I am having trouble with an apparently straightforward cloning. I need to excise a fragment of approximately 1.7 kb from a 4.8 kb parent vector and join it to a 5.7 kb destination vector. I could not find any proper restriction sites for a cohesive ligation so I had to blunt both the fragment and the destination vector. The enzymes I choose to excise the fragment and open the vector all leave 5' overhangs.
I use NEB's quick blunting kit (T4 polymerase) so I think my fragments are not "polished", but filled (beacause T4 5'->3' polymerase activity). Then, I CIP treat the blunted vector, but not the 1.7 kb fragment, in order to reduce the background in the subsequent ligation. I perform an overnight, 16 ºC, ligation at different molar ratios (1:3, 1:6, 1:10, 1:20) in a final volume of 10 ul. Then, I pick 2 ul of the ligations and transform DH5a competent bacteria, but I dont see any clones. My positive and transformation controls are OK. I repeated the transformation with 4 ul of the ligation, with the same results.
This is annoying me a lot, ¿any idea of what is going wrong? Thanks in advance.
Edited by litos, 18 August 2009 - 12:22 AM.