cloning confusion - HELP!!
num208jn at MBDS.NRC.CA
Tue Nov 19 22:21:09 EST 1991
In article <1991Nov19.214417.26673 at dartvax.dartmouth.edu>, Carine.Tischmann at dartmouth.edu (Carine Tischmann) writes:
>Thanks for taking the time to read and respond to our posting. We are
>confident about our ampicillin levels. When plating out competent
>cells on amp+ and amp- plates, we get growth on the amp- plates but no
>growth on the amp+ plates as predicted. Furthermore, we have made up
>several liquid broth solutions and several different batches of plates,
>all of which have produced the same results. The plates and the broth
>have been made from the same initial broth solution.
Have you eliminated the possibility that you have feeder colonies..
which (I believe) arise from breakdown of antibiotic in the plates...
allowing Ap^s colonies to grow.
Before inoculation of broths, do you purify the colonies by streaking
or patching? To avoid the carryover of feeder colonies after
transformation, I usually streak transformants 16/plate... and
inoculate the broths with the single colonies which arise from this.
(I also use carbenicillin to reduce the number of feeder colonies).
Just a thought...
John Nash. Nash at biologysx.lan.nrc.ca (preferred) or num208jn at mbds.nrc.ca
Institute for Biological Sciences, National Research Council of Canada,
==> Disclaimer: All opinions are mine, not NRC's! <==
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