I am trying to insert a fragment in the .THT vector, which was cut with two RE : NdeI and BamHI. Then double cut vector is gel purified and "ligated" with insert using T4 ligase. After transforming ligated vector, it was digested again to see if the insert will be present on the gel. And the result for this was negative. So we tried to CIP treat vector before ligation and that did not work either.
Then we decided to vary ratio of DNA:insert (1:1, 1:3, 1:57, 1:15) and none of those worked. So I will be very grateful if you make any suggestion how to make this ligation to work.
Thanks in advance.