Reference:Engelke,D.R.etal.Anal.Biochem.191:396-400(1990).Pluthero,F.G.etal.NAR.21:4850-4851(1993). Day1 1)Inoculatetwo2LflasksofTB/amp(500ml)with15mlofanovernightofTaqbugs.Thesevolumesmaybescaleddown. 2)GrowtoanOD600of0.6(approxmidlog) 3)AddIPTGto0.5mM(0.119gm/litre),growo/nbutnotformorethan16hrs. Day2 N.B:Allthefollowingproceeduresshouldbecarriedoutoniceoratleast4°C. 4)Collectcellsbycentrifugation(3.5K/15mins/4°C)andresUSPendin40mlbufferA. 5)AddanequalvolumeofbufferB(45-50ml)andincubateat75°Cfor1hr,withperiodicmixing. 6)Centrifuge(8K/15mins/4°C). 7)Add1.86mgofKCl/mlofsupernatant. 8)Aliquotequalvolumesofsupernatantintoeachof2x250mlcentrifugetubes(preferablyconical)containing75mlofwashedSigmaDP-1cationexchangeresin(packedvolume).Thisshouldbewashed2xwithsterilewaterand4xwithicecoldBufferB. 9)Vortextubeswellandincubateonashakingplatform(30mins/4°C). 10)Centrifuge(approx3K/2min/4°C)topelletresinanddiscardsupernatant. 11)Washresin4xwith100-200mloficecoldbufferB,removesupernatantbyaspiration. 12)Elute3xwithonepackedbedvolumeoficecoldbufferC. 13)Add30gm(NH4)2SO4/100mlofeluatewhilestirringrapidly. N.B:Atthispointitisofgreatadvantageifyouuseconicalorroundbottomedtubes.i.e.50mltubesforthe8x50rotor.Priortothisthesamplemaybehandledin250mlcentrifugebottlesforeaseofuse. 14)Centrifugeat12-15Krpmfor10minsat4°C. 15)Resuspendpellet(weaklytranslucent)in25-35mlofbufferC. 16)Dialise2xagainst2Lofdialysisbuffer(6-18hrs/4°C). Day3 17)TitrebyassayingserialdilutionscfcomercialTaq. 18)AliquotconcentratedTaqpolymeraseandstoreat-20°C. Reagents Terrificbroth(TB);perlitre12gmtryptone24gmyeastextract4mlglycerol(autoclaved).100ml0.17MKH2PO4(2.31gm/100ml)/0.72MK2HPO4(12.54gm/100ml);Autoclavedseparately. BufferA(Require100ml)50mMTris(pH7.9)1mMEDTA50mMDextrose BufferB/100ml(Require1000ml)20mMHepes(pH7.9)2ml(1M)1mMEDTA1ml(0.1M)0.5%Tween-200.5ml0.5%NP-400.5ml0.5mMPMSF50mMKCl BufferC/500ml(Require500ml)20mMHepes(pH7.9)10ml(1M)1mMEDTA5ml(0.1M)0.5%Tween-202.5ml0.5%NP-402.5ml0.5mMPMSF200mMKCl DialysisBuffer/2L(Require2000ml)20mMHepes(pH7.9)40ml(1M)1mMEDTA4ml(0.5M)0.5mMPMSF100mMKCl50%glycerol1L1mMDTT DilutionBufferRequiredtodiluteTaq20mMHEPES(pH7.9)0.1mMEDTA100mMKCl50%glycerolHome-madeTaqPolymerasePurification
Ialsohavethebacteriacontainingtheclone.ItappearstoproducelotsofTaqandisquitestable.Theproceeduretakes4daysstartto(15000unitsofTaq)finish.TheTaqalsoappearsverystableandreliable.Imade15mlsayearagoanditstillworksfine.
