Blunt end PCR product ligation - (Jan/06/2012 )
On the onset, I wish everyone of you happy n prosperous new year 2012..
Now coming to my problem.
I am trying to ligate 2 PCR generated blunt end products. Subsequently, this ligated product has to be cloned in pJET (again blunt end ligation). However, I am not getting ligated product in the first place (no ligation of blunt end PCR products). Any suggestion, how to proceed with this?
I am not sure about the primers which I used for PCR i.e, whether it was phosphorylated or not. Will it have any impact on ligation effiiciency. If yes, then should I go for phosphorylated primers or can I phosphorylate my PCR products directly.
Looking forward for reply.\
The blunt ends must end up being phosphorylated. You can do this by ordering phosphorylated primers, by treating the primers with PNK, or by waiting and treating the pcr product with PNK. Your pcr must also produce blunt ends, which enzymes such as
Taq or mixes containing Taq will not do. You will have to use a proof reading enzyme, or blunt the product after PCR. You will also have the problem that the ligation is not selective, so if your two fragments are A and B, you will get AB, BA, AA, and BB fragments. Even if you phosphorylate only one end of one fragment, you will get AB and AA, for example.
I think this is a poor general strategy, unless you have absolutely no choice,