常见溶液与缓冲液配方

APPENDIX2LABORATORYSTOCKSOLUTIONSANDEQUIPMENTAPPENDIX2ACommonStockSolutions,Buffers,andMediaRECIPESThissectiondescribesthepreparationofbuffersandreagentsusedinthismanualforcellculture,manipulationoftissue,andcellbiologicalmethods.Whenpreparingsolutions,usedeionizedordistilledwaterandreagentsofthehighestavailablegrade.Sterilization—byfiltrationthrougha0.22-µmfilterorbyautoclaving—isrecommendedformostsolutionsstoredatroomtemperatureandisessentialforcellcultureapplications.Wherestorageconditionsarenotspecified,storeupto6monthsatroomtemperature.Discardanyreagentthatshowsevidenceofcontamination,precipitation,ordiscoloration.Acid,concentratedstocksolutionsSeeTableA.2A.1.Acidprecipitationsolution1MHCl0.1MsodiumpyrophosphateNucleicacidscanalsobeprecipitatedwitha10%(w/v)solutionoftrichloroaceticacid(TCA);however,thisrecipeischeaper,easiertoprepare,andjustasefficacious.Ammoniumhydroxide,concentratedstocksolutionSeeTableA.2A.1.Ammoniumacetate,10MDissolve385.4gammoniumacetatein150mlH2OAddH2Oto500mlAmmoniumsulfate,saturated76gammoniumsulfate100mlH2OHeatwithstirringtojustbelowboilingpointLetstandovernightatroomtemperatureCurrentProtocolsinCellBiology(1998)A.2A.1-A.2A.10Copyright©1998byJohnWiley&Sons,Inc.TableA.2A.1MolaritiesandSpecificGravitiesofConcentratedAcidsandBasesaAcid/baseMolecularweight%byweightMolarity(approx.)Specificgravity1Msolution(ml/liter)Aceticacid(glacial)60.0599.617.41.0557.5Ammoniumhydroxide35.02814.80.9067.6Formicacid46.03909823.625.91.2051.2242.438.5Hydrochloricacid36.463611.61.1885.9Nitricacid63.017015.71.4263.7Perchloricacid100.4660729.212.21.541.70108.882.1Phosphoricacid98.008514.71.7067.8Sulfuricacid98.079818.31.83554.5aCAUTION:Handlestrongacidsandbasescarefully.A.2A.1LaboratoryStockSolutionsandEquipmentATP,100mM1gATP(adenosinetriphosphate)12mlH2OAdjustpHto7.0with4MNaOHAdjustvolumeto16.7mlwithH2OStoreinaliquotsindefinitelyat−20°CBase,concentratedstocksolutionsSeeTableA.2A.1.BSA(bovineserumalbumin),10%(w/v)Dissolve10gBSA(e.g.,Sigma)in100mlH2O.Filtersterilizeusingalow-protein-binding0.22-µmfilter.Storeindefinitelyat4°C.Lower-concentrationstocksolutions(e.g.,1%),whichareusefulforvariousapplications,canbemadebydiluting10%stockappropriatelywithsterilewater.BSAisavailableinvariousformsthatdifferinfractionoforigin,preparation,purity,pH,andcost;themostcommonlyusedisfractionV.Usetheformthatisappropriatefortheapplication;thismayneedtobeoptimizedempirically.CaCl2,1M147gCaCl2⋅2H2OH2Oto1literCarbonatebuffer1.6gNa2CO3(15mMfinal)2.9gNaHCO3(35mMfinal)0.2gNaN3(3.1mMfinal)H2Oto1literAdjusttopH9.5CAUTION:Sodiumazideispoisonous;followappropriateprecautionsforhandling,storage,anddisposal.CMF-DPBS(calcium-andmagnesium-freeDulbecco’sphosphate-bufferedsaline)8.00gNaCl(0.137M)0.20gKCl(2.7mM)2.16gNa2HPO4⋅7H2O(8.1mM)0.20gKH2PO4(1.1mM)H2Oto1literStoreatroomtemperatureDEPC(diethylpyrocarbonate)-treatedsolutionsAdd0.2mlDEPCto100mlofthesolutiontobetreated.ShakevigorouslytodissolvetheDEPC.AutoclavethesolutiontoinactivatetheremainingDEPC.CAUTION:WearglovesanduseafumehoodwhenusingDEPC,asitisasuspectedcarcinogen.ManyinvestigatorskeepthesolutionstheyuseforRNAworkseparatetoensurethat“dirty”pipetsdonotgointothem.DonottreatsolutionscontainingTriswithDEPC,asTrisinactivatestheDEPC.DMEM(Dulbecco’smodifiedEaglemedium),supplementedDulbecco’smodifiedEaglemedium,high-glucoseformulation(seeAPPENDIX2B;e.g.,LifeTechnologies),containing:5%,10%,or20%(v/v)FBS,heatinactivated(optional;seerecipebelow)1%(v/v)nonessentialaminoacids2mML-glutaminecontinuedCurrentProtocolsinCellBiologyA.2A.2CommonStockSolutions,Buffers,andMedia100U/mlpenicillin100µg/mlstreptomycinsulfateFiltersterilizeifanythingnonsterilehasbeenaddedStoreupto1monthat4°CDMEMcontainingthissetofadditivesissometimescalled“completeDMEM.”Thepercentageofserumusedisindicatedafterthemediumname—e.g.,“DMEM/5%FBS.”Absenceofanumberindicatesnoserumisused.DMEMisalsoknownasDulbecco’sminimumessentialmedium.Ham’sF-12nutrientmixture(APPENDIX2B;availablecommercially,e.g.,fromLifeTechnolo-gies),issometimesaddedtoDMEM;theresultingmediumisknownasDMEM/F-12.Becauseofthehigherbicarbonatecontent,DMEMrequires∼10%CO2tomaintainpH7.4.Culturemediacontainingglutamineandpenicillinshouldbewarmedto37°Casfewtimesaspossiblesincecomponents,especiallyglutamine,degraderapidlyat37°C.DPBS(Dulbecco’sphosphate-bufferedsaline)8.00gNaCl(0.137M)0.20gKCl(2.7mM)0.20gKH2PO4(1.1mM)0.10gMgCl2⋅6H2O(0.5mM)2.16gNa2HPO4⋅7H2O(8.1mM)0.10ganhydrousCaCl2(0.9mM)H2Oto1literDTT(dithiothreitol),1MDissolve1.55gDTTin10mlwaterandfiltersterilize.Storeinaliquotsat−20°C.EDTA(ethylenediaminetetraaceticacid),0.5M(pH8.0)Dissolve186.1gdisodiumEDTAdihydratein700mlwater.AdjustpHto8.0with10MNaOH(∼50ml;addslowly).Addwaterto1literandfiltersterilize.Begintitratingbeforethesampleiscompletelydissolved.EDTA,eveninthedisodiumsaltform,isdifficulttodissolveatthisconcentrationunlessthepHisincreasedtobetween7and8.FBS(fetalbovineserum)Thawpurchasedfetalbovineserum(shippedondryiceandkeptfrozenuntilneeded).Store3to4weeksat4°C.IfFBSisnottobeusedwithinthistime,asepticallydividei