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332Mitochondrial DNA Analysis Using PCR
Storage: See Page 3 for specific storage instructions
ExPERiMENT OBjECTivE:
The objective of this experiment is for students to isolate human mitochondrial DNA and amplify two separate regions of the mitochondrial chromosome
using the polymerase chain reaction.
This experiment is designed for DNA staining with InstaStain® Ethidium Bromide.
Mitochondrial DNA Analysis Using PCR2
xxx332EDVO-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 10
Laboratory Safety 11
Module I: Isolation of DNA from Human Hair 12
Module II: Amplification of the Mitochondrial Regions 14
Module III: Separation of PCR Reactions by Agarose
Gel Electrophoresis 16
Study Questions 18
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
Mitochondrial DNA Analysis Using PCR
EVT 2010-04-12K
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FAX: (301) 340-0582 • email: [email protected]
332EDVO-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. Mitochondrial Primer mix -20°C Freezer
C. 200 base pair ladder -20°C Freezer
D. Control DNA -20°C Freezer
E. Tris buffer -20°C Freezer
F. Proteinase K Room temperature
G. Chelating Agent Room temperature
Reagents & Supplies
(Store all components below at room temperature)
• UltraSpec-Agarose™
• ElectrophoresisBuffer(50x)
• 10xGelLoadingSolution
• InstaStain®EthidiumBromide
• InstaStain®Blue
• MicrocentrifugeTubes
• PCRtubes(0.2 ml - for thermal cyclers with 0.2 ml template)
• Calibratedtransferpipets
• Waxbeads(for waterbath option or thermal cyclers without heated lid)
Experiment # 332 contains material for up to 25 human DNA typing reactions.
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
Mitochondrial DNA Analysis Using PCR4
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Please have the following information ready:
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Technical ServiceDepartment
Requirements
• Thermalcycler(EDVOTEKCat.#541highlyrecommended)
or three waterbaths*
•Horizontalgelelectrophoresisapparatus
•D.C.powersupply
• Balance
• Microcentrifuge
• Waterbath(56°C)
• UVTransilluminatororUVPhotodocumentationsystem
• UVsafetygoggles
•Automaticmicropipets(5-50µl)withtips
•Microwave,hotplateorburner
•Pipetpump
•250mlflasksorbeakers
•Hotgloves
• Disposablevinylorlatexlaboratorygloves
• Icebucketsandice
•Distilledordeionizedwater
Online Orderingnow available
Visit our web site for information about EDVOTEK’s complete line of “hands-on” experiments forbiotechnology and biology education.
*If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three waterbaths. However, a thermal cycler as-sures a significantly higher rate of success.
5Mitochondrial DNA Analysis Using PCR
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Info
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Mitochondrial DNA Analysis
Mitochondria(pluralformitochondrion)aretheenergy-producingorgan-elles of the cell. Mitochondria are generally oblong or egg-shaped. Both plant and animal cells possess mitochondria. The number of mitochondria percellvariesdependingonthecelltype,rangingfromonlyafewinskincells to thousands in skeletal muscle cells.
FattyAcid
Oxidation
CitricAcid Cycle
Fat & SugarByproducts
ATP
Energyfor cell
Electr
on Tran
spor
t Cha
in
A
TP S
ynth
ase
Outer membrane
Inner membrane
Matrix
Cristae
RibosomesDNA
Figure 1: Structure of Mitochondrion
Figure 2: Site of metabolic pathways that convert sugars and fats to energy.
Unlikeotherorganelles,mi-tochondria have two sepa-rate membranes. The outer membraneisfairlyporous,possessing a protein called porin.Theinnermembrane,however,ishighlyimperme-able to ions and is enriched inarare,negativelychargedphospholipid known as cardiolipin. The inner membrane is highly convo-luted,withinfoldingscalledcristae(Figure1)thatgreatlyincrease the total membrane surface area. The inner membrane also contains the enzymes that catalyze cel-lularrespiration,theprocesswhereby energy is produced for the cell.
