HELP WITH TRANSFORM./COMP.CELLS!!!

Rachelle Leger ax041 at FreeNet.Carleton.CA
Mon Dec 12 10:35:57 EST 1994


In a previous posting, Zhixiang Hu (xiang at scripps.edu) writes:
> In article <stauth-081294142709 at darenmac.usgmrl.ksu.edu>,
> stauth at crunch.usgmrl.ksu.edu (Darren Stauth) wrote:
> 
>> 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.
>> 
> 
> I have a simlilar problem with electroplation. We do get seperate
> transformants on the plate. However, only one out of ten clones can grow
> in liquid media or new plates. The cells are DH12. Plasmids are pBR322
> derivatives. One of my colleagues has the same problem with BL21(DE3) and
> pET vectors.
> Does anybody have a sound explaination?


I had the same problem and found out the ampicillin was too old (the
powder). After preparing plates from a new bottle of ampicillin the 
problem disappeared.

Regards,

Rachelle Leger



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