336 Determining QuickPlant™ Genetics Using PCR...A PCR Experimental Success Guidelines 28 B...

Updated

Revised

and

The Biotechnology Education Company ®

EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com

EVT 2010-04-19

EDVO-Kit #

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.

http://www.edvotek.com

Determining Quick Plant™ Genetics Using PCR2

xxx336EDVO-Kit #

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

EVT 2010-04-19

3

EDVOTEK - The biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com

FAx: (301) 340-0582 • email: [email protected]

336EDVO-Kit #

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


mailto:[email protected]


xxx336EDVO-Kit #


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

Online Orderingnow available

Visit our web site for information about EDVOTEK’s complete line of “hands-on” experiments forbiotechnology and biology education.

Mon - Fri 9 am - 6 p

m E

T

(1-800-338-6835)

EDVO-TECH SERVICE

1-800-EDVOTEK

Mon - Fri9:00 am to 6:00 pm ET

FAX: (301) 340-0582Web: www.edvotek.comemail: [email protected]

Please have the following information ready:

• Experiment number and title• Kit lot number on box or tube• Literature version number (in lower right corner)• Approximate purchase date

Technical ServiceDepartment

*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.


http://www.edvotek.comemail:




5Determining Quick Plant™ Genetics Using PCR

336EDVO-Kit #

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

backg

rou

nd

Info

rmatio

n

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.



6



EDVO-Kit #b

ackg

rou

nd

Info

rmat

ion


• 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).



336EDVO-Kit #



backg

rou

nd

Info

rmatio

n


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.



8



EDVO-Kit #Th

e Ex

per

imen

t


Figure 2: Polymerase Chain Reaction

3'5'

3'5'

5'3'

5'3'

5'

5'3'3'5'

5'3'

5'5'

Denature 94°C

5'

Extension72°C

3'5'

Separation of two DNA strands

=

Primer 1=

Primer 2=

5'3'5'

Anneal 2 primers 40°C - 65°C

3'5'5'

5'5'

3'5'5'

5'

5'3'

5'

5'5'

5'3'

5' 3'

5' 3'

5'3'

5'3'

5'3'

5'

5' 3'

Cyc

le 1

Cyc

le 2

Cyc

le 3

Target Sequence

5'3'

5' 3'

5' 3'



336EDVO-Kit #



The Exp

erimen

t

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



10



EDVO-Kit #Th

e Ex

per

imen

t

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.



336EDVO-Kit #



The Exp

erimen

t

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.



12



EDVO-Kit #Th

e Ex

per

imen

t

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.



336EDVO-Kit #



The Exp

erimen

t

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.



14



EDVO-Kit #Th

e Ex

per

imen

t

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.



336EDVO-Kit #



The Exp

erimen

t

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.




16



EDVO-Kit #Th

e Ex

per

imen

t

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



336EDVO-Kit #



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



xxx336EDVO-Kit #




EVT 2010-04-19

19



336EDVO-Kit #

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.

Online Orderingnow available

Visit our web site for information about EDVOTEK’s complete line of “hands-on” experiments forbiotechnology and biology education.

Mon - Fri 9 am - 6 p

m E

T

(1-800-338-6835)

EDVO-TECH SERVICE

1-800-EDVOTEK

Mon - Fri9:00 am to 6:00 pm ET

FAX: (301) 340-0582Web: www.edvotek.comemail: [email protected]

Please have the following information ready:

• Experiment number and title• Kit lot number on box or tube• Literature version number (in lower right corner)• Approximate purchase date

Technical ServiceDepartment








20



EDVO-Kit #Th

e Ex

per

imen

t

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)



336EDVO-Kit #



Instru

ctor’s G

uid

e


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.



22



EDVO-Kit #In

stru

cto

r’s

Gu

ide


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.



336EDVO-Kit #



Instru

ctor’s G

uid

e

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



24



EDVO-Kit #In

stru

cto

r’s

Gu

ide

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.



336EDVO-Kit #



Instru

ctor’s G

uid

e

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


Please refer to the kit insert for the Answers to

Study Questions


EVT 2010-04-19

27



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




28


336EDVO-Kit #

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




29

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.



30


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




31

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.



32


336EDVO-Kit #

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)




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™



34


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


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


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


336 Determining QuickPlant™ Genetics Using PCR...A PCR Experimental Success Guidelines 28 B...

Documents

Transcript of 336 Determining QuickPlant™ Genetics Using PCR...A PCR Experimental Success Guidelines 28 B...