The space inside the inner membrane is known as the matrix(Figure1).Withinthematrixandinnermembrane,the chemical reactions that produce energy for the cell take place. As shown in Figure2,sugarsandfattyacids,brokendowntotwocarbonunits,enteraseriesof reactions known as the citric acid or Krebs cycle. Sugars are broken down in the cytoplasm while fatty ac-ids are broken down in the mitochondria by a process knownasß(beta)oxidation.The citric acid cycle gener-ates electrons that enter the
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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
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332Mitochondrial DNA Analysis Using PCR
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Mitochondrial DNA Analysis
D-Loop
Ribosomal
RNA genes
Complex IGenes
Cytochrome B
Complex IGenes
Complex IVGenes
ATP
Synthase
PCRProducts
82789199
11688
12360
0/16569
Figure 3: Genetic map of mitochondrial DNA
I
II
IIIFigure 4: Patterns of inherited maternal mito-chondrial diseases
electrontransportchain,aclusterof protein complexes that reside in the inner membrane of the mitochondria. In the final step ofenergyproduction,protonsgenerated by the electron trans-portchainflowthroughapumpknownasATPsynthase,drivingtheproductionofATP,thepri-mary energy-containing molecule used in biological systems. This final energy-producing process is known as oxidative phosphoryla-tion.
The DNA present in the matrix is distinct from the DNA found in the cell’s nucleus. Mitochon-drialDNA(mtDNA)iscontainedinasinglecircularchromosome,showninFigure3,thathasbeencompletely sequenced. The mito-chondrial chromosome contains 16,569basepairsofDNAand37 genes. MtDNA encodes 13 polypeptides,allofwhicharesubunits of the respiratory chain complex. This is shown on the mapinFigure3,illustratingthe
7Mitochondrial DNA Analysis Using PCR
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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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
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Mitochondrial DNA Analysis
locations of genes that encode proteins in complexes I and IV of the electron transportchain.Asshown,mtDNAalsoencodesmitochondrialribosomalRNAandtheATPsynthase,inadditiontocytochromeB,anotherconstituentof the electron transport chain. MtDNA encodes only part of the electron transport chain; nuclear DNA encodes the remaining complex subunits. One peculiarity is that mitochondrial protein synthesis uses a slightly differ-ent genetic code than cytoplasmic translation. As all cells possess only one nucleusbutseveralhundredorthousandmitochondria,mtDNAispresentin great excess over nuclear DNA in most cells. This relative abundance of mtDNA is taken advantage of by forensic investigators after obtaining crime scene specimens that are degraded or otherwise insufficient for nuclear DNA PCRanalysis.TheD-loop(Figure3)hasahighdegreeofvariabilitybetweenindividualsandcanbesequencedtodemonstratevariations.MtDNAtyping,however,cannotbeusedtoconclusivelylinksuspectstocrimescenes;rather,it is used to include or exclude suspects for further scrutiny.
Duringthepasttwentyyears,anever-increasingnumberofdiseaseshavebeen shown to be due to mitochondrial dysfunction. These disorders result when mitochondrial ATP generation is insufficient to meet energy needs in a particular tissue. Because muscle and nerve cells contain large numbers ofmitochondria,theseorgansystemsaremostaffectedbymitochondrialdysfunction. Mitochondrial diseases may be due to mutations in mtDNA genes or mutations in nuclear genes that encode mitochondrial enzymes. DiseasescausedbymtDNAmutationsincludethemyopathies,diseasesthataffectvariousmusclesandencephalomyopathies,whichcausebothmuscularandneurologicalproblems.Huntington’schorea,adevastatingdiseasethatresultsindementiaandlossofmotorcontrol,iscausedbydefectsinoxida-tive phosphorylation and has been mapped to a mutation in nuclear DNA encoding a non-mitochondrial protein. Other diseases such as Alzheimer’s andParkinson’sdiseaseinvolvemitochondrialabnormalities,althoughitisunclear how these abnormalities relate to disease pathology. Mitochondria alsoappeartoplayrolesinagingandinprogrammedcelldeath,alsoknownas apoptosis. Sincemitochondriaarepresentinthecytoplasm,theyareinheritedindepen-dentlyfromthenucleus.Afemaleeggcellpossessesover10,000mitochon-dria,whileaspermcellhasveryfew.Thusduringfertilization,mitochon-drial DNA is inherited almost exclusively from the mother. Although a small amountofpaternalmtDNAispresentinthefertilizedegg,thisDNAappearsto be selectively destroyed by the newly fertilized egg. This pattern of inheri-tance of mtDNA is known as maternal inheritance. Maternal inheritance is indicatedwhenalloffspring,maleandfemale,ofthemotherareafflictedwithaspecificcondition(Figure4).Theseverityofanyparticularmito-chondrialdisorderishighlyvariable,dependingonthenumberofmutatedmitochondriainheritedfromthemother(Figure5).
