methods to prevent digestion by REs?

Steve Rodems smrodems at students.wisc.edu
Sat Apr 23 09:15:48 EST 1994


In article <9404221408.AA29527 at venus.med.pitt.edu>, bsh at MED.PITT.EDU
(Basavaraju Shankarappa) wrote:

> Hi folks:
> 	We have a situation with one of our clones where we thought the 
> restriction site was an unique one, but now it turns out that there is an 
> extra one in the vector.  Now we need  a method that will prevent 
> digestion by restriction enzyme at one site while allowing it to cut at
> the other site.  

Partial digests are easy to perform.  I think Maniatas has a section on it.
 I've done several just like they say and they work great.  You just put
your DNA in a tube with maybe 1/3 of the normal conc. of digest buffer in a
total volume of about 130-140 microliters and then take 15 microliter
aliquots and put in separate tubes.  Then add 0.5-1.0 microliters of enzyme
to the 1st tube (which should actually contain 30 microliters), mix, and
transfer 15 microlites to the next tube, etc.  This essentially keeps the
DNA conc the same in each tube but dilutes out the enzyme.  Incubate for
0.5-1.0 hr and run on a gel.  You will see most or all of the sites cut in
the first couple tubes, partials in the middle to last tubes.  If you want
to permanently get rid of the other site you can take the partial product
where just one site is cut and fill-in or add linker (if its blunt) and
re-ligate.  Then screen for knock out of the site you want.

I think this method is easier than taking timepoints after the addition of
enzyme. 
-- 
Steve "Some day I will get the hell out of Wisconsin" Rodems

"Then I am here for the Lee family renioun ... shur-wajo-shur"



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