Plasmid instability - why and how do i check? (Jul/18/2006 )

Hi,

Have a very basic and stupid question..

What all can lead to plasmid instability?

How do i check whether that is the case.. i have been getting very drastically changing protein yields from my clone? Cud it be that the plasmid is lost? I do have Amp marker, but growth on LB and LB + Amp are not helping enough to conclude.. can i do something else?

Please let me know.. Thanks lot!

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there are 2 things you should consider.
one is the antibiotic. Amp tends to lose effectiveness after 18hrs. beyond 18hrs the amps would be quenched and you would see tiny secondary colonies on your plate around the orig. so try to pick colonies or broth at around 18hrs.

second factor is a toxic product. if the gene product it toxic, typically some bact will die off and others will tend to throw away the insert. the abs/presence of this could be checked by rest digestion/ PCR/sequencing.

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