SexA I: digestion problems

M. Alexeyev malexeyev at biost1.thi.tmc.edu
Thu Oct 12 21:44:17 EST 1995


In article <elena-1210951645070001 at bhmlabci.chem.purdue.edu>,
elena at chem.purdue.edu (Lena Polovnikova) wrote:

> I need to use SexAI site in my subcloning procedure. SexAI is very
> expensive ($1 a unit) and only BMB sells it. So I purchased the enzyme,
> and it does not cut my plasmid, neither alone, nor together with Bgl II
> (the sites are ~1.3kb away from each other, and the buffer is compatible,
> Bgl II cuts perfectly well). I did get partial digest on the PCR fragment
> that I PCRed from my plasmid (~300bp, site is in the middle), but I used a
> lot of enzyme. I sequenced my plasmid, and the site is there. The other
> plasmid with SexAI site is being cut OK. So I do not know what's going on
> (2ndary structure?) and what can be done (unfortunately, I cannot use any
> other site for my purposes). If anyone has any experience with SexAI or
> has similar experiences with other enzymes, your help will be greatly
> appreciated. Please, e-mail me or post here.
> 

Lena,
Recognition sequence for SexAI is ACCWGGT. It overlaps CCWGG site that is
being methylated by E. coli dcm methylase. PCR fragment is not methylated,
however, it could be not clean enough to be digested completely. Is it
possible that your other plasmid has been isolated from dcm- strain? I
beleive NEB provides dcm- strain free of charge.

Good luck,
M.Alexeyev



More information about the Methods mailing list