HELP! - cloning problem
MSRA at novo.dk
Wed Nov 4 02:25:36 EST 1998
We have a strange cloning problem (and totally frustrated!). After
the blue/white selection, we tried insert check by colony PCR. Here,
most of the case, there is no band amplified, even though we selected
white colonies. We also tried minipreping plasmids and checked with
restriction enzyme cuts. Plasmids are there, but we get different
sizes of digested fragments - total size of appeared fragments are
shorter than what we expect.
Our vector is 5-6 kb (pBAD and pMAL) and the insert is 2 kb. Yes, I
have to add that this problem started ever since we tried to clone in
the 2 kb insert to these different vectors. We tried two kinds of
cells, DH12S and JM109, by electroporation and heat shock,
respectively. But both showed similar results - while colonies,
plasmids are there, but wrong ones.
I wonder whether some kind of plasmid recombination happened here...?
However, the DH12S cell is recombinant deficient strain (recA1-).
Does anyone out there know whether it's worth trying other cells such
Any comments and suggestions are greatly appreciated. Please help us!
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