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质粒稳定性

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PlasmidstABIlitytestImmediatelybeforeindcution,itisadvisabletotesttheculturetodeterminethefractionofcellsthatstillcarrythetargetplasmid.Thisinvolvesplatingofcellsonfourdifferentplates.

Plate	cellsthatgrowontheseplate
LBplate	allviablecells
LBplate+antibiotic	cellsthatstillcarrytheplasmid
LBplate+IPTG(1mM)	cellsthathavelosttheplasmidormutantsthathavelosttheabilitytoexpressthetargetgene
LBplate+antibiotic+IPTG(1mM)	onlymutantsthatretaintheplasmidbuthavelosttheabilitytoexpressthetargetgene

Remarks

InthepresenceofIPTG,cellscarryingaproteinproductionplasmiddonotgrowbecausehavededicatedalltheirresourcestotheproductionoftherecombinantproteininsteadofcellmaintainance.
InthepresenceofthepLysSvector,IPTGalsopreventscolonyformationexceptwithcertainvector(suchaspET-3andsomevectorscarryingtheT7lacpromoter).InthepresenceofpLysE,IPTGusuallydoesnotpreventcolonyformationunlessthetargetproteinistoxic.

Inatypicalcultureusefulforproducingtargetprotein,almostallcellswillformcoloniesbothontheLBplateandontheLBplate+antibiotic;lessthan2%ofthecellswillformacolonyontheLBplate+IPTG;andlessthan0.01%willformacolonyontheLBplate+antibiotic+IPTG.

Withunstabletargetplasmids,thefractionofcellsthathavelosttheplasmidwillbereflectedbyanincreaseincoloniesontheLBplate+IPTGandadecreaseontherLBplate+antibiotic.

Protocol

1.	ImmediatelybeforeinductionwithIPTG(atOD₆₀₀isapprox.0.6),takea100-mlaliquotofthecellculture.
2.	MakeaserialdilutionofthecellsUSPension,includinga10⁵and10⁶dilution.
3.	Platecellsatadilutionof10⁵ontheLBplate+IPTGandontheLBplate+IPTG+antibiotic.
	Platecellsatadilutionof10⁶ontheLBplateandontheLBplate+antibiotic.
4.	Incubatetheplatesovernighta37°C.
5.	Countthenumberofcoloniesoneachplate.

Reference:pETSystemManual,8thed.,1999(Novagen).

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