sflamant at pasteur.fr
Mon Sep 10 07:29:00 EST 2001
Hi, Peter. I usually use this kit from Invitrogen with success. As it was
mentionned by others, you first have to check that the polymerase you use is
able to generate overhanged extremities. The classical Taq polymerase is
suitable for such an aim. Secondly, be sure that your PCR product is
correct, it is not necessary to purify it if you have only one product.
Finally, just for information, I do the ligation reaction exactly as
mentionned by Invitrogen: 10 minutes at room temperature, then in ice, and
that's all. If the problem comes from the transformation reaction, you may
do the experiment in cold room, especially when you mix the ligation
reaction with the bacteries. Then let stand them on ice in cold room for
20-30 minutes, and do the thermic shock (30'' at 42°C, then on ice). Maybe
you should spread all the transformation reaction mix at the end, you can do
that on more than one LB-Amp plate, if you prefer. Good luck.
Peter Guo wrote:
> Hi, friends:
> I tried to clone a PCR product. However, after transform the Top10
> compentence cells, I did not get clones. The PCR is good. I phosphoralted
> the 5' end of the primer. I used Invitragen Company's kits. Ligation
> condition is at 16 degree centigrade. I think it should be at 4 degree
> centigrade. Is this correct?
> Would somebody tell me how to treat the problem.
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