Thefollowingisalistofthesolutionsandchemicalsrequiredthroughoutthelaboratorymanual.Itisorganizedalphabetically,andindividualexerciseslistthematerialsneededforthatexercise.Formanysolutions,directionsaregivenforamolarsolutionandtheuserislefttodilutetotheappropriateconcentrationfortheirneeds.Therearemanyvendorsofthechemicalslisted,andmanyofthesolutionscanalsobepurchasedpre-mixed.

Aceticacid(MW60.05)

Glacialaceticacidis99.6%(w/v)aceticacid,andis17.4M.

1M	Add57.5mlofglacialaceticacidto800mlofwaterandthenmaketo1literwithwater.
0.05N	Add2.87mlofglacialaceticacidto800mlofwaterandthenmaketo1literwithwater.
0.9M	Add54mlofglacialaceticacidto800mlofwaterandthenmaketo1literwithwater.
7%(w/v)	Add70mlofglacialaceticacidto800mlofwaterandthenmaketo1literwithwater.
45%(w/v)	Add450mlofglacialaceticacidto500mlofwaterandthenmaketo1literwithwater.

AceticAcid/Butanol/Water(15:60:25)

Combine150mlofglacialaceticacid,600mlofnbutanoland250mlofwater.

Aceto-orcein

Add2.0gramsoforceinto45mlofglacialaceticacid.Bringtoaboilandcontinuetoheatuntilcompletelydissolved.Coolandadd55mlofdistilledwater.Filterpriortouse.Note:Someearlyinvestigatorsaddedaofanironsalt(suchasferriccitrate)asamordant.Ittendstoincreasetheintensityoftheaceto-orceinstain.Thesamereactioncanbehadbychoppingplantmaterialwithanoldersteel(notstainless)razorblade.

Acidalcohol

Add1.0mlofconcentratedHClto100mlof70%(v/v)ethylalcohol.

AcidOrcinolReagent(0.1%FeClin10%HCl)

Add0.1gramsofFeClto50mlof10%(v/v)HClandmaketo100mlwith10%HCl.

AcrylamideSolutions

AcrylamidesolutionsforPAGEaregivenastotalconcentrationofacrylamide(acrylamide+bisacrylamide)andtheamountofcrosslinker(bisacrylamide).ThisislistedastheT:Cratio.Forexample,a10%gel(10%T:5%C)wouldcontainatotalof10gramsofacrylamideper100ml,andwouldbecomposedof5gramsofacrylamideand5gramsofbisacrylamide.Usually,astocksolutionof30%acrylamideisproducedcontaining0.8%bis-acrylamide.Manyinvestigatorsuse30gramsofacrylamideplus0.8gramsofbis-acrylamideper100mlofwater,but29.2gramsofacrylamideplus0.8gramsofbiswouldbetechnicallycorrect.Inpractice,itmakeslittledifferencesincethegelsaredilutedto10%orless.The30%stocksolutionisfilteredthrougha0.45µfilterandstoredat4°Cinthedark.Foruse,thestocksolutionisdilutedwithanappropriatebuffer(usuallya2XTris-HCl).Thestocksolutionisstableforaboutonemonth.Discardafterthisperiod.

Acrylamidesintheirmonomericformareneurotoxic.Polymerizeallacrylamidesolutionspriortodisposal.

SDS,-mercaptoethanolandatrackerdye(bromophenolblue)areaddedatvariouspoints.RefertoChapter4formorecompletedetails.

Alcianblue(MW1300)

0.001M

Dissolve0.13gramsofAlcianBlue8GX(Sigma#A-2899)in100mlofwater.

AlcoholOrcinolReagent(10%orcinolin95%ethanol)

Dissolve1.0gramoforcinolin95%ethanoltoafinalvolumeof10ml.

AlkalineDistilledWater

AddonepelletofNaOHto1literofdistilledwater.

AlkalineSolutionforG-banding

Dissolve2.8gramsofNaOHand6.2gramsofNaCltoafinalvolumeof1literwithwater.

-AminosalicicAcid(PASMW175.1)

6%(w/v)

Dissolve6.0gramsofPAStoafinalvolumeof100mlwithwaterorbuffer.

Ammoniumacetate(MW77.08)

0.1M	Add7.708gramsofAmmoniumacetatetoafinalvolumeof1literofwater.

Ammoniumpersulfate(MW228.2)

10%(w/v)

Dissolve1.0gramsofammoniumpersulfatetoafinalvolumeof10mlwithwater.Mixfresh,priortouseasacatalystforPAGE.Normally,about50µlofammoniumpersulfateisaddedtoeach15ofgelsolutionforpolymerization.Dissolve0.13gramsofAlcianBlue8GX(Sigma#A-2899)in100mlofwater.

Ammoniumsulfate(MW132.14)

2%(w/v)	Add2gramsofammoniumsulfatetoafinalvolumeof100mlwater.
4.1M(sat.)0.001M	Dissolve542gramsofammoniumsulfatetoafinalvolumeof1liter.

n-Amylalcohol(PentanolCHOHMW88.15)

Density=0.8144grams/ml

0.38M

Theamylalcoholcanbeweighed(33.5grams)ormeasuredvolumetricallybyusingthedensity.Thatis,33.5grams0.8144grams/mlor41.1mlofn-amylalcohol.Weighormeasuretheappropriateamountanddilutetoafinalvolumeof1literwithwater.

Amylase,bufferedpH7.0

1%(w/v)

Dissolve0.5gramsofamylasetoafinalvolumeof50mlwith0.01Msodiumphosphatebuffer,pH7.0.

Ascorbicacid(MW176.12)

2mM	Dissolve35.2mgofascorbicacidtoafinalvolumeof100mlwithwater.

ATP(Adenosinetriphosphate,MW507.21)

5mM	Dissolve254mgofATPtoafinalvolumeof100mlwithwaterorbuffer.Dissolve35.2mgofascorbicacidtoafinalvolumeof100mlwithwater.

Baker"sFormalin

Add1.0gramofcalciumchloride,1.0gramofcadmiumchlorideand10mlofconcentratedformalinto75mlofwater.Maketoafinalvolumeof100mlwithwater.

Benzoicacid(MW122.12)

8mM	Dissolve98mgofbenzoicacidtoafinalvolumeof100mlwithwaterorbuffer.

Bis-acrylamide(N,N"-Methylene-bis-acrylamide)

Crosslinkerforacrylamidegels.RefertoAcrylamidesolutionsorChapterFourformoredetails.

