PHEM(500mls)2x18.14gPipes6.5gHepes3.8gEGTA0.99gMgSO₄pH7.0w/KOHPBS(5xin500mls)20.45gNaCl0.465gKCl10.142gNa₂HPO₄*7H₂O0.545gKH₂PO₄pH7.2MountingMedia20mMTrispH8.00.5%N-propylgallate90%GlycerolStoreat4oCPMBuffer*100ml1MPIPES(100mMPIPESpH=6.9)2ml1MMgSO₄,(2mMMgSO₄)2ml0.5MEGTA(1mMEGTA,)distilledwaterto900mladjustpHto6.9distilledwaterto1literstoreat4oCValap*Put50gVaseline,50glanolin,and50gparaffin(allavailablefromFisher)ina1LPyrexbeakerHeaton"low"settingonahotplate,stirringoccasionally,untilallcomponentsaremeltedandwellmixedPourintoseveralsmallscrew-capjars(~50mlcapacity)Storeatroomtemperature20xEnergyRegenerationSystem*150mMcreatinephosphate(Boehringer#127574)2mlof100mMMgATPStock(20mMATP,20mMMgSO₄)40ml0.5MEGTA(2mMEGTA)distilledwaterto10mlDisburseinto100mlaliquotsandstoreat-20oCIsolationBuffer*20ml5MNaClStock(0.1MNaCl)4mlof1MMgSO₄Stock(4mMMgSO₄)2mlof0.5MEDTA(1mMEDTA)10mlof1MHEPESStock(10mMHEPES)distilledwaterto900mladjustpHto7.0distilledwaterto1literstoreat4oCHighSaltBuffer*120mlof5MNaClStock(0.6MNaCl)4mlof1MMgSO₄Stock(4mMMgSO₄)2mlof0.5MEDTA(1mMEDTA)10mlof1MHEPESStock(10mMHEPES)distilledwaterto900mladjustpHto7.0distilledwaterto1literstoreat4oC10xColumnBuffer*500ml1MPIPESStock(250mMPIPES)40mlof0.5MEGTAStock(10mMEGTA)40mlof1MMgSO₄Stock(5mMMgSO₄)distilledwaterto1800mladjustpHto6.7distilledwaterto2litersStoreat4oCHomogenizationBuffer*300mlPMbuffer52.3mgPMSF(1mMPMSF)3gleupeptin(10mg/mlleupeptin)300mgpepstatinA(1ug/mlpepstatinA)3mgTAME(10ug/mlTAME)mixwelltodissolve,useimmediatelyPMGBuffer*80ml1MPIPESStock(80mMPIPES)2ml1MMgSO₄Stock,(2mMMgSO₄)2ml0.5MEGTAStock(1mMEGTA,)600mlGlyceroldistilledwaterto900ml,mixwelladjustpHto6.9distilledwaterto1literstoreat4oCPBS*8.18gNaCl(140mMNaCl)0.186gKCl(2.5mMKCl)0.218gKH₂PO₄(1.6mMKH₂PO₄)2.15gNa₂HPO₄(15mMNa₂HPO₄)distilledwaterto1literStoreatroomtemperature1MMgSO₄Stock*24.074gMgSO₄distilledwaterto200mlstoreatroomtemperature1MHEPES,pH=7.0Stock*119.15gHEPES(freeacid)distilledwaterto400mladdsolidNaOHafewpelletsatatimewhilemixinguntilthepHis~6.8addconcentratedNaOHdropwisetoachievepH=7.0distilledwaterto500mlsterilefilterandstoreat4oC1MPIPES,pH=6.9Stock*151.2gPIPES(freeacid)distilledwaterto400mladdsolidNaOHafewpelletsatatimewhilemixinguntilthepHis~6.7addconcentratedNaOHdropwisetoachievepH=6.9distilledwaterto500mlsterilefilterandstoreat4oC0.5MEDTAStock*16.81gEDTA(SodiumSalt)distilledwaterto90mlAdjustpHto7.0Distilledwater100mlStoreatroomtemperature0.5MEGTAStock*19.02gEGTA(SodiumSalt)distilledwaterto90mlAdjustpHto7.0Distilledwaterto100mlStoreatroomtemperature1Mdithiothreitol(DTT)*1.542gdithiothreitoldistilledwaterto10mldisburseinto500mlaliquotsandstoreat-20oC1MKClStock*74.55gKCldistilledwaterto1literstoreatroomtemperature10MNaOHStock*40gNaOHdistilledwaterto100mlstoreatroomtemperature100mMMgGTPStock*checkformulaweightofthelotofGTPyouhave,determinetheamountrequiredfor10mlofa100mMsolution.add8.5mldistilledwatertothedeterminedamountadd1mlof1MMgSO₄StockadjustpHto7.0,distilledwaterto10mldisburseinto200mlaliquots,storeat-20oC100mMMgATPStock*checkformulaweightofthelotofATPyouhave,determinetheamountrequiredfor10mlofa100mMsolution.add8.5mldistilledwatertothedeterminedamountadd1mlof1MMgSO₄StockadjustpHto7.0distilledwaterto10mldisburseinto200mlaliquots,storeat-20oC5MNaClStock*292.2gNaCldistilledwaterto1literstoreatroomtemperature3.6mMBrefeldinA*10mgBrefeldinA(availablefromEpicenterTechnologiesMADIson,WI)absoluteethanolto12mlstoreat-20oC30xNaF*378mgNaF(0.9MNaF)distilledwaterto10mlStoreat-20oC30xAlCl₃*20mg(1.5mMAlCl₃)distilledwaterto100mlStoreatroomtemperature30mMMgGTP-g-S*10mgGTP-g-Stetralithiumsalt(availablefromBoehringerMannheim#220467)18ml1MMgSO₄Stock(30mMMgSO₄)add572mldistilledwater(to592ml)Storeat-20oC150mMMgAMP-PNP*25mgAMP-PNP(availablefromBoehringerMannheim#102547)90ul1MMgSO₄Stock(150mMMgSO₄)add225mldistilledwater(to315ml)Storeat-20oC100mMSodiumorthovanadate*1.839gSodiumorthovanadatedistilledwaterto8mlinascrewcaptubeadjustpHto10ifsolutionisyellow,placeinboilingwateruntilclearrecheckpH,repeatasnecessaryadjusttofinalconcentrationbycheckingA265nm,ext.coeff.=2925M-1cm-1andaddingdistilledwaterasneededstoreat-20oC3MKI*4.98gKIadddistilledwaterto10mlstoreatroomtemperature0.5MNa₂CO₃*531mgNa₂CO₃adddistilledwaterto10mlstoreatroomtemperatureLBBrothIn1literofddH₂O,combine:10gramsBacto-tryptone5gramsBacto-yeastExtract5gramsNaClStiruntildissolved.

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