Partial RE digestion - please advise!

John Dixon jpcd0 at mole.bio.cam.ac.uk
Tue Feb 20 09:02:27 EST 1996


In article <U10784.15.0011F447 at UICVM>, U10784 at UICVM (Victor Levenson) wrote:

> Hi, bionetters;
> 
> I have a problem with a partial digestion and I need your input.
> 
> Plasmid has 2 sites for BsmBI, one of them in the fragment that I need.

snip

> I can get a partial, but the majority of the cuts are at the wrong site; I 
> get a lot of uncut stuff, a considerable amount of linear form and some 
> double-cut fragments, second digestion (with another enzyme) reveals
that most 
> cuts are at the wrong site.  

> Is there any way around this crap?  

Presumably you wish to clone a fragment cut at one of the BsmB1 sites and
one other unspecified enzyme, which cannot be resolved on a gel from the
fragment cut at the other BsmB1 site and the unspecified enzyme (if they
*are* different sizes you could cut with the unspecified enzyme first and
then partial with BsmB1).

As BsmB1 is a hapaxoterministic enzyme you should be able to clone the
fragment you want from your partial digest even if the majority of the
band you purify consists of DNA that has been cut at the wrong BsmB1 site.
Unless you are very unlucky (256-1) the 5' overhang generated by the BsmB1
on the wrong fragment will not be compatible with the BsmB1 site that you
have in whatever you are cloning into (I assume you know that the BsmB1
site that you are cloning into is compatible with the BsmB1 site at the
end of the insert you do want). In fact if you bung in lots of your cut
vector to your ligation you will probably be able to clone the piece you
want without even gel purifying the bands away from the original vector.

Hope that makes sense! e-mail me if not, Good Luck,


John

-- 
John Dixon                                        Lab 44 (1223) 334131
Wellcome/CRC Institute                            Fax 44 (1223) 334134
Department of Genetics
Cambridge University    
United Kingdom                           e-m: jpcd0 at mole.bio.cam.ac.uk



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