Ligation problems on agarose-isolated fragments

Anne Bokman bokmana at aa.wl.com
Thu Feb 1 08:34:55 EST 1996


As the subject of the message says, we are having major problems with ligations:

I am trying to ligate a 450bp fragment into pET29b, using NdeI and BglII
as restriction sites, but other people in our lab are experiencing the
same problems with bigger and smaller inserts and different vectors. 

We have tested, and eliminated, the ligase, the ligase buffer, the water
and the transformation method.  Everything seems to point to a problem
occuring from the gel isolation.
I have tried to Phenol/Chloroform extract, EtOH precipitate the gel
isolated fragment and vector, to run them on commercially available
purification columns (QIAGEN) but to no avail...

I am desperate and hoped that someone out there can help us.  I am open to
any suggestions or advice you might have.

Thanks,

A Bokman



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