C.Lu at cellbio.duke.edu
Tue Jan 27 13:35:05 EST 1998
It seems to me that the quality of NdeI and BamHi from NEB is gettng worse
and worse. I constrcuted dozens of constructions using NdeI or NdeI and
BamHi sites, no problem at all. Some stranges happened about month ago, I
can not cut these constructions again under the conditions I used before.
Since then no sucess has been reported using these two REs for cloning in
our lab. We ordered new REs, still can not get satisfied digestions. The
vector I am using is pET.
In article <svetlov-ya02408000R2601981541390001 at news.doit.wisc.edu>,
svetlov at oncology.wisc.edu (Vladimir Svetlov) wrote:
> In article <34CC3B71.6D1F at lille.inra.fr>, odonohue at lille.inra.fr wrote:
> > Vladimir Svetlov <svetlov at oncology.wisc.edu> wrote:
> > > NdeI-BamHI doubles I've used their B buffer. People around here often
> > >complain about NEB NdeI, so there you go.
> > Ian A. York wrote:
> > > For what it's worth, I've cloned maybe a dozen products using NdeI from
> NEB. Until I read this newsgroup, I had no idea NdeI was supposed to be a
> problem; I don't remember doing anything special.
> > I find that Nde 1 is bad for PCR cloning unless you have lots of bases
> > before the CATATG site. Otherwise, in normal digestions it should work
> > well. Having said that in our lab. we are having lots of problems with
> > Nde 1 at the moment. We use Eurogentec's enzyme.
> > Mike
> I made a dozen or so clones by PCR using NdeI and BamHI sites imbedded into
> primers (for pET33 cloning). In order to make NdeI to cut at the ends, I
> include exactly the sequence that was in the NEB catalog's table
> GGAATTC[CATATG]. The primer gets longer but works like a charm. 2 bucks
> worth of extra sequence but lotsa convenience.
> Among the complaints I heard about NEB NdeI was that it's often
> contaminated with exonucleases. As I said, I never used NEB enzyme, and can
> not confirm nor refute this claim. Use Boehringer and be happy. No
> affiliation, just a positive experience.
> Vladimir Svetlov
> McArdle Lab for Cancer Research
> Dept. Oncology
> 1400 University Ave.
> Madison, WI 53706
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