BiuretReagent

Add1.50gramsofCuSO·HOand6.0gramsofsodiumpotassiumtartrateto500mlofwater.Separatelymake300mlof10%(w/v)NaOHbydissolving300gramsofNaOHtoafinalvolumeof300mlwithwater.Combinethetwosolutionsina1litervolumetric,swirltomixandmakeupto1literwithwater.Storethefinalsolutioninadark,plasticbottle.Discardifblackorredprecipitateforms.

BovineSerumAlbumin(BSA)

TherearemanygradesofBSAavailableandcareshouldbetakenwhenusingthisprotein.Forroutineproteinconcentrationstandards,a96-99%purefraction(Sigma#A2153)maybeused.Fortissueculture,RIA,ormolecularweightstandardization,BSAshouldbeoptainedwhichisextractedandpurifiedspecificallyforthatpurpose.

1%(w/v)

Dissolve0.5gramsofBSAtoafinalvolumeof50mlinwaterorbuffer.

BradfordProteinAssay

ThisprocedureusesanabsorbanceshiftinanacidicCoomasieBluesolution.ItiscommerciallyavailablefromPierceChemicalCompany,Rockford,IllinoisasProteinAssayReagent,Cat.#23200.Itcontainsmethanolandsolubilizingagentsandisveryreliable.Ifyouwishtomakeyourown,dissolve100mgofCoomasieBrilliantBlueG-250in50mlof95%ethanol.Add100mlof85%phosphoricacid,andbringtoafinalvolumeof1literwithdistilledwater.

Phosphoricacidisextremelycorrosive.Handlewithcare.

Bromophenolblue(Sodiumsalt,MW692.0)

0.001(w/v)

Dissolve1mgofBromophenolblue,sodiumsalt(Sigma#B7021)toafinalvolumeof100mlwitheitherwaterorbuffer.

n-Butanol(CHOHMW74.12)

Density=0.8098grams/ml

1.1M	Thebutanolcanbeweighed(81.5grams)ormeasuredvolumetricallybyusingthedensity.Thatis,81.5grams0.8098grams/mlor100.7mlofn-butanol.Weighormeasuretheappropriateamountanddilutetoafinalvolumeof1literwithwater.

C.elegansRingers

Thisisabasicsalinesolutionfornematodes.Dissolve11.36gramsofNaHPO·7HO,3.0gramsofKHPO,0.5gramsofNaCland1.0gramofNHCltoafinalvolumeof1liter.AdjustthepHto7.0.Maybeautoclavedforsterilization.

Calciumchloride(MW110.99)

0.0033M

Dissolve0.522gramsofcalciumacetatetoafinalvolumeof1literwithwaterorbuffer.

Calciumchloride(MW110.99)

0.001M	Dissolve0.111gramsofanhydrouscalciumchloridetoafinalvolumeof1literwithwaterorbuffer.
0.08M	Dissolve8.879gramsofanhydrouscalciumchloridetoafinalvolumeof1literwithwaterorbuffer.
2%(w/v)	Dissolve2gramsofanhydrouscalciumchloridetoafinalvolumeof100mlwithwaterorbuffer.

cAMP(Adenosinemonophosphate,cyclicMW329.22)

0.001M(1mM)

Dissolve33mgofcAMPtoafinalvolumeof100mlwithwater,bufferormedia.

Carnoyfixative

Combine10.0mlofglacialaceticacidwith60.0mlofabsoluteethylalcoholand30.0mlofchloroform.

Chloroplasthomogenizationbuffer

To400mlofdistilledwater,add30.058gramsofsorbitol,2.23gramsofsodiumpyrophosphate,0.407gramsofmagnesiumchloride,and0.176gramsofascorbicacid.AdjustthepHto6.5withHClanddilutetoafinalvolumeof500ml.

ChloroplastsUSPensionbuffer

To400mlofdistilledwater,add30.058gramsofsorbitol,0.372gramsofEDTA,0.102gramsofmagnesiumchlorideand5.958gramsofHEPESbuffer.AdjustthepHto7.6withNaOHanddilutetoafinalvolumeof500ml.

Chromalumgelatin(Subbingsolution)

Dissolve5.0gramofgelatinin1literofboilingwater.Coolandadd0.5gramsofpotassiumchromealum(CrK(SO)·12HO).Storeinrefrigerator.Touse,dipcleanslidesintothesolutionanddryinverticalpositioninadustfreelocation.

Citricacid(HCHO·HOMW210.14)

0.1M	Dissolve21.01gramsofcitricacidtoafinalvolumeof1liter.

Citratebuffer(Sodiumphosphate-Citratebuffer)

0.001M
PH4.8	Add493mlof0.2MNaHPOto507mlof0.1Mcitricacid.
PH3.6	Add322mlof0.2MNaHPOto678mlof0.1Mcitricacid.
PH4.2	Add414mlof0.2MNaHPOto586mlof0.1Mcitricacid.
PH5.4	Add557.5mlof0.2MNaHPOto442.6mlof0.1Mcitricacid.
PH6.0	Add631.5mlof0.2MNaHPOto368.5mlof0.1Mcitricacid.
PH6.6	Add727.5mlof0.2MNaHPOto272.5mlof0.1Mcitricacid.
PH7.2	Add869.5mlof0.2MNaHPOto130.5mlof0.1Mcitricacid.
PH7.8	Add957.5mlof0.2MNaHPOto42.5mlof0.1Mcitricacid.

CobaltousNitrate(MW182.96)

2%(w/v)

Dissolve2.0gramsofcobaltousnitrate(hexahydrateisverysoluble)toafinalvolumeof100mlwithwater.Keepwellclosedinacoolplace.

Colcemid

10µg/ml

Dissolve10gofcolcemidpermlofsalineorculturemedium.

Coomasieblue(CoomasieBrilliantBlueR250)

0.25%(w/v)0.001M

Dissolve2.50gramsofCoomasieBrilliantBlueR250toafinalvolumeof1literwith20%(w/v)trichloroaceticacid(TCA).Someinvestigatorsusea0.25%solutionofCoomasieBlueinmethanol-water-glacialaceticacid(5-5-1).

Coppersulfate(CuSO5HOMW249.68)

0.5%(w/v)

Dissolve0.13gramsofAlcianBlue8GX(Sigma#A-2899)in100mlofwater.

0.5%(w/v)

Dissolve0.5gramsofcoppersulfatetoafinalvolumeof100mlwithwater.

