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.
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