Hi,

Which restriction enzymes were used in your previous approaches? Is it a sticky end or blunt end ligation? Have you used controls for the transformation and the cPCR? Some more details would be beneficial to provide decent advices.

Hi Boas, I am using KpnI/BamHI sequential digestion, its sticky end ligation. Yes, i did a control for transformation, no prob for competent cells. I didnt do any control for cPCR as I expected efficiency of T7/T7t should be able to get my insert without any problem. I am wondering whether there is any ways to know if RE digest product cut correctly?

Hi there,

Single digest on the circular plasmid is a good control for activity as it's easy to make the difference between circular and linearized plasmid on agarose gel. Is the insert generated by PCR? If yes how far from the ends are RE sites (there should be at least three bases to ensure maximum cutting activity). Do you run ligation controls alongside the ligation? These controls are ligation with digested vector alone (without ligase) to evaluate the presence of intact plasmid in the mixture and digested plasmid alone(with ligase) to evaluate selfligation event frequency which derives directly to single digest event.

Dear Tang,

Try running the miniprep of the transformants on a gel and check for the right size. If it is at the size of the vector alone, that would explain your negative results in PCR. Secondly, it can also be deduced that the problem might be with your 1.3 kb fragment. 

Cheers