E.coli library

mcgeed at HAL.HAHNEMANN.EDU mcgeed at HAL.HAHNEMANN.EDU
Fri Sep 16 16:19:10 EST 1994


	Hi there!   I have over 500 transformants of E. coli strain DH5-alpha,
containing a 6kb plasmid vector with inserts of 4-6kb (this is the optimal size 
for the vector).  The inserts are from the gonococcus.  I realize that there
aren't enough colonies for a complete library and I'm working on increasing the
transformation efficiency.... I strongly suspect that my ligation reaction (not
ethanol precipitated) is inhibiting my electroporation by at least a factor of
ten.   (I get > 100,000 transformants [a lawn] with 1 ug undigested vector, so the
electroporation itself is working; when I digest the vector with a restriction
enzyme and then re-ligate it, the number of transformants drops down to less
than 1000 per ug of DNA).  Next time I will phenol:chloroform and ethanol
precipitate my ligation mix before electroporating.  Has anyone else out there
experienced this problem with "unclean" ligation mixes in an electroporation?
	On a different note, over 90% of my transformants were extremely tiny
after a full 24 hours of growth on selective media; I guess the E. coli are
"sick" from this foreign DNA.  I would like to keep these buggers alive for
several days to a week for screening purposes.  I assume that even under this
sickened state that they will survive in the cold?  I'm also concerned that if
I pool these colonies for overnight growth into 50 groups of 10 (for example),
that some colonies will overgrow the rest and my library will be grossly
under-represented (even if I would have had several thousand transformants, I
will still have the problem that most are growing poorly).  Is my concern
warranted??  Any comments??

	Thanks a lot and good luck with your own studies!




David J. Mcgee
MCGEED at hal.hahnemann.edu




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