Pvu II partial digest conditions needed

plxsrt at pln1.nott.ac.uk plxsrt at pln1.nott.ac.uk
Mon Mar 24 07:25:29 EST 1997


obrien at pharm.med.upenn.edu (:-Peter) wrote:

>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  

Can't you use PCR or shuttle the cloning through another vector?
Partials have a habit of being very messy.  If you must use partials
and are worried about star activity just scale up the volume.   Most
genomic libraries are made by partlal Sau3a digests of 100's of
micrograms of DNA.  Promega's own Protocol and Applications guide
gives a protocol that you could adapt to your needs.

Good luck

Simon T








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