Having trouble cutting pET19b with Nde I and BamHI - (Mar/07/2002 )
Ok I am trying to cut pET 19b with NdeI and BamHI in order to clone an insert with the same ends. The problem is that everytime time i perform a ligation using cut pET 19b as a control i seem to get lots of colonies back. The same is true if i ligate the cut vector, despite the ends being incompatible. I have cloned many genes into these same two sites in the same vector and have never had this problem before. I digest for three hours with NdeI and Two hours with BamHI. Can anyone help me as i am going insane. Thanks
Try CIP treat your digested pET19
Are you sure the digestion was complete?
I was not successful doing this except with NEB buffers and enzymes (with pET21a). Cut 1st with NdeI in the BamHI unique buffer + BSA, check that it is linearized, and then cut with BamHI.
I have had similar problems in the past, and would:
1) Digest plasmid with Nde1 in its buffer
2) Run on agarose gel + extract cut DNA
3) Digest DNA with BamH1
4) Alkaline phosphatase (Shrimp/calf) treat vector
5) Purify DNA
6) Run a little on a gel to check DNA is still there
Provided the correct restriction enzyme buffers are used, this seems to work every time for me !!!!