SacI near end of PCR product

Mikhail Alexeyev malexeye at JAGUAR1.USOUTHAL.EDU
Wed Nov 12 12:12:47 EST 1997


 Koen De Smet wrote:


> I did a PCR with primers containing a SacI and a XbaI site in them for
> subsequent cloning. I have done similar PCR+cloning experiments with
> other restriction enzyme sites, so have a technique that usually works.
>
> But this was the first time I used SacI as a RE site, and the cloning
> didn't work (twice); that is, no colonies at all. There could be several
> explanations, some of which I already tested or will investigate in the
> next few days, but I suspect that SacI didn't cut near the end of the
> PCR product.
>
> I normally rely on the NEB catalogue to figure out how many extra
> nucleotides to add at the end of the primer to get good cleavage. But
> they only show data for one extra nucleotide for SacI, which cuts only
> 10% overnight. So I added four and hoped for the best.
>
> Now my question is: has anybody out there used SacI sites in their
> primers, and what was the result?
>
> (I know, I may have to resort to cloning my product in pUC or
> pBluescript without digesting, and then cut it out again...)
>

You may have a better luck with Ecl136II- a SacI isoschizomer that
generates blunt ends upon cutting.  It is less sensitive to salts and is
available from NEB.

M.A.




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