SacI near end of PCR product
brett at BORCIM.WUSTL.EDU
Wed Nov 12 13:37:45 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.
One better - SstI, which cuts identically to SacI but is much more
salt tolerant. I think we buy it from BRL.
Washington University - St Louis
brett at borcim.wustl.edu
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