1.Transfercellstoa15mltube.2.Add5mlTBS-toflasks,shake&pourintotubes.3.Spindownthecells@1KRPMfor5".4.ResUSPendin1mlTBS-.5.Transferto1.5mltubes.6.Spindown@14KRPMfor2-3",removesupernatant.7.Resuspendin100µlbufferI(250mMTrispH7.8/5mMEDTA).8.Freeze-thaw3x(10"freeze-10"thaw).9.Spindowndebris@14KRPMfor5".10.Transfer100µlsupernatanttoanewsetoftubes.11.Setnewsetoftubesandadd10µlofsupernatantand50µlbufferIineach.12.Add20µlof5mMchloramphenicolinbufferI(or20µlofbufferIforablank).13.Incubate5min@37C14.Makethefollowingmix:0.75µlof4mMacetylCoA2.0µl750µMHCl(10X)16.25µlH2O1.0µl3Hacetyl-CoA(Timesnumberofsamples)15.Add20µlofmixintoeachtube(reaction&blank).16.Incubatefor2hrs@37C.17.Add500µloftoluenetoeachtube.18.Vortexfor10sec.19.Spindownfor1min.20.Remove400µlintoscintillationtubes.21.Add10mlofscintillationliquid.22.Countitfor2min.
(totalcpm"sinreactionis210000-230000)