Coppertartrate/carbonate(CTC)

Dissolve0.5gramsofcoppersulfateand1.0gramofpotassiumsodiumtartratetoafinalvolumeof100mlwithwater.Combine1.0mlofthissolutionwith50mlof2%NaCOin0.1NNaOH.Mustbemadefresh,priortouse.Stocksolutionsarestable.

CrystalViolet

Dissolve0.1gramsofcrystalvioletand0.25mlofglacialaceticacidtoafinalvolumeof100mlwithwater.

DCMU(3-(3,4-Dichlorophenyl)-1,1-DimethylureaMW233.1)

1x10M0.001M	Dissolve2.3mgDCMUtoafinalvolumeof100mlwithwaterorbuffer.
5x10M0.001M	Dilutethe1x10-Msolution1/200priortouse.

Dichlorophenolindophenol(DCPIPMW290.1)

0.0025M	Dissolve73mgofDCPIPtoafinalvolumeof100mlwithwaterorbuffer.
0.0001M	Dissolve2.9mgofDCPIPtoafinalvolumeof100mlwithwaterorbuffer.

Dinitrophenol(DNPMW184.11)

18.4mg%

Dissolve18.4mgof2,4-dinitrophenoltoafinalvolumeof100mlwithwaterorbuffer.

Dischediphenylaminereagent

Dissolve500mgofdiphenylaminein49mlofglacialaceticacid.Add1.0mlofconcentratedHCl.

Dithiothreitol(Cleland"sReagentMW154.3)

0.01M

Dissolve154mgofdithiothreitoltoafinalvolumeof100mlwithwaterorbuffer.DithiothreitolisavailablefromSigmaChemicalCo.,St.Louis,Cat#D0632.Dithioerythritolmaybesubstituted.

DOPA(3-(3,4-Dihydroxyphenyl)-L-alanineMW197.19)

8mM	Dissolve158mgofL-DOPAtoafinalvolumeof100mlwithwaterorbuffer.NotethatthemaximumsolubilityofDOPAinwateris165mg/100ml(8.3mM).

EDTA(EthylenediaminetetraaceticacidMW292.24)

1M

Dissolve292.24gramsofEDTA,freeacidtoafinalvolumeof1liter.IfthemoresolubledisodiumsaltofEDTAisused,adjusttheweightaccordingly.ThepHcanbeadjustedwithaceticacidorNaOH.Forcorrespondingconcentrationdilutions,multiplytheweightingramsbythedesiredmolarity.Forexample,for10mMEDTA,multiply292.24X0.010toobtain2.92gramsofEDTAperliter.

EGTA(EthyleneGlycol-bis(aminoethylEther)N,N,N",N"-TetraaceticAcidMW380.4)

1mM	issolve380mgofEGTAtoafinalvolumeof1literwithwaterorbuffer.

Eosin

0.5%(w/v)

Dissolve0.5gramsofEosinYin100mlofwater.

Ethanol(CHOHMW46.07)

Density=0.7893gm/ml

50-95%(v/v)

Since95%ethylalcoholislessexpensiveandeasiertostorethanabsolute,thesedilutionsshouldbemadewith95%ethylalcohol.Unlessotherwisestated,denaturedalcoholworksaswellasthemoreexpensivenon-denatured.Asimplewaytomakethe%solutionistousetheappropriateamountof95%ethanolanddiluteto950mlinsteadof1liter.Forexample,tomakea50%(v/v)solution,measureout500mlof95%ethylalcoholanddilutetoafinalvolumeof950mlwithwater.Fora70%solution,measure700mlofethylalcoholanddiluteto950mlwithwater.Absoluteethanolshouldbeuseddirectlyas100%ethanol.Itisimportantforhistologythatthisbetruly100%.Sinceitishydroscopic(itabsorbswaterfromtheair),donotassumeitisabsoluteunlessitissealedortreatedtoensurenowater.Totest,addadroptoasampleofxylol.Ifanycloudinessoccurs,thealcoholisnotabsolute.

8.5M

Theethanolcanbeweighed(391.6gramsofabsolute,412.2gramsof95%(v/v)ormeasuredvolumetricallybyusingthedensity.Thatis,391.6grams0.7893grams/mlor496.1mlofabsoluteethanol.Using95%,412.2grams0.7893grams/mlor522.2ml.Weighormeasuretheappropriateamountanddilutetoafinalvolumeof1literwithwater.

Ethanol-aceticacidfixativeforhistology(3:1)

To75mlofabsolutealcohol,add25mlofglacialaceticacid.Mustbemadefresh,justpriortouse.

FetalCalfSerum(FCS)

WhileitispossIBLetoprepareyourownserumfromwholeblood,itiseasier(andsafer)topurchaseFCSfromareputablesupplier.Commercialsourcesarefreeofmycoplasma,pre-sterilizedandcontrolledforthepresenceofantibodies.Thereareanumberofserumsubstitutesavailableonthemarketandthesemaybelessexpensivewhenstorageisconsidered.SuppliersincludeGibco,FlowLaboratories,KCBIOLOGicalandSigmaChemicalCo.

Folin-CiocalteuReagent

Thisisusuallypurchasedpremixed,sinceitisdifficulttomake.Alsoknownas2NFolin-phenolreagent.

Giemsastain

Prepareastocksolutionbydissolving3.8gramsofgiemsapowderin25mlofglycerin.Heatgentlywithstirringforabout2hoursat60°C.Coolandadd75mlofmethanol(neutral,acetonefree).Foraworkingsolution,dilutethestocksolution1/10withwaterbeforeuse.Forchromosomebanding,combine5.0mlofstockGiemsa,3.0mlofabsolutemethanol,3.0mlof0.1Mcitricacidand89mlofdistilledwater.AdjustthepHofthesolutionto6.6withNaHPO.

Glucose(MW180.16)

10%(v/v)

Dissolve10gramsofD-glucose(dextrose)inafinalvolumeof100mlwithwaterorbuffer.

Glutaraldehyde(GTA)

5%	GTAisusuallysuppliedasa25%or50%(w/v)solution.Itisusedforelectronmicroscopefixationasa5%solutioninabuffer.Forroutineuse,add20mlof25%GTAto80mlof0.2Msodiumcacodylatebuffer,pH7.4.

Glycerol(MW92.09)

10%(v/v)	To10mlofglycerol(glycerine)addenoughwatertomakeafinalvolumeof100ml.
8M	Weigh73.67gramsofglycerolandaddtoafinalvolumeof100ml.Alternatively,measure499.1mlofglycerolandmaketoafinalvolumeof1liter(thedensityofglycerolatroomtemperatureis1.476)withwaterorbuffer.For8MglycerolinMTbuffer,makea2XMTbufferforuseasthediluent.

