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about heatshock transformation - (Nov/08/2007 )

I used CaCl2 competent DH5a cell transformed with plasmid via heatshock procedure, but I did it by mistake, before 42 C heatshock, LB medium added, so when heatshock, the volume was too big and also with extra ions, I think which can be bad for transformation. After 37 C overnight incubation, single colonies grew up, but no positive when PCR verification, I was surprised why sigle colonies could grew up on medium containing antibiotic.



check the efficiency of the Atb in your LB/agar preps (incubate some water or any biological sample and check for growing, or put a diluted transformed bacteria and look for satelite colonies...)
change your Atb stock, or increase its concentration
if plates were prepared many weeks ago, or badly stored, Atb would not work loonger