ToexaminemitochondrialDNA,thepolymerasechainreaction(PCR)isusu-allyemployed.PCR,inventedin1984,hasgainedwidespreaduseandits
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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
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332Mitochondrial DNA Analysis Using PCR
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Mitochondrial DNA Analysis
inventor,KaryMullis,wasawardedaNobelPrizein1994.Theenormousutility of the PCR method is based on its ease of use and its ability to allow the amplification of small DNA fragments. The essence of the PCR method(Figure6)istheuseofanen-zyme known as Taq polymerase. This enzyme,purifiedfromabacteriumisolatedfromhotsprings,isstableatvery high temperatures.
InthefirststepofPCR,knownasde-naturation,theDNAcomplementarystrandsareseparatedat94degrees,while the polymerase remains stable. Inthesecondstep,knownasan-nealing,thesampleiscooledtoatemperature in the range of 42°C to 65°C. to allow hybridization of small (15-30nucleotide)syntheticoligo-nucleotides,knownas“primers”,tothe target to be amplified. In the thirdstep,knownasextension,thetemperature is raised to 72°C and the DNA polymerase then adds nucleo-tides to the primers to complete each new complementary strand of the target. These three steps constitute one“cycle”.Thisprocessistypicallyrepeatedfor25to50cycles,amplify-ingthetargetexponentially(Figure6).PCRisperformedinaninstrumentknownasathermalcycler,whichisprogrammed to heat the sample at the designated temperature for each step and then rapidly change temper-atures for the following step.
Mother with mildor no symptoms
number of mitochondria
increases
Matureoocyte
Fertilizationof oocyte
Child withseveredisease
Child withmild
disease
Child withno
disease
80%mutant
50%mutant
20%mutant
+ + +
mutant
normal
nucleus
Figure 5: Mitochondrial inheritance
Inthisexperiment,studentswillexaminetheirmtDNAfromhaircells.Todothis,PCRisusedtoamplifytwoseparateregionsofthemitochondrialchromosome,showninFigure3.AmplificationoftheseregionswillresultinPCRproductsof921and672basepairs.FollowingPCR,theamplifiedDNAisthen subjected to gel electrophoresis and staining for visualization.
9Mitochondrial DNA Analysis Using PCR
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Mitochondrial DNA Analysis
Figure 6: 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'
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5'3'
5'
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Cyc
le 1
Cyc
le 2
Cyc
le 3
Target Sequence
5'3'
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5' 3'
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332Mitochondrial DNA Analysis Using PCR
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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 objective of this experiment is for students to isolate human mitochon-drial DNA and amplify two separate regions of the mitochondrial chromo-some using the polymerase chain reaction.
BRiEf DESCRiPTiON Of ExPERiMENT:
Inthisexperiment,studentsexaminetheirmtDNAfromhair.Todothis,PCR is used to amplify two separate regions of the mitochondrial chromo-some.AmplificationoftheseregionswillresultinPCRproductsof921and672basepairs.FollowingPCR,theamplifiedDNAisthensubjectedtogelelectrophoresis and staining for visualization.
This experiment has three modules:
I. Isolation of DNA from hair II. PCR Amplification of the Mitochondrial region III. Separation of PCR Reactions by 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
11Mitochondrial DNA Analysis Using PCR
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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.
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 reagents or biological materials in the laboratory.
Wear gloves and safety goggles
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332Mitochondrial DNA Analysis Using PCR
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Module i: isolation of DNA from human hair
It is critical that there is a sufficient volume of cells to obtain enough DNA to yieldpositiveDNAfingerprintingresults.Tomaximizesuccess,carefullyreadand follow all experimental instructions.
WARNING!Use only screw-cap tubes when boiling for DNA isolation. Do not use snap-top tubes when boiling.
1. Isolate3-4hairscontainingasheath,abarrel-shapedstructure(oftenwhiteincolor)encirclingtheshaftnearthebaseofthehair(seefigureatright).Ifnecessary,sheaths can be cut from the remainder of the hair shaft.
Shaft
Sheath
Root
HUMANHAIR
The preferred source is hairs from the eyebrows. They are short in length and can be readily placed in the bottom of the tube.
2. Place the hairs in the bottom of a 1.5 ml screw-cap tube.
3. Obtain the lysis solution from your instructor.
This lysis solution contains 25 mM Tris-HCl pH 8.0, 10% chelating agent, 50 µg/ml proteinase K. The chelating agent removes Mg (required by DNA degrading nucleases and DNA polymerases). The small beads need to be suspended in the buffer prior to delivery to the cell suspension. Proteinase K will digest proteins found in solution.