Glycine(MW75.07)

0.192M

Dissolve1.44gramsofGlycinetoafinalvolumeof100mlwithwaterorbuffer.

Gram"siodine

Dissolve0.33gramsofiodineand0.67gramsofpotassiumiodidetoafinalvolumeof100mlwithwater.

HEPES(N-[2-Hydroxyethyl]piperazine-N"-[2-ethanesulfonicacid]MW238.3)

50mM	Dissolve11.92gramsofHEPES,freeacidtoafinalvolumeof1liter.Ifhemisodiumsaltisused,adjustweightaccordingly.Donotusesodiumsaltsunlessspecified.Hemisodiumsaltcontains0.5molesofsodiumforeachmoleofHEPES.
10mMpH7.6	Dissolve2.38gramsofHEPES,freeacidin900mlofwater.AdjustthepHwithNaOHorHClto7.6.Adjustthefinalvolumeto1literwithdistilledwater.

HydrochloricAcid(HClMW36.46)

ConcentratedHClhasamolarityofapproximately11.6.HClisagas,whichissolubleinwaterandwhichcomesintheformofconcentratedreagentgradeHCl.Thissolutionisapproximately36-38%(w/v)HCl.Tomakea1Nsolution,add86mlofconcentratedHClto800mlofwateranddilutetoafinalvolumeof1liter.For0.1N,dilutethe1Nbyafactorof10.For%solutions,notethatliquidHClisonly38%HCl,thusa1%solutionwouldrequire2.6mlofconcentratedHCl(1/.38)perfinalvolumeof100ml.

JanusGreenB

0.01%(w/v)

Dissolve10mgofJanusGreenBin2-3mlofabsoluteethanol.Dilutetoafinalvolumeof100mlwithwater.

KnudsonMedia

KnudsonX4Stock:

Ca(NO)·4HO	4.0grams
(NH)SO	2.0grams
MgSO·7HO	1.0gram
DistilledHO	1.0liter

B5MinorElements:

HSO	0.5ml
MnCl·4HO	2.5grams
HBO	2.0grams
ZnSO·7HO	50mg
CoCl·6HO	30mg
CuCl·2HO	15mg
NaMoO·2HO	25mg
DistilledHO	1.0liter

FerricCitrate:

FeCHO·5HO	2.5grams
DistilledHO	100ml

StockPhosphate:

KHPO	25grams
DistilledHO	100ml

1XMedia	Add250mlofKnudsonX4to750mlofdistilledwater.Add0.5mlofB5MinorElements,0.5mlofStockPhosphate,and0.4mlofFerriccitrate.AdjustthepHto5.5withHCl,add15gramsofagarandheattodissolve.Autoclaveandpourintoplates.<br>
Note:	2.50gramsofsucrosemaybeaddedpriortoadjustmentofthepH,ifdesired.Itisnotnecessaryforgerminationofspores,butaddsanorganicsourceformutantsandabnormalferngrowths.Italsoincreasestheneedforsubsequentaseptictechnique.

KrebsPhosphateRingers(KPR)

Prepareeachofthefollowingseparately:

0.90%(w/v)NaCl1.15%(w/v)KCl1.22%(w/v)CaCl3.82%(w/v)MgSO·HO0.1Mphosphatebuffer,pH7.4(17.8gramsofNaHPO·HO+20mlof1NHCl,dilutedto1liter)

Tomix,combine200mlofNaCl,8mlofKCl,6mlofCaCland2mlofMgSO.Carefully,andwithconstantstirring,add40mlofphosphatebuffer.

LPSbuffer(LowerPadSolutionbuffer)

Dissolve1.5gramsofKCl,0.5gramsofMgCl,and0.5gramsofsteptomycinsulfatein500mlofwater.Add40mlof1Mphosphatebuffer,pH6.5anddiluteto1literwithwater.

Magnesiumchloride(MgClMW95.23)

1mM	Dissolve95.2mgofmagnesiumchlorideperfinalvolumeof1liter.
4mM	Dissolve0.381gramsofmagnesiumchlorideperfinalvolumeof1liter.
10mM	Dissolve0.952gramsofmagnesiumchlorideperfinalvolumeof1liter.
0.1M	Dissolve9.523gramsofmagnesiumchlorideperfinalvolumeof1liter.
Note:	Asinglestocksolutionof1MMgClcanbemixedbydissolving95.23gramsofmagnesiumchloridetoafinalconcentrationof1literwithwater,andalldilutionsmadeappropriatelyfromthisstocksolution.

Magnesiumsulfate(MgSOMW120.39)

5%(w/v)

Dissolve5.0gramsofmagnesiumsulfatetoafinalvolumeof100mlwithwaterorbuffer.

Mayer"shematoxylin

Purchasecommerciallyormixwitheitherofthefollowingprocedures:

A.Dissolve1.0gramofhematoxylinin10mlofabsoluteethanol.Dissolve20gramsofpotassiumalum(KAl(SO)·12HO)in200mlofwater.Inachemicalhood,withprotectionagainstexplosion,bringthepotassiumalumsolutiontoaboilandaddhematoxylin/ethanolmixture.Continuetoboilforapproximately1minute.Add0.5gramsofmercuricoxideandcoolrapidly.Add0.5mlofglacialaceticacid.Filterbeforeuse.Thismixtureisstableforabouttwomonths.

B.Alternatively:Dissolve5.0gramsofhematoxylinin50mlofabsoluteethanolandaddto650mlofwarmwater.Heatgentlyuntilthehematoxylindissolvesandthenadd300mlofglycerin,0.3gramsofsodiumiodateand20ml.ofglacialaceticacid.Coolandmakevolumeupto1literwithdistilledwater.Filterbeforeuse.

<>>-Mercaptoethanol(MW78.13)

0.5M	Density=1.2grams/ml.Useeither3.91gramsOR3.26mlofmercaptoethanolinafinalvolumeof100mlofwaterorbuffer.
5%(w/v)	Use5.0gramsor4.167mlinafinalvolumeof100mlofwaterorbuffer.

MES(2-(N-Morphilino)ethanesulfonicacidMW195.2)

0.1M	Dissolve1.952gramsofMEStoafinalvolumeof100mlwithwaterorbuffer.

