Q:Does Gene Clean interfere with ligation?

Richard R. Hardy hardy at mighty.fccc.edu
Tue Mar 7 14:09:01 EST 1995


In article <3jghpq$a6a at hydra.acs.ttu.edu>, Z3C20 at ttacs3.ttu.edu (Yi,
Xiaoming) wrote:

> Hi, netters,
> 
> I have been trying to ligate a double digested PCR fragment onto a double
> dogested vector. Since i was using a very long primer(500bp), the yield was
> low. So, I did not do EtOH precipetation after I did Gene Clean to the gel
> slice. I found no transformants on the plate. I am thinking the DNA I had
> contained a little "New wash" solution(about 1/6), it interfered with the
> digestion or lugation, since I could see a light band on the agarose gel
when I
> ran half of my PCR reaction, I assume I had enough DNA for transformation.
> 
> Thank You.
> 
> Xiaoming Yi
> z3c20 at ttacs.ttu.edu

I've had this problem with "New wash" inhibiting ligations; first try and
get every last bit off by respinning and carefully removing the residue;
then speed-vac the beads for a short time (5min); maybe lowers your yield
of DNA, but it will ligate... Good luck.

-- 
R. Hardy
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
(215) 728-2463



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