4. Mix the lysis solution by vortexing or pipeting up and down. Before the chelatingagentsettles,quicklyremove150µlandaddittothetubecontaining the hair.
5. Make sure the hair sheaths are completely submerged in solution and
are not stuck on the sides of the tube.
6. Place the tube in a 56°C waterbath for 15 minutes.
7. Remove the tube from the waterbath and allow it to cool for 30 sec-onds.
8. Vortex the tube for 15 seconds.
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9. Checkagainthatthehairsheathsarecompletelysubmergedinsolu-tion and are not stuck on the sides of the tube.
10. Placethetubeinafloatandplaceinboilingwaterfor10minutes.
Boiling is required to obtain cell lysis. The DNA is not degraded by boiling and nucleases will not degrade the DNA because the Mg is chelated (trapped).
11. Remove the tube from water and cool on ice for 2 minutes.
Module i: isolation of DNA from human hair, continued
WARNING!Make sure you are using a screw-cap tube for boiling your sample. Do not use snap-top tubes when boiling.
12. Vortex the tube for 10 seconds.
13. Spin in a microcentrifuge for 30 seconds. Centrifuge the cell suspension carefully.
If a microcentrifuge is not available, allow the chelating agent to settle in the tube for 3 minutes.
14.Carefullyremove50µlofsupernatantandtransferittoaclean0.5mlmicrocentrifuge tube. Discard the tube with the pellet.
Transfer the DNA (supernatant) to a new microcentrifuge tube very care-fully. It is the step prior to the PCR reaction. If any chelex beads (as few as a couple) are transferred, they can easily trap the Mg cation which is required by the Taq DNA polymerase as a cofactor for catalysis.
15. Place the tube containing the supernatant on ice.
16. Proceed with steps as outlined in Module II: Amplification of the Mito-chondrial regions.
OPTiONAl STOPPiNG POiNT
The supernatant may be stored at -20°C until the experiment is continued.
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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.
Module ii: Amplification of the Mitochondrial Regions
PCR REACTiON:
1. TransferthePCRReactionpellet™totheappropriatesizedtube(e.g.0.5mlor0.2ml)foryourthermalcycler.
2. Label the tube containing the PCR reaction pellet™ with your initials.
3. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube.
4. Add the following to the pellet:
Mitochondrialprimermix 20.0µl HairDNA(supernatant) 5.0µl
5. Gently mix the PCR reaction tube and quickly spin it in a microcentrifuge to collect all the sample at the bottom of the tube. Make sure the PCR reaction pellet™ is completely dissolved.
6. Ifyourthermalcyclerisequippedwithaheatedlid,proceeddirectlytopolymerase chain reaction cycling.
Ifyourthermalcyclerdoesnothaveaheatedlid,orifyouarecyclingmanuallywiththreewaterbaths,addonewaxbeadtothetubebeforeproceeding to polymerase chain reaction cycling.
COnTrOl rEACTIOn (OpTIOnAl):
One control reaction can be prepared for the entire class by a student or the instructor.
7. TransferthePCRreactionpellet™totheappropriatesizedtube(e.g.0.5mlor0.2ml)foryourthermalcycler.
8. TopreparethePCRControlreaction,addthefollowingtothepellet:
Mitochondrialprimermix 20.0µl ControlDNA 5.0µl
9. Gentlymixthecontroltubeandquicklyspinitinamicrocentrifugetocollect all the sample at the bottom of the tube. Make sure the PCR reaction pellet™ is completely dissolved.
10. Ifyourthermalcyclerisequippedwithaheatedlid,proceeddirectlytopolymerase chain reaction cycling.
Ifyourthermalcyclerdoesnothaveaheatedlid,orifyouarecyclingmanuallywiththreewaterbaths,addonewaxbeadtothetubebeforeproceeding to polymerase chain reaction cycling.
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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.
Module ii: Amplification of the Mitochondrial Regions, continued
POlyMERASE ChAiN REACTiON CyCliNG
11. Forautomaticcycling,eachstudentshouldplacehis/herPCRtube(andtheoptionalcontrolreaction)intheprogrammedthermalcycler.Followthe same cycling schedule if you are manually cycling in three water baths.
