Problems cloning adaptor
triolo at mcbsgi.bio.sunysb.edu
Tue Sep 12 14:59:11 EST 1995
In article <bjanssen-1209951033460001 at hall-pmac8100.tamu.edu>,
bjanssen at bio.tamu.edu (Bart Janssen) wrote:
Try simply cutting your vector and not phosphatasing it. Then ligate
in the presence of massive amounts of adaptor, several micrograms. This
is my way of doing this type of cloning, and I've had success with it.
If your kinase is being inhibited by some sort of salts or
contaminants in your adaptor preparation then phosphatase treatment of the
vector is only going to decrease your cloning efficiency. I may be wrong,
but I think that some kinases may be inhibited by salts left over from
ethanol precipitation of nucleic acids with amonium acetate as the salt.
You didnt say what salt you used though.
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