Restriction Enzyme Digestion at the End of a Vector

Hi all,

Has anyone ever tried a restriction enzyme digest where the site is literally on the end of a vector? I’m trying to do a cloning experiment right now, cloning a 2.8kb insert into pET14b and the two multiple cloning sites on my vector that I must use (to prevent cutting my insert) actually share a base so that after my first digestion there isn’t a single base pair before my next RE site. I’m cutting with XhoI then BamHI sequentially.

MCS:
CTCGAGGATCC
GAGCTCCTAGG

after cutting:

CTCGA GGATCC
G AGCTCCTAGG

The GGATCC sequence (BamHI) is my second site. On New England Biolabs website for cleavage close to the end of DNA fragments it doesn’t even have information about cutting with ZERO base pairs before your RE site which I’m assuming means that it’s probably not going to work too well.

I’ve tried the transformation a couple of times. My first was an attempt with dephosphorylation and got nothing but I didn’t perform a positive control and the buffer I was using ended up causing trouble for other members of my lab, so I decided to try again.

When I tried running ligating the insert directly into the sequentially cut vector without dephosphorylating it I had more colonies on my negative control (vector with no insert ligated) than I did on my actual experiments although the transformation efficiency wasn’t great (around 3×10^5).

I tried it again last night with dephosphorylation and I again got nothing on all of my plates except for the positive control.

I’m thinking that this approach needs to be abandoned or at least modified because it looks like the second digest with BamHI just isn’t working. Does anyone have any good ideas about how to increase the chance of a cut in such inoptimal conditions? I ran with BSA for three hours and 10 units BamHI per ug of plasmid DNA.