I have tried cloning with both PCR purification and gel purification of my amplified PCR product. I have used insert: vector ratio 3:1 and 8:1. I also have difficulty in amplifying my gene sometimes, my amplified PCR product is getting consumed in putting up A-tailing and ligation reactions multiple times but after transformation in E. coli Top 10 cells, the transformed colonies do not contain my desired insert. I also want to verify if my A-tailing is working or not but I do not know how to do it.
It is usually a problem with the T tails of the vector. Over time, pGEM-t can loses them and causes self-ligation with the blunt ends. This is something that usually happens when the pGEM-T is old. Try with a new kit, you will see how the problem is solved
Hi there,
I agree with Luis. If your vector has actually lost T tails then you may try blunt end ligation provided your insert has blunt ends at some point...
phosphatase treatment generally helps in eliminating self ligation. You can try that.
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