Cloning problems-Help!!

zimprich zimprich at
Sun Nov 28 12:24:44 EST 1999

Dear Colleagues,

In the last few months, i have a very big cloning problem,.
I try to get a AOC1 (5´overhang CC!TGAGG), ECO R1 (also 5´overhang
G!AATTC) fragment into a  vector also cut with AOC1, ECO R1.  It sounds
quite easy, but it is a nightmare.
I tried it over a dozen times, but i only get  very few colonies after
transformation, which were all wrong. I did all kinds of control
experiments; so let us assume that Ligation, Transformation etc. do work
in my hands.
Is it possible the some restriction enzyme overhangs get lost, or need a
special treatment for ligation?
I purify my DNAs over agarose gels and clean them up using Quiagen

Any ideas

Thanks a lot


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