I had some issues with a cloning since a couple of months. I explain: I want to insert a ~6000pb gene into a mamalian plasmid. The PCR from cDNA went well I have my insert (even if it's not really a thight band) and I purify it using a Qiagen kit. then I digested the insert and the vector with BglII/NotI and proceed to the ligation. I got some colonies but everytime that I made some analytical digestion to confirm it I don't have the right "size".
So I tried the TA cloning and no colony @all....
So I tried ro change the strain DH5alpha, TOP10 and pirHC, the growth temp (37 and 30°C).. and each time nothing (I mean no positive clone);
So I tried to just ligate directly into the expression vector and I got the same things.....
Did someone have an idea to sucess?