svetlov at oncology.wisc.edu
Mon Jan 26 16:41:39 EST 1998
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.
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.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
More information about the Methods