Flumoxing ligation problem

Andrew Doherty A.Doherty at Bris.ac.uk
Wed Dec 18 04:22:57 EST 1996


Can any one out there give me any sort of reasonable explanation for my
problem, which is this. We've been trying to do a nice simple,
straightforward mutagenesis by PCR, amplifying a fragment of coding
sequence using mutant primers and replacing the equivalent section of
native sequence. The primers also contain the necessary restriction
sites (AccIII - 5'; SphI - 3'), but the amplified fragment will not
ligate back into the coding sequence. So to test the system, we cut out
the band from our receptor CDS which is to be relaced with the mutant
fragment, (cut with AccIII and SphI)and tried to religate this fragment
back in, and get NO COLONIES!!!!!! The E.coli are fine, and transform
quite happily with the un-messed-about-with plasmid. SO WHAT THE HELL IS
GOING ON?@#!!@##@

Any ideas will be most welcome

Yours confusedly
Andy D
Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK

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