Methanol(CHOHMW32.04)

Density=0.7914grams/ml

22M	Themethanolcanbeweighed(704.9grams)ormeasuredvolumetricallybyusingthedensity.Thatis,704.9grams0.7914grams/mlor890.7mlofmethylalcohol.Weighormeasuretheappropriateamountanddilutetoafinalvolumeof1literwithwater.

Methanol/AceticAcid(forfixingproteinsinacrylamidegels)

45%:12%

Add120mlofglacialaceticacidto450mlofmethanolanddilutetoafinalvolumeof1literwithwater.

Methanol/AceticAcid(fordestainingorfixingproteinsinacrylamidegels)

5%:7%

Add70mlofglacialaceticacidto50mlofmethanolanddilutetoafinalvolumeof1literwithwater.

Methylgreen

0.2%(w/v)

Dissolve0.2gramsofmethylgreentoafinalvolumeof100mlwith0.1Macetatebuffer,pH4.2.Acetatebuffer(0.1MpH4.2)ispreparedbydissolving.361gramsofsodiumacetate(trihydrate)inapproximately80mlofwater.Add.42mlofglacialaceticacidandadjustthevolumeto100mlwithwater.

Microtubulebuffer(MTbuffer)

Dissolve19.52gramsofMESin800mlofdistilledwater.Add0.380gramsofEGTAand47.62gramsofMgCl.AdjustthepHto6.4withHClorNaOHanddilutetoafinalvolumeof1literwithdistilledwater.

Minimumessentialmedium(MEM)

Forallpurposesofthismanual,MEMreferstoEagle"sMEM.Whileitispossibletomixthismedium,itisinfinitelyeasier(andlessexpensive)topurchasethemediapre-mixedfromanynumberofcommercialsources(Gibco,Flow,KCBiological,SigmaChemical).TheingredientsarelistedinTable12.1.Itisessentialthatchemicalsofthehighestpurityareusedthroughout.

NGagar(NematodeGrowthagar)

Dissolve3.0gramsofNaCl,2.5gramsofpeptone,and17gramsofagarinafinalvolumeof1liter.Boiltodissolvetheagar,autoclavetosterilize.

Meanwhile,prepareseparatesterilesolutionsof:

1MCaCl2mg/mluracil10mg/mlcholesterolinethanol1MPotassiumphosphatebuffer,pH6.01MMgSO

Usingpropersteriletechnique,cooltheagarsolutionslightlyandadd1mlofCaCl,1mlofuracil,0.5mlofcholesterol,25mlofphosphatebuffer,and1mlofMgSO.Swirltomixallingredientsandpourplates.

-Nitrophenylphosphate(MW263.1)

0.05M	Dissolve1.32gramsof-nitrophenylphosphatetoafinalvolumeof100mlwithwaterorbuffer.
0.8%(w/v)	Dissolve0.8gramsof-nitrophenylphosphatetoafinalvolumeof100mlofwaterorbuffer.
Note:	SigmaChemicalCo.,St.Louis,suppliesthiscompoundasSigma104,PhosphataseSubstrate,Cat.#104-0.

Osmiumtetroxide(OSOMW254.2)

1%

OsmiumtetroxideisagaswhichisusedinsolutionforEMpreservation.Itisbestpurchasedinsealedvialsof2mlof4%OsO/4/.Foruse,add6.0mlofwaterorbuffertothe2.0mlof4%osmiumtetroxide.Sealinatightlysealedcontainer,wrappedwithaluminiumfoilandkeepintherefrigerator.UseofafumehoodismandatorywhenusingOsO/4/.Osmiumtetroxidewillrapidlyfixthenasalpassagesandexposedcorneaifnotproperlyvented.Itshouldbehandledwithextremecare.

Perchloricacid(PCAMW100.47)

2%(w/v)

Dissolve2.0gramsofPCAtoafinalvolumeof100mlwithwaterorbuffer.

Percoll

ColloidalPVPcoatedsilica.AvailablefromSigmaChemicalCo.,St.Louis.Cat.#P1644.

PeriodicAcid(PeriodateUsedforPASreaction)

Dissolve0.6gramsofperiodicacidin100mlofwaterandadd0.3mlofconcentratednitricacid.

Phenazinemethosulfate(PMSMW306.34)

Mutagenandirritant.

0.033%(v/v)

Add33µlofphenazinemethosulfateto90mlofwaterorbufferandmakeupto100mlfinalvolume.Mustbemadeimmediatelypriortouse.

Phenolmixture

Combine555mlofaqueousphenol(or500gramsofphenolcrystalsplus55mlofwater)with70mlofmcresol.Add0.5gramsof8-hydroxyquinoline.

PhenolwillcausesevereburnsandreADIlydissolvesallplasticandrubbercompounds.Useextremecautionwhenhandlingthiscompound.

-Phenylenediameneoxalate(PPDOMW198.18)

0.02%(w/v)

Dissolve20mgofPPDOtoafinalvolumeof100mlwithwaterorbuffer.

Phosphatebufferedsaline(PBS)

Mix100mlCa/Mgfree10XPBSAwith800mlofdistilledwater.Separately,dissolve0.1gramofmagnesiumchlorideand0.1gramofanhydrouscalciumchloridetoafinalvolumeof100mlwithwater.Withconstantstirring,slowlyaddthemagnesium/calciumchloridesolutiontothedilutedPBSA.Ifaprecipitateforms,startover,andaddslowerwithcontinuousstirring.

Ca/MgfreePhosphatebufferedsaline-10X(10XPBSA)

Dissolve80gramsofNaCl,2.0gramsofKCl,15.0gramsofDibasicsodiumphosphateand2.0gramsofMonobasicpotassiumphosphatein1literofdistilledwater.Thismakesa10XsolutionofCa/Mgfreephosphatebufferedsaline.Dilute1:10priortouse.Storeinarefrigerator.

Phosphatebufferedsaline-Tween20(4.8)

MixPBSandadd0.1%(v/v)Tween20.

Phytohemaglutinin(PHA)

Availableaskidneybeanlectin.Itistypicallyusedasastocksolutionof10-20g/mlinbalancedsaltsolution.Fortissuecultureitmustbecoldsterilizedpriortouse.

Potassiumchloride(KClMW74.55)

1M	Dissolve74.55gramsofKCltoafinalvolumeof1literwithwaterorbuffer.Forotherconcentrations,multiplytheweightbytherequiredmolarity.Forexample,for0.150M(150mM),use0.150X74.55,or11.183gramsofKClin1literofwaterorbuffer.Usehalfasmuchtoobtain0.075Mforkaryotyping.

Potassiumcyanide(KCNMW65.11)

8mM	Dissolve52mgKCNtoafinalvolumeof100mlwithwaterorbuffer.

