Pvu II partial digest conditions needed

:-Peter obrien at pharm.med.upenn.edu
Sun Mar 23 16:35:00 EST 1997


I've got a 2.1kb insert in pGEM3Z that has one Pvu II site at position
1170 of the insert.  I want to cut this site, but avoid the two sites that
flank the MCS (a tall order, I know).  My reason for this is to blunt
clone a linker into a doubly blunt-digested plasmid (the ther site being a
unique NAE I site located upstream by about 560 bases).

I've used the following strategy:  First I cut Nae I to linearize the
plasmid.  Then I cut for short times (3-5 min) with Pvu II.  This allows
me to isolate a band that is about 560 bp shorter than the linearized
vectoron an agarose gel.  This can only occur from digestion of the Nae I
site and the Pvu II site at 1170.  All other combinations give different,
separable bands.

THE PROBLEM: Yield. The site I want gets cut, and I can get some DNA of
the proper length, but I need quite a bit to get enough to blunt clone the
linker into the plasmid. 

Pvu II has STAR activity (per the NEB catalog), so I'm afraid to encourage
low Pvu II activity with high DNA concentrations, using the wrong buffer
to reduce activity etc.  All I can think to do is to do the digest at room
temp or colder.  to get enough DNA to play with.

Any suggestions for increasing the partial digest yield?

Thanks for listening :-)

:-Peter  








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