Help subcloning large PCR product
ANDREI.POPOV at bbsrc.ac.uk
Tue Sep 26 06:33:25 EST 1995
>gel purify all final products on 1% Seakem agarose prior to ligation and
>ligate using 100-200 ng vector with a 1:1, 1:5, and 1:10 molar vector:
>insert ratio in a 10 ul rxn. I use NEB T4 ligase and ligation and
>transformation controls work fine.
I guess this should go to FAQ as these problems have been
discussed billions of times here.
In short, I think you are using a huge excess of both your vector
and insert. Check ligation on agarose: I bet you will see
a high MW smear.
I cloned a 2 kb PCR product into the HincII site of pBS w/o any polishing,
killing Taq, proteinasing, vector CIPing etc....
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