Hi, I've been trying to clone three PCR fragments into a vector using Gibson cloning.
All three fragments amplified well with the chimeric primers, and used in the reaction onto a pUC19 background.
However I failed to get any transformants. I did PCR analysis from vector:fragment 2 and fragment2:vector and obtained strong bands at the predicted size for both fragments indicating the presence of the desired product. After speccing the sample the DNA concentration was quite high (~3000ng/ul) so I tried first diluting before transformation into both DH5a chemically competent cells, and DH10B electrocompetent cells and plating on Amp plates, but again both these transformations failed. Next I used plasmid safe exonuclease to digest remaining linear DNA, and then cleaned the reaction through a column, eluting in 30ul water. Speccing of this sample indicated I had a much reduced concentration (8ng/ul). I repeated the PCR test on this cleaned template and again obtained both bands at correct size, but still the reaction failed to transform.
It seems to me the product has been generated (as only circular DNA would survive the plasmidsafe digestion, and the PCR reactions were successful) but the lack of transformation is very puzzling. Do you have any advice?