ZAP-Express excision problem

smoothies channels at sghms.ac.uk
Fri Feb 27 10:00:55 EST 1998


Hi all

I am sure this problem has already been discussed but I would be
interested to read new suggestions.
I am starting from a single (pure) lambda ZAP-Express plaque. This clone
is a positive as checked by hybridisation and PCR with specific test
primers. So I go ahead with in vivo excision following the Stratagene
protocol. I get a suspension of excised phagemids which also gives me a
positive signal with the test primers.
I infect host cells (XLOLR) and I get the expected KAN-resistant
colonies. I perform PCR on 28 of them, with the same test primers and
none is positive !
I know this sort of thing has happened to other people. After
sequencing, the insert turns out to be some piece of rubbish. Has
anybody got an idea of what is going on ? (There has been no confusion
in the sample number and PCR controls were included at all times).
Thank you very much for any help - even if it is obvious.

Claire Fenech  - email: c.fenech at sghms.ac.uk
Dpt of Pharmacology
St George's Hospital Medical School
London - UK




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