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1、质粒稳定性测试Immediatelybeforeindcution,itisadvisabletotesttheculturetodeterminethefractionofcellsthatstillcarrythetargetplasmid.Thisinvolvesplatingofcellsonfourdifferentplates.PlatecellsthatgrowontheseplateLBplateallviablecellsLBplate+antibioticcellsthatstillcarrytheplasmidLBplate+IPTG(1mM)cellsthathavel
2、osttheplasmidormutantsthathavelosttheabilitytoexpressthetargetgeneLBplate+antibiotic+IPTG(1mM)onlymutantsthatretaintheplasmidbuthavelosttheabilitytoexpressthetargetgeneRemarksInthepresenceofIPTG,cellscarryingaproteinproductionplasmiddonotgrowbecausehavededicatedalltheirresourcestotheproductionofther
3、ecombinantproteininsteadofcellmaintainance.InthepresenceofthepLysSvector,IPTGalsopreventscolonyformationexceptwithcertainvector(suchaspET-3andsomevectorscarryingtheT7lacpromoter).InthepresenceofpLysE,IPTGusuallydoesnotpreventcolonyformationunlessthetargetproteinistoxic.Inatypicalcultureusefulforprod
4、ucingtargetprotein,almostallcellswillformcoloniesbothontheLBplateandontheLBplate+antibiotic;lessthan2%ofthecellswillformacolonyontheLBplate+IPTG;andlessthan0.01%willformacolonyontheLBplate+antibiotic+IPTG.Withunstabletargetplasmids,thefractionofcellsthathavelosttheplasmidwillbereflectedbyanincreasei
5、ncoloniesontheLBplate+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).