Potassiumphosphate,monobasic(KHPOMW136.09)

0.01M

Dissolve1.36gramsofmonobasicpotassiumphosphatetoafinalvolumeof1literwithwater.

Potassiumphosphate,dibasic(KHPOMW174)

0.01M

Dissolve1.74gramsofdibasicpotassiumphosphatetoafinalvolumeof1literwithwater.

Potassiumphosphatebuffer

0.01MpH7.4

Prepare500mlof0.01MKHPOand500mlof0.01MKHPOPlacetheK2HPOontoamagneticstirrerandinsertapHelectrode.AddtheKHPOslowlytoadjustthepHto7.4.

Potassiumhydroxide(KOHMW56.10)

0.5N	Dissolve28.05gramsofKOHtoafinalvolumeof1literwithwater.
10%(w/v)	Dissolve10gramsofKOHtoafinalvolumeof100mlwithwater.Storeinaplasticcontainer.

Potassiumsodiumtartrate(RochellesaltKNaCHO·4HOMW282.23)

n-Propanol(CHOHMW60.11)

Density=0.8035grams/ml

3M	Then-propanolcanbeweighed(180.3grams)ormeasuredvolumetricallybyusingthedensity.Thatis,180.3grams0.8035grams/mlor224.4mlofn-propanol.Weighormeasuretheappropriateamountanddilutetoafinalvolumeof1literwithwater.

ProteinBuffer

Dissolve1.46gramsofKHPOand0.92gramsofK2HPOin80mlofdistilledwater.Add2.5gramsofcrystallineserumalbuminandadjustthevolumetoafinal100mlwithwater.

PyroninY(acetone)

0.6%(w/v)

Dissolve0.6gramsofpyroninYin100mlofacetone.

Ribonuclease

0.1%(w/v)

Dissolve10mgofpancreaticribonucleasetypeAin10mlofwaterorbuffer.Useforenzymetreatmentofhistologicalsectionsbyfloating0.5-1.0mlofthissolutionontothesection,withtheslidesetintoacoveredpetriplate.

Safranin

Dissolve2.5gramsofSafraninOin10mlof95%ethanolanddiluteto100mlwithwater.

Saline(NaCl)

0.85%(w/v)

SalinereferstoasolutionofNaCl,withthemostcommonusageforthatwhichisisotonictomammalianbloodcells,notablea0.85%or0.9%solution.Tomix,dissolve8.5gramsofNaCltoafinalvolumeof1literwithwater.

Salinecitrate(1/10dilutionofSSC)

Dissolve0.878gramsofNaCland0.294gramsofsodiumcitratetoafinalvolumeof1literwithwater.

Salinecitratebuffer(SSC)

20X

Itiscommontopreparethisbufferasastock20Xsolution,tobedilutedto2X,1Xor0.1Xpriortouse.Topreparea20Xstocksolution,dissolve175gramsofNaCland88gramsofsodiumcitratein900mlofwater.AdjustthepHto7.0with1NHClandbringtoafinalvolumeof1liter.Foruse,asa1XSSC,dilute1part20Xstockwith19partsdistilledwater.Fora2XSSC,dilute1part20Xstockwith9partswater.

Schiff"sReagent

Dissolve0.8gramsofbasicfuchsinin85mlofdistilledwater.Add1.9gramsofsodiummetABIsulfiteand15.0mlof1NHCl.Placethesolutioninseparatoryfunnelandshakeat2hourintervalsforaperiodofapproximately12hours.Add200gramsofactivatedcharcoal,shakefor1minuteandfiltertheclearsolution.Ifthesolutionisstillpink,addanother100gramsofcharcoalandshakeforanadditionalminute.Filterandstoreinadarkbottle.Solutionshouldbeclear(nopinkcoloration)foruse.

Scottsolution

Dissolve2.0gramsofsodiumbicarbonateand20.0gramsofmagnesiumsulfateinwatertoafinalvolumeof1liter.Addapinchofthymoltoretardthegrowthofmolds.

SDS

RefertoSodiumlaurylsulfate.

1XSDS-ElectrophoresisRunningBuffer

Dilute5XTris-Glycinebufferto1Xandadd1.0gramofSDSperliterof1XTris-Glycine.ThepHshouldbe8.3afterdilution.

2XSDSSampleBuffer

Dissolve1.52gramsofTrisbase,2.0gramsofSDS,20mlofglycerin,2.0mlof-mercaptoethanoland1mgofbromophenolbluetoafinalvolumeof100mlwithwater.

SiliconizedPipettes

Pasteurpipettescanbesiliconizedbysoakingtheminabeakercontaining5%(v/v)dichlorodimethylsilaneinchloroformforabout1minute.Remove,drainandrinseseveraltimeswithdistilledwater.Bakethepipettesat180°Cfor2hoursandcoolbeforeuse.

Dichlorodimethylsilaneandchloroformarebothtoxicandvolatile.Useonlyinproperfumehoodandkeepallflamesawayfromworkarea.Insurethatallsiliconeandchloroformareremovedfromglasswarebeforeplacinginanoven.

Silvernitratesolution(forelectrophoresisstaining)

Dissolve0.15gramsofNaOHin150mlofwater.Add3.5mlofconcentratedNHOHandbringtoavolumeof200ml.Separately,dissolve2.0gramsofsilvernitrateinafinalvolumeof10ml.Withconstantstirring,add8.0mlofthesilvernitratetothe200mlofNaOH/NHOH.Thissolutionshouldbepreparedimmediatelypriortouse,andusedwithin30minutes.

Disposeofthissolutionwithcopiousflushing.Itbecomesexplosiveupondrying.

SMagarmedium(SlimeMoldmediumofSussman)

Dissolve10.0gramsofglucose,10.0gramsofpeptone,1.0gramofyeastextract,1.0gramofMgSO,1.5gramsofKHPO,1.0gramofK2HPOand20.0gramsofagartoafinalvolumeof1liter.Heattodissolvetheagar,autoclaveanddispensetopetriplates.

Sodiumacetate(MW82.04)

1M	Dissolve82.04gramsofsodiumacetatetoafinalvolumeof1literwithwaterorbuffer.
0.02M	Dissolve1.64gramsofsodiumacetatetoafinalvolumeof1literwithwaterorbuffer.

Sodiumacetatebuffer

1MpH5.7

To925mlof1Msodiumacetate,add75mlof1Maceticacid.

