problem in ligation and cloning - (Jan/23/2010 )
i am trying to clone a PCR product (1.4kb) by ligation to zero blunt PCR cloning vector (Invitrogen) and transformation into TOP10 cells (Invitrogen). The PCR product amplified by pfu polymerase is purified by PCR clean up kit (Qigen) shows good concentration with no non-specific products. I have followed the manufacture instructions. No colonies appear on overnight incubation. The other PCR product (1.3kb) is getting cloned. Just wondering what is going wrong.
Please suggest possible trouble shooting.
Do I have this right? ...
Your PCR produces two products - 1.3 and 1.4 kBP respectively, you then take your PCR and clean it up using a kit, clone, and the final product is the 1.3 kBP variant?
Smaller fragments preferentially clone over larger fragments (due to reaction sepped energetics), though the difference between 1.3 and 1.4 kBP should be not a lot in terms of the energentics.
I suggest trying to extract preferentially the 1.4 band, using gel extraction and then try cloning.