tk at pasteur.ai.mit.edu
Tue Nov 23 17:12:08 EST 1999
We're having trouble with transformations of the Clonetech DSRed and I
was wondering if others are having similar problems.
We transform (into DH5a), and mostly get white colonies. We grow them
for a while and sometimes a few isolated colonies turn distinctly red;
we then streak these colonies out, and the result is again a set of
white colonies. As far as we can tell, the medium has adequate
ampicillin (100ug/ml), so the cells are unlikely to be losing the
plasmid. The fact that a few distinct colonies are red suggests that
at least some of the plasmids are the correct one. We've done a
plasmid dna prep (white colonies, the only ones we can reliably
produce), and are preparing to sequence it. My best guess is that the
protein is toxic, and we are selecting for plasmids which eliminate
expression of the toxic protein.
Any other ideas? Similar experiences?
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