Initial Denaturation 25 cycles @ Final Extension
94°Cfor4min. 94°Cfor1min. 72°Cfor5min. 55°C for 1 min. 72°C for 2 min.
12. Afterthecyclesarecompleted,add5µlof10xGelLoadingSolutiontothe sample and store on ice until ready for electrophoresis.
13. Proceedtoinstructionsforpreparinga1.0%agarosegel(7x14cm)andseparating the PCR products by electrophoresis.
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Module iii: Separation of PCR Reactions by 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. Place the bed on a level surface and pour the cooled agarose solution into 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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Module iii: Separation of PCR Reactions by Agarose Gel Electrophoresis
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 DNA ladder 2 Control(optional) 3 Student#1 4 Student#2 5 Student#3 6 Student#4
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.
+-Black Red
Sample wells
Reminder:
Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.
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Answer the following study questions in your laboratory notebook or on a separate worksheet.
1. What are the three energy-producing sets of chemical reactions that take place inside the mitochondrion?
2. How are mitochondria different from other organelles inside the cell?
3. Is it possible for a child to be healthy if his/her father is affected with a mitochondrial disease? From an unaffected mother? Why or why not? What might be some symptoms of such a disease?
4. IfacrimescenesampleistoodegradedfornormalDNAprofiling,areanyfurtheranalysespossible?Ifso,whatassay(s)couldbeperformed?
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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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:
I. Isolation of DNA from hair II. PCR Amplification of the Mitochondrial region III. Separation of PCR Reactions by 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(25cycles)willtakeabout90-110minutesorcanbepro-cessed overnight and held at 4°C.
3. 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)inadvanceorhavethestu-
dentspreparetheirown,allowapproximately30-40minutesforthisprocedure.Generally,20minutesofthistimeisrequiredforgelsolidi-
fication.Seesection“OptionsforPreparingAgaroseGels”whichfollows.
4. The approximate time for electrophoresis willvaryfrom1-5hours.Generally,thehigherthevoltageapplied,thefasterthesamplesmigrate.However,dependinguponthe apparatus configuration and the distance betweenthetwoelectrodes,individualelec-trophoresis units will separate DNA at differ-ent rates. Follow manufacturer's recommen-dations. 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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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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Notes to the instructor:
GEl STAiNiNG AND DESTAiNiNG AfTER ElECTROPhORESiS Afterelectrophoresis,theagarosegelsrequirestaininginordertovisualizethe separated DNA samples. This experiment features a proprietary stain called InstaStain®.
InstaStain® Ethidium Bromide (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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pre-lab preparations
MODUlE i: iSOlATiON Of DNA
1. Add100µlofTrisbuffer(E)tothetubeofProteinaseK(F)andallowthesampletohydrateforseveralminutes.Afterthesampleishydrated,pipet up and down several times to thoroughly mix the material.
2. Prepare and dispense the Lysis Solution:
• TransfertheentireamountofProteinaseKsolutiontothetubecon-tainingthechelatingagent(G).
• Addanadditional4mlofTrisbuffer(E)tothechelatingagentandinvert the tube several times to obtain a uniform suspension.
• Quicklydispense300µlinto13tubes.Capandmixthechelatingagent in between each aliquot.
• Labelthese13tubes“lysissolution”.Eachtubewillbesharedbyastudent pair.
Suggestion: Cut the end of the pipet tip to avoid clogging of the pipet with beads in the chelating agent.
Each Student should receive:
• One1.5mlscrewcapmicrotesttube • Onecalibratedtransferpipet
Reagents to be Shared by Two Students:
• 300µlLysisSolution
Warning !!
Remind students to only use screw-cap tubes when boiling DNA isolation samples. The snap-top tubes can potentially pop open and cause injury.
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332Mitochondrial DNA Analysis Using PCR
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mODulE II - AmplIFICATIOn OF mITOChOnDrIAl rEGIOnS
Each Student should receive:
• OnetubecontainingaPCRreactionpellet™ • One0.5mlmicrotesttube
Reagents to be Shared by Two Students:
• 50µl MitochondrialPrimerMix • 20µl 10xGelLoadSolution
Notes and Reminders:
• AccuratetemperaturesandcycletimesarecriticalforPCR.Apre-runforonecycle(approx.3to5 minutes) is recommended to check that the thermal cycler is properly programmed.
• Forthermalcyclersthatdonothaveatopheatingplate,itisnecessarytoplacealayerofwaxabove the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix entitled "Preparation and Handling PCR Samples with Wax ".
