HELP WITH TRANSFORM./COMP.CELLS!!!

Duncan Clark Duncan at genesys.demon.co.uk
Mon Dec 12 10:21:47 EST 1994


In article: <D0KEw2.2uG at phibred.com>  bipin dalmia <dalmiabk at phibred.com> writes:
> 
> 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.

Alternatively try 80mg/ml methicillin (Sigma) + 20mg/ml ampicillin. 
Works great on plates but I have had unusual results in liquid. 
Methicillin is also cheaper than carbenicillin.

Duncan
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