Sodiumazide(MW65.02)

0.01M	Dissolve0.065gramsofsodiumazidetoafinalvolumeof100mlwithwater.
0.39%(w/v)	Dissolve0.39gramsofsodiumazidetoafinalvolumeof100mlwithwaterorbuffer.

Sodiumbarbitol

0.2%(w/v)

Dissolve0.2gramsofsodiumbarbitoltoafinalvolumeof100mlwithwater.

Sodiumbicarbonate(NaHCOMW84.0)

0.1M	Dissolve0.84gramsofNaHCOtoafinalvolumeof100mlwithwaterorbuffer.

Sodiumcacodylatebuffer

0.2MpH7.4

Preparea0.2Msolutionofcacodylicacid,sodiumsalt(MW159.91).Dissolve3.20gramsofcacodylicacid,sodiumsalttoafinalvolumeof100mlwithwater.AdjustthepHto7.4withHCL.

Cacodylicacidcontainsarsenic.Handleproperly.

Sodiumcarbonate(MW106.0)

2%(w/v)	Dissolve2.0gramsofsodiumcarbonatetoafinalvolumeof100mlwith0.1NNaOH.
0.1NNaOH	UsedforLowryProteinassay.

Sodiumchloride(MW58.44)

M	Foramolarsolutionofsodiumchloride,dissolve58.44gramsofNaCltoafinalvolumeof1literwithwaterorbuffer.Forcorrespondingdilutions,multiplytheweightbythemolarityrequired.Forexample,for0.05M,multiply58.44by0.05or2.92grams/liter.
%	For%solutions,theyareinvariablyw/v.Fora1%(w/v)solution,dissolve1.0gramofNaCltoafinalvolumeof100mlwithwaterorbuffer.Multiplytheweightbyacorrespondingchangein%forotherconcentrations.
200,300,400mOsM	OsmolesforNaClarecalculatedastwicethemolarconcentration.Thus,a200mOsMsolutionwouldbe.100MNaCl.Likewise,300mOsMwouldbe.150Mand400mOsMwouldbe.200MNaCl.

Sodiumcitrate(MW294.10)

0.09M

Dissolve2.65gramsofsodiumcitratetoafinalconcentrationof100mlwithwater.

Sodiumcitrate/formaldehyde(forsilverstainedproteins)

Dissolve5.0gramsofsodiumcitratein800mlofwater,add5.0mlofconcentratedformalin(37%formaldehydesolution)anddiluteto1literwithwater.

Sodiumdeoxycholate(Deoxycholicacid,sodiumsaltMW392.58)

0.15%(w/v)

Dissolve150mgofdeoxycholicacid,sodiumsalttoafinalvolumeof100mlwithwater.

Sodiumdithionite(NaSO·2HOMW242.16)

.1mg/ml

Dissolve10mgofsodiumdithionitein100mlofwaterjustpriortouse.Alternatively,toreduceasolution,thedrypowdercanbeaddedasneeded.Sodiumdithioniteshouldbestoredat-20°C.

Sodiumfluoride(NaFMW42.0)

0.1M	Dissolve4.2gramsofNaFtoafinalvolumeof1literwithwater.

Sodiumlaurylsulfate(SDSorSLSMW288.38)

0.1%(w/v)	Dissolve0.1gramsofSDStoafinalvolumeof100mlwithwaterorbuffer.Mixbygentlestirring,donotshake.
10%(w/v)	Dissolve10gramsofSDStoafinalvolumeof100mlwithwater.

SDSshouldnotbeinhaledinitspowderform.Whenweighing,useamask,orbetter,ahood.

Sodiummalonate(MW104.0)

0.6M	Dissolve2.49gramsofmalonicacid,sodiumsalt,toafinalvolumeof25mlwithwaterorbuffer.

Sodiumperchlorate(NaClO·HOMW140.47)

1M	Dissolve14.01gramsofsodiumperchloratetoafinalvolumeof100mlwithwaterorbuffer.

Sodiumphosphate,monobasic(NaHPOHOMW137.99)

1M	Dissolve14.01gramsofsodiumperchloratetoafinalvolumeof100mlwithwaterorbuffer.
0.01M	Dissolve1.38gramsofmonobasicsodiumphosphatetoafinalvolumeof1liter.

Sodiumphosphate,dibasic(NaHPO·7HOMW268.07)

1M	Dissolve268.07gramsofdibasicsodiumphosphatetoafinalvolumeof1liter.
0.2M	Dissolve53.61gramsofdibasicsodiumphosphatetoafinalvolumeof1liter.
0.01M	Dissolve2.68gramsofdibasicsodiumphosphatetoafinalvolumeof1liter.

Sodiumphosphatebuffer

Thesearethemostcommonbuffersusedinbiology.TheyareproducedbyaddingequimolarsolutionsofKHPOandNaHPO.EqualvolumesofthetwowillyieldapHof7.0,whilesodiumphosphatewillincreasethepH.IncreasedvolumesofpotassiumphosphatewilldecreasethepH.ThepHcanbeadjustedfrom5.4to8.2.IfapHof7.0-8.2isdesired,startwith500mlofsodiumphosphateandaddpotassiumphosphatewhilestirringandmonitoringthepHwithapHmeteruntilthedesiredpHisreached.IfapHof5.4-7.0isdesired,startwith500mlofpotassiumphosphateandaddsodiumphosphateuntilthedesiredpHisreached.Typically,themolarityofthebufferwillrangefrom0.01to0.1M.UsetheappropriatemolarityofKHPOandNaHPO.Thatis,if0.05Mbufferisdesired,use0.5MKHPOand0.5MNaHPOasdirectedabove.

Sodiumpotassiumphosphatebuffer

RefertoSodiumphosphatebuffer.

Sodiumpyrophosphate(NaPO·10HOMW446.06)

10mM	Dissolve0.446gramsofNaPO·10HOtoafinalvolumeof100mlwithwater.

Sodiumsuccinate(MW270.16)

0.6M	Dissolve16.2gramsofsuccinicacid,sodiumsalttoafinalvolumeof100mlwithwaterorbuffer.

Sorbitol(MW182.17)

0.33M

Dissolve60.12gramsofsorbitoltoafinalvolumeof1literwithwaterorbuffer.

Sorensonphosphatebuffer

Refertosodiumphosphatebuffer.

0.2MpH7.5

Dissolve24.14gramsofNaHPOand4.08gramsofKHPOin800mlofwater.Dilutetoafinalvolumeof1liter.

Subbingsolution(slides)

Refertochromalumgelatin.