• ThreewaterbathscanbeusedforPCRifathermalcyclerisunavailable.Theexperimentwillrequire great care and patience. Samples will require wax layers. See appendices entitled "Poly-merase Chain Reaction Using Three Waterbaths" and "Handling samples with wax overlays".
mODulE III - SEpArATIOn OF pCr rEACTIOnS By ElECTROPhORESiS
1. Students will share gels in this experiment. Each 1.0% gel should be loaded with the 200 base pair ladder and samples from 4 or 5 students. The control PCR reaction can also be loaded in one of the wells.
2. Aliquot30µlofthe200base-pairladder(C)intomicrocentrifugetubes.Distribute one tube of ladder per gel.
pre-lab preparations
1. Thawthemitochondrialprimermix(B)andplaceitonice.Dispense50µlinto13tubes.Labelthese13tubes“MitoPrimer”.Distributeonetube for each pair of students
2. Dispense20µlof10xGelLoadingSolutionforeach student pair.
3. There is material for one control PCR reaction. You may wish to perform the control reaction in your thermal cycler before the students try their reactions.
4. The Thermal cycler should be programmed for the following cycles.
Initial Denaturation 25 cycles @ Final Extension
94°Cfor4min. 94°Cfor1min. 72°Cfor5min. 55°C for 1 min. 72°C for 2 min.
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Experiment Results and Analysis
Idealized Schematics
Photo of Gel Results
Long Gel
Short gel
Students' PCR products should show two bands with lengths of 672 and 921basepairs.
Note: Depending on the PCR conditions used,adiffuse,small-molecularweightband,knownasa"primerdimer",maybepresentbelowthe200bp 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.
1200bp
1000bp
800bp
600bp
400bp
200bp200 b
ase p
air ladd
er
Stud
ent #1
Stud
ent #2
Stud
ent #3
Stud
ent #4
Stud
ent #5
200 base
pair lad
der
Stud
ent #1
Stud
ent #2
Stud
ent #3
Stud
ent #4
Stud
ent #5
1200 bp1000 bp800 bp600 bp400 bp200 bp
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Study Questions and Answers
1. What are the three energy-producing sets of chemical reactions that take place inside the mitochondrion?
The three energy-producing reaction cascades that occur in the mito-chondrion are:
a) thecitricacid,orKrebs,cycle b) fattyacid(beta)oxidation;and c) theelectrontransportchain(oxidativephosphorylation)
2. how are mitochondria different from other organelles inside the cell?
Mitochondria have a double membrane and also have their own unique DNAchromosome,RNA,andribosomes.
3. Is it possible for a child to be healthy if his/her father is affected with a mitochondrial disease? From an unaffected mother? Why or why not? What might be some symptoms of such a disease?
Mitochondrialdiseasesareinheritedmaternally,exclusivelyfromthemother.Therefore,achildcannotinheritamitochondrialdiseasefromanafflictedfather.Itispossible,however,forachildtobestrickenwitha mitochondrial disease inherited from a healthy mother who carries a smallnumberofdiseasedmitochondria,ifthesedefectivemitochondriaare incorporated into the oocyte that becomes fertilized. Mitochondrial disordersprimarilyaffectthemuscularandnervoussystems,astheseorgans possess large numbers of mitochondria. Typical symptoms of muscle disorders include muscle wasting and exercise intolerance. Ner-voussystemdisorderscanresultinheadaches,seizures,andhearingandvisual impairment.
4. If a crime scene sample is too degraded for normal DnA profiling, are any further analyses possible? If so, what assay(s) could be performed?
If a crime scene specimen is too degraded for standard DNA finger-printing(whichisperformedonnuclearDNA)itmaystillbepossibletoanalyze the mitochondrial DNA. This further analysis is possible because most cells typically possess many copies of mitochondrial DNA but only one nuclear DNA copy.
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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
• MaterialSafetyDataSheets
Appendices
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PCR Experimental Success Guidelines
EDVOTEK experiments which involve the extraction and amplification of DNA for fingerprinting are extremely relevant,excitingandstimulatingclassroomlaboratoryactivities.Theseexperimentshavebeenperformedsuc-cessfullyinmanyclassroomsacrossthecountry,butdorequirecarefulexecutionbecauseofthesmallvolumesused.Thefollowingguidelinesoffersomeimportantsuggestions,remindersandhintsformaximizingsuccess.
