Nde I restriction enzyme

Andrew Doherