne
Yes
Ir
rita
tio
n
U
nkn
ow
n
Stro
ng
oxi
diz
ing
ag
ents
, str
on
g a
cid
s
Ru
bb
er
C
hem
ical
saf
ety
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
Sod
ium
Ch
lori
de
10/1
0/06
CA
S #
7647
-14-
5
No
Dat
a
No
dat
a
No
dat
a
No
dat
a
2.16
5
801°
C
No
dat
a
Solu
ble
Cle
ar li
qu
id
----
----
No
n-c
om
bu
stib
le--
----
----
Use
ext
ing
uis
hin
g m
edia
ap
pro
pri
ate
to s
urr
ou
nd
ing
fir
e co
nd
itio
ns.
No
ne
No
neED
VO
TE
K®
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
ED
VO
TE
K®
Extr
acti
on
Bu
ffer
10/1
0/06
5144
-89-
860
-00-
457
-09-
0
100°
C
No
data
No
data
1.02
0
100°
C
No
data
Yes
Clea
r liq
uid
Wat
er s
pray
Wea
r SC
BA a
nd p
rote
ctiv
e cl
othi
ng t
o pr
even
t co
ntac
t w
/ ski
n an
d ey
es.
Emit
s to
xic
fum
es u
nder
fir
e co
ndit
ions
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
extr
emel
y de
stru
ctiv
e to
tis
sue
of t
he m
ucou
s m
embr
anes
and
upp
er r
espi
rato
ry t
ract
, eye
s an
d sk
in.
X Stro
ng o
xidi
zing
age
nts,
pro
tect
fro
m m
oist
ure
Carb
on m
onox
ide,
car
bon
diox
ide,
nit
roge
n ox
ides
, hy
drog
en b
rom
ide
gas
X
N/A
Safe
ty s
how
er a
nd e
ye b
ath,
fac
e sh
ield
Was
h th
orou
ghly
aft
er h
andl
ing,
kee
p ti
ghtl
y cl
osed
.
Yes
Yes
Yes
Har
mfu
l if
swal
low
ed, i
nhal
ed, o
r ab
sorb
ed t
hrou
gh s
kin.
Mat
eria
l is
Imm
edia
tely
flu
sh e
yes
or s
kin
w/ c
opio
us a
mou
nts
of w
ater
for
at
leas
t 15
min
utes
whi
le r
emov
ing
Evac
uate
are
a. C
over
wit
h dr
y lim
e or
sod
a as
h-pi
ck u
p. k
eep
in a
clo
sed
cont
aine
r an
d ho
ld f
orw
aste
dis
posa
l.
Dis
solv
e or
mix
the
mat
eria
l w/ c
ombu
stib
le s
olve
nt a
nd b
urn
in a
che
mic
al in
cine
rato
req
uipp
ed w
ith
an a
fter
burn
er.
Vent
ilate
are
a an
d w
ash
spill
sit
e af
ter
mat
eria
l pic
k up
is c
ompl
ete
Obs
erve
all
fede
ral,
stat
e, a
nd lo
cal l
aws.
Use
onl
y in
a c
hem
ical
fum
e ho
od. W
ear
appr
opri
ate
NIO
SH/M
SHA
apr
rove
d re
spir
ator
.
safe
ty g
love
s
saf
ety
gogg
les