DNA ExTRACTiON AND SAMPlE PREPARATiON:
1. Sufficient Cells: It is critical that there are sufficient cells to obtain enough DNA that will yield positive DNA fingerprinting results. Cell sources include human,plant,drosophilaandbacterialcells.Withoutenoughcells,therewill not be enough DNA template for the PCR reaction.
2. hair Cells:Atleastfour(4)hairfolliclesareneeded.Thepreferred source ishairfromeyebrows.Useonlyhairscontainingasheath,abarrel-shapedstructure(oftenwhiteincolor)encirclingtheshaftnearthebaseofthehair(seefigureatleft).Centrifugethehairfolliclestothebottomofthemicro-centrifuge tube to ensure direct contact with the reagents used.
3. Chelating Agent:ChelatingagentremovesMg(requiredbyDNA-degradingnucleasesandDNApolymerases).Thesmallbeadsmustbesuspendedinthebufferpriortodeliverytothecells(i.e.,mixthechelatingagentjustbeforeyoutransferittothetubecontainingthecells).
4. Boiling: The boiling step for 10 minutes is required to obtain cell lysis. Boil-ing will not degrade the DNA and nucleases will NOT degrade DNA in the absence of Mg.
5. Centrifugation: Centrifuge the cell suspension carefully after cooling. If the pelletloosens,repeatthisstep.Thesupernatantshouldbeclear,notcloudy,and the pellet should be solid at the bottom of the tube. Repeat centrifuga-tionforalongerperiodoftime,ifnecessary.
6. DNA Transfer: Transfer the DNA to a new microcentrifuge tube very care-fully.ItisthesteppriortothePCRreaction.Ifanychelatingagentbeads(asfewasoneortwo)aretransferred,theycaneasilytraptheMgrequiredbythe Taq DNA polymerase as a cofactor for catalysis. As an additional precau-tion,centrifugethesupernatantasecondtime.
Shaft
Sheath
Root
HUMANHAIR
Appendix A
Important Note
Remember: Any carry-over of chelating agent to the PCR reaction will not yield results.
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332EDVO-Kit #
PCR Experimental Success Guidelines(continued)
Appendix A
ThE PCR REACTiON
9. 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.
10. 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.
11. Oil or Wax: Forthermalcyclersthatdonothaveatopheatingplate,there-action in the tubes must be overlaid with oil or wax to prevent evaporation.
12. manual Water Bath pCr: Three water baths can be used as an alternative to athermalcyclerforPCR,butresultsaremorevariable.Samplesrequireoilor wax layers. This method requires extra care and patience.
GEl PREPARATiON AND STAiNiNG
13. 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.
14. Electrophoretic separation: The tracking dye should travel at least 6 cm from the wells for adequate separation before staining.
15. Staining:Higherconcentrationgels(>0.8%)mayrequireadditionalstainingtoobtainclear,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.
16. 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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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°Cfor1minute 50°C for 1 minute 72°C for 1 minute
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 the experiment instructions. On the final cycle the 72°C incubation can be extended to 5 minutes.
6. Afterallthecyclesarecompleted,thePCRsampleispreparedforelectro-phoresis.
Appendix B
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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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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
31
332EDVO-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.
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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
32
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
332EDVO-Kit #
ForDNAanalysis,therecom-mended electrophoresis buffer is Tris-acetate-EDTA,pH7.8.Theformula for diluting EDVOTEK (50x)concentratedbufferisonevolume of buffer concentrate to every49volumesofdistilledordeionized 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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
33
332EDVO-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 the 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. Allow the gel to completely solidify. It will become firm and cool to the touch after approxi-mately 20 minutes. Then proceed with preparing the gel for electrophoresis.
60˚CNote: 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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
34
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
332EDVO-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.Moistenthegelwith a few drops of electrophoresis buffer.
2. Wearinggloves,removetheclearplasticprotec-tivesheet,andplacetheunprintedsideoftheInstaStain® 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
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 © 2000, 2001, 2003, 2004 2005, 2007, 2008, 2010 EDVOTEK, Inc., all rights reserved EVT 2010-04-12K
35
332EDVO-Kit #
Staining and visualization of DNA
iNSTASTAiN® BlUEOnE-STEp STAInInG AnD DESTAInInG
Agarose gels can be stained and destained in one easy step with InstaStain® Blue cards. This one-step method can be completed in approximately3hours,orcanbeleftovernight.
