HELP WITH TRANSFORM./COMP.CELLS!!!

bipin dalmia dalmiabk at phibred.com
Fri Dec 9 17:38:26 EST 1994


In article <stauth-081294142709 at darenmac.usgmrl.ksu.edu> Darren Stauth,
stauth at crunch.usgmrl.ksu.edu writes:
>We recently started making competent cells using DMSO and PEG3350 and we
>were excited at its ease and efficiency.  We are using DH5-alpha cells
and
>transforming TA ligations.  However, we are now running into the problem
of
>cell lawns appearing on our transformation plates containing amp.  First
>the transformants appear and then a few hours later the lawn becomes
>visible.  We have tested the amp and the cells both are fine.  Is the
>treating of the cells to make them competent somehow causing them to be
>resistant to amp?  Has anybody experienced this problem?   Any
suggestions
>welcome.

the 'satellite' colony effect that you are seeing is due to the
ampicillin around the true transformants getting degraded by some
secreted b-lactamase and/or inactivated by the lowering of pH when cells
grow. you may try higher amp. concentration (upto 200 ug/mL) or switch to
the more expensive carbenicillin. carbenicillin is less sensitive to low
pH and in my experience never gives satellite colonies. use carbenicillin
at 100 ug/mL.

bip



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