competent cells and ampicillin

[Sasha Kraev]

Just a suggestion: when you are plating out on ampicillin plates, the number
of cells plated is at least 1000 fold higher then the number of resistant 
colonies. Cells that do not grow ( ampicillin is bacteriostatic but not 
entirely killing E.coli ), however, appear later when ampicillin is degraded
by a transformant colony. So, if the number of actual transformants is very low
like it is in blunt-end ligations, one tends to plate larger part of the 
transformation mixture, thus increasing the number of the cells surviving the
ampicillin background. So, the background must be proportional to the portion
of the transformation mixture plated on one plate AND to the extent of 
ampicillin dying-out due to aged preparation, old plates etc. The problem 
should be more frequent in electroporation, where a large number of cells is 
used in one experiment. In my hands, plating more then an equivalent of 1 ml 
log-culture cells on one 90-mm plate always resulted in quickly appearing 
background colonies, no matter fresh or old ampicillin was used. 
Hope this helps. Sasha