Hi,

I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.  The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful  in subcloning (T/A vector) and getting my insert at 1.2kb after  double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how  when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there.