3' overhang ligation

lhyatt at hkucc.hku.hk lhyatt at hkucc.hku.hk
Mon Aug 23 01:16:01 EST 1993


Hi.

I have been following the discussions on ligations, and would like the advice
of all those ligation experts. I am doing a cohesive end ligation of a 3kb
fragment in a 3kb vector, but with 3' overhang (KpnI). I have repeated this
several times, with phosphatased vector, and always with the same result: all
colonies are religated vector (or linear vector, if my concentration of DNA is
too high). There was a post a week or so ago about ligations with 3' overhangs,
but i have lost it. Is there a special trick to these ligations? I have also
tried doing the same insert and vector with 5' overhang (Asp718), but the
enzyme does not give good digests in my hands (I have tried 2 batches, and the
NEB equivalent from Fermantas in Lithuania, Acc65 I). At the moment, none of my
ligations seem to work, and so it may be the incompetence of the
scientist.....

I would be very grateful for any advice, experience of similar problems,
attempts to use Asp718 and isozymes etc.

Lucy
LHYATT at HKUCC.HKU.HK

All the usual disclaimers apply.



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