Ligation using TA cloning - (Aug/18/2008 )
I am having problems with ligation after TA cloning. Here is how it goes. First, I cut the puc19TA vector with xcm, which generates T-overhangs. Then I PCR amplify (with Taq DNA polymerase) my insert which is about 1.45kb. The cut vector is about 2.6kb. I then ran the pcr product in an agarose gel and excised the desired band and extrated the DNA from the gel. After which, I ligated the vector and the insert using molar ratios from 1:1 to 1:5. But none gave colonies containing the ligated product.
I then read that the 5' base of the primers used in PCR can affect the addition of the A overhangs to the 3' ends of the pcr products. So, i used a pair of primers which have A and G at the 5' ends respectively. A and G can vastly increase the efficiency of addition of A overhangs. However, this method too did not results in desired colonies. So, I do not know what the problem is.
The cells are competent and I perform ligation at 4 deg overnight.
I would try this without gel purifying your vector or PCR product. Gel purification solves some problems, but causes several others. With a good PCR reaction, you should not need to gel purify the product. It's less work, also. I'd guess that a shorter, 15C ligation would work. Make sure your ligation buffer still has ATP (or add 1 mM final ATP to make sure).