Partial RE digestion - please advise!
malexeyev at biost1.thi.tmc.edu
Thu Feb 15 14:39:27 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.
> >I tried to get a partial by modifying:
> >1. plasmid:enzyme ratio
> >2. temperature
> >3. time of the digestion
> >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
> >cuts are at the wrong site. Today I tried digestion in the presence of
> >works great for DNaseI - and I could not get ANY cuts!
When and if everything fails (like trying to linearise plasmid before
BsmBI digest, titration EtBr, etc.) you may try to PCR the fragment that
you need out or (if fragment is large and risk of introduction of mutation
by PCR is considerable) try RecA-assisted cleavage of the site that you
need to cleave
Weiner MP. Felts KA. Simcox TG. Braman JC.
A method for the site-directed mono- and multi-mutagenesis of
Gene. 126(1):35-41, 1993 Apr 15.
Sorry, this is as much as I can think of.
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