Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Fri Feb 19 13:01:59 EST 1999
In article <7ah8kn$e7i$1 at unix2.glink.net.hk>, Andrew Leung
<leungkc at glink.net.hk> writes
> Specifically, I cut both my pcr product and my favorite vector with two
>restriction enzymes. Then, I perform ligation reaction which is supposed to
>give transformant of having insert in right orientation. Now, I can't get
>any transformant. Since all control experiments were OK, I guess the problem
>resides on ligation parameters.
> Is there any suggestion in improving my ligation efficiency?
What everyone is forgetting is that Taq, other PCR enzymes and
nucleotides can survive simple phenol extractions and ethanol pptations.
You then cut with and RE and the Taq gap fills the overhang. It just
reduces the cloning efficiency. In theory I suppose you should get blunt
ended ligation if the overhangs are 100% filled in.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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