质粒稳定性测试Immediatelybeforeindcution,itisadvisabletotesttheculturetodeterminethefractionofcellsthatstillcarrythetargetplasmid.Thisinvolvesplatingofcellsonfourdifferentplates.PlatecellsthatgrowontheseplateLBplateallviablecellsLBplate+antibioticcellsthatstillcarrytheplasmidLBplate+IPTG(1mM)cellsthathavelosttheplasmidormutantsthathavelosttheabilitytoexpressthetargetgeneLBplate+antibiotic+IPTG(1mM)onlymutantsthatretaintheplasmidbuthavelosttheabilitytoexpressthetargetgeneRemarksInthepresenceofIPTG,cellscarryingaproteinproductionplasmiddonotgrowbecausehavededicatedalltheirresourcestotheproductionoftherecombinantproteininsteadofcellmaintainance.InthepresenceofthepLysSvector,IPTGalsopreventscolonyformationexceptwithcertainvector(suchaspET-3andsomevectorscarryingtheT7lacpromoter).InthepresenceofpLysE,IPTGusuallydoesnotpreventcolonyformationunlessthetargetproteinistoxic.Inatypicalcultureusefulforproducingtargetprotein,almostallcellswillformcoloniesbothontheLBplateandontheLBplate+antibiotic;lessthan2%ofthecellswillformacolonyontheLBplate+IPTG;andlessthan0.01%willformacolonyontheLBplate+antibiotic+IPTG.Withunstabletargetplasmids,thefractionofcellsthathavelosttheplasmidwillbereflectedbyanincreaseincoloniesontheLBplate+IPTGandadecreaseontherLBplate+antibiotic.ProtocolImmediatelybeforeinductionwithIPTG(atOD600isapprox.0.6),takea100-mlaliquotofthecellculture.1. 56Makeaserialdilutionofthecellsuspension,includinga10and10dilution.2. Platecellsatadilutionof105ontheLBplate+IPTGandontheLBplate+IPTG+antibiotic.Platecellsatadilutionof106ontheLBplateandontheLBplate+antibiotic.3. Incubatetheplatesovernighta37C.Countthenumberofcoloniesoneachplate.Reference:pETSystemManual,8thed.,1999(Novagen).