Sucrose(MW342.3)

1.0M	Dissolve34.2gramsofsucrosetoafinalvolumeof100mlwithwaterorbuffer.Forothermolarities,multiplytheweightbytherequiredmolarconcentration.Forexample,for0.25Msucrose,weight34.2x0.25or8.55gramstoafinalvolumeof100ml.
40%(w/v)	Dissolve40gramsofsucrosetoafinalvolumeof100mlwithwaterorbuffer.Dilutethissolutionforlowerpercentrequirements.Ifusingforsucrosedensitygradients,thesucroseshouldhave0.1mlofdiethylpyrocarbonateadded,thesolutionbroughttoaboilfor3-5minutesandcooledbeforeuse.ThiswilleliminateRNAase,whichwouldotherwisebeacontaminantofthesolution.Storeallsucrosesolutionsinarefrigerator

SulfuricAcid(H2SO4MW98.08)

Caution:Sulfuricacidisextremelycausticandwillcausesevereburns.Itmustalwaysbeaddedtothewater,whenmakingdilutions.Uponadditiontowateroralcohol,heatwillbegeneratedwhilethesolutionwillcontractinvolume.Useextremecareinhandlingthisacid.

ConcentratedH2SO4is17.8Mor35.6N.1.09NAdd30.6mlofconcentratedsulfuricacidslowly,withconstantstirring,andwithadequateprotectionfromsplashes,toapproximately800mlofwater.Coolandmakeupvolumeto1literwithwater.

Sulfurousacid(forFeulgenReaction)

Add1.0mlofconcentratedHCland0.4gramsofsodiumbisulfiteto100mlofdistilledwater.Thissolutionshouldbemadefreshpriortouse.Itdoesnotstorewell.

Swabbingdetergent

Fortissueculturepurposes,useanon-toxicdetergentdesignedforsurgicalscrubbing.e.g.Phisohex,Betadineorequivalent.Formostroutineswabbing,70%(v/v)ethanolissufficientandhastheadvantagethatitwillleavenoresidue.

TEMED(N,N,N",N"-tetramethylethylenediamine)

CatalystforPAGE.Usedirectlyandadd10lTEMEDper15mlofgelsolution.

Toluidineblue

0.1%(w/v)

Dissolve0.1gramsoftoluidinebluein10mlofethanolandaddwaterorcitratebuffer(pH6.8-7.2)toafinalvolumeof100ml.

Trichloroaceticacid(TCACCl3COOHMW163.4)

Extremelycausticacid.Handlewithcare.

72%(w/v)

Dissolve72gramsofTCAtoafinalvolumeof100ml.TCAishydroscopicandwillreadilyabsorbwater.Thesolidcrystalswillbecomeliquidifthestockbottleisplacedinwarmwater,withaloosecap(meltingpoint57-58°C.Itiseasiertohandleasaliquid.Storageofsolutionsgreaterthan30%(w/v)arenotrecommendedasdecompositionisrapid.Thereforethesesolutionsshouldbemadeasneeded.

Trisbuffer

TherearemanyvariationsonthebasicTris-HClbuffercombination,mostofwhicharecommerciallyavailable.SolutionswithEDTAareknownasTEbuffers,whilesolutionswithEDTAandaceticacidareknownasTAEbuffers.Theterminologyvarieswiththeauthor,withTrisbufferbeingusedtomeanTris-HClsolutions.SigmaChemicalCo.,St.Louis,carriesafulllineofthebuffersmarketedunderthetradenameofTrizma(baseandHCl).ThebasicbufferisacombinationofTris(tris(hydroxymethyl)aminomethane)andHClacid.ThesearesometimesreferredtoasTris-baseandTris-HClsolutions.TrisbuffersshouldnotbeusedbelowapHof7.2oraboveapHof9.0.Trisbuffersarealsoextremelytemperaturesenstive.Directionsaregivenforroomtemperature(25°C).ThepHwilldecreaseapproximately0.028unitsforeachdegreedecreaseintemperature.

1M

Dissolve121gramsofTrisin800mlofdistilledwater.AdjustthepHwithconcentratedHCl.Dilutetoafinalvolumeof1liter.LowerrequiredmolaritiescanbedilutedfromthisstockormixedascombinationsoflowermolaritiesofTrisandHCl.ItisimportanttomeasurethepHatthetemperatureandmolaritythatwillbeusedinthefinalanalysis.

Tri-Glycinebuffer

5X	Dissolve15.1gramsoftrisbaseand72.0gramsofglycinetoafinalvolumeof1liter.Foruse,dilute1part5Xbufferwith4partswater.

Trypanblue

0.2%(w/v)

Dissolve0.2gramsoftrypanbluetoafinalvolumeof100mlwithwater.

Trypsin

0.25%	0.25%Dissolve0.25gramsofcrudetrypsininPBSAtoafinalvolumeof100ml.Coldsterilizebyfiltration.Alternatively,purchasepre-dilutedcrudetrypsin,soldas1:250whichispre-sterilizedaswell.
Note:	Whenusingtrypsinfortissuedisaggregation,itmustbesubsequentlyinhibitedbytheuseofserumintheculturemedia,orbytheadditionofsoyabeantrypsininhibitor.

Trypticasesoybroth

Add17.0gramsoftrypticasepeptone,3.0gramsofphytonepeptone,5.0gramsofsodiumchloride,2.5gramsofdipotassiumphosphateand2.5gramsofglucoseto1literofwater.AdjustthepHto7.3,andautoclave.

Tween20or80(Polyoxyethylenesorbitanmono-oleate)

1%(v/v)

Add1.0mlofTweento90mlofwater.Mixanddilutetoafinalvolumeof100mlwithwater.NotethatTweenisextremelyviscousandcaremustbetakentoaccuratelypipette1.0ml.Wipetheoutsideofthepipettebeforedispensing.

Uranylacetate(MW424.19)

5%(w/v)

Dissolve5.0gramsofuranylacetatetoafinalvolumeof100mlin50%(v/v)ethanol.Storeinthedarkatroomtemperature.Allowatleast24hoursfortheuranylacetatetocompletelydissolve.Thissolutionwillkeepforabout3months.

Urea(MW60.06)

2.5M	Dissolve15.02gramsofureatoafinalvolumeof100mlwithwaterorbuffer.
10M	Dissolve60.06gramsofureatoafinalvolumeof100mlwithwaterorbuffer.
14M	Dissolve84.08gramsofureatoafinalvolumeof100mlwithwaterorbuffer.

Viabilitystain

RefertoTrypanblue