1. Remove the 7 x 7 cm agarose gel from its bed and completely submersethegelinasmall,cleantraycontaining75mlofdis-tilledordeionizedwater,orusedelectrophoresisbuffer.Theagarose gel should be completely covered with liquid.
Examples of small trays include large weigh boats, or small plastic food containers
2. Gentlyfloata7x7cmcardofInstaStain®Bluewiththestainside(blue)facingtheliquid.
3. Let the gel soak undisturbed in the liquid for approximately 3 hours.Thegelcanbeleftintheliquidovernight(coverwithplasticwraptopreventevaporation).
4. Afterstaininganddestaining,thegelisreadyforvisualizationand photography.
Wear gloves and safety goggles
Do not stain gel(s) in the electrophoresis apparatus.
InstaStain™
InstaStain™
One Step Stain and Destain
Appendix G
Storage and Disposal of instaStain® Blue Cards and Gels
• Stainedgelsmaybestoredintherefrigeratorforseveralweeks.Placethegelinasealableplastic bag with destaining liquid.
DO NOT FREEZE AGAROSE GELS!
• UsedInstaStain®cardsanddestainedgelscanbediscardedinsolidwastedisposal.
• Destainingsolutionscanbedisposeddownthedrain.
Gels stained with InstaStain® Blue may fade with time. Re-stain the gel to visualize the DNA bands.
36
332EDVO-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
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ata
No
dat
a
No
dat
a
No
dat
a
Inso
lub
le -
co
ld
W
hit
e p
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der
, no
od
or
N.D
. = N
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No
dat
a
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.D.
N.D
.
Wat
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pra
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, car
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Poss
ible
fir
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d w
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exp
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d t
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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
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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
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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
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Mec
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G
en. d
iluti
on
ven
tila
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n
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
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e C
loth
ing
or
Equ
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Wo
rk/H
ygie
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Pra
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es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
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osi
tio
n o
r B
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du
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Yes
Sp
lash
pro
of
go
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les
Imp
ervi
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s cl
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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®
37
332EDVO-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 sp
ecif
ied
Stro
ng
oxi
diz
ing
ag
ents
Toxi
c fu
mes
of
carb
on
mo
no
xid
e, c
arb
on
dio
xid
e, n
itro
gen
oxi
des
X
In
com
pat
icle
s
Yes
Y
es
Y
es
Irri
tati
ng
to
mu
cou
s m
emb
ran
es
No
dat
a
No
ne
spec
ifie
d
No
dat
a
Skin
/Eye
s: Im
med
iate
ly f
lush
wit
h c
op
iou
s am
ou
nts
of
wat
er f
or
15 m
in. I
nh
alat
ion
: R
emo
ve t
o f
resh
air,
if n
ot
bre
atin
g g
ive
arti
fici
al r
esp
irat
ion
, if
dif
ficu
lty
bre
ath
ing
giv
e o
xyg
en
In
ges
tio
n:
Was
h o
ut
mo
uth
wit
h w
ater
. C
all p
hys
icia
n.
Wea
r su
itab
le p
rote
ctiv
e cl
oth
ing
. Sw
eep
up
an
d p
lace
in s
uit
able
co
nta
iner
fo
r la
ter
dis
po
sal.
Ob
serv
e al
l fed
eral
, sta
te, a
nd
loca
l reg
ula
tio
ns
Kee
p t
igh
tly
clo
sed
in a
co
ol,
dry
pla
ce
Avo
id c
on
tact
Yes
N
on
e
No
N
on
e
Yes
Ch
em p
roo
f g
og
gle
s
Eye
was
h
Wea
r p
rote
ctiv
e cl
oth
ing
an
d e
qu
ipm
ent
to p
reve
nt
con
tact
.
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®
Ch
elat
ing
Ag
ent
Imin
od
iace
tic
Aci
dC
AS
#142
-73-
4
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
Solu
ble
Wh
ite
flu
ffy
gra
nu
les
(hyg
rosc
op
ic),
od
orl
ess
No
dat
a
N.D
. = N
o d
ata
N.D
.
N.D
.
Dry
ch
emic
al, c
arb
on
dio
xid
e, w
ater
sp
ray
or
reg
ula
r fo
am
Wea
r N
IOSH
/MSH
A a
pp
rove
d S
CB
A a
nd
fu
ll p
rote
ctiv
e eq
uip
men
t.
No
ne
spec
ifie
d
10/1
0/06
38
332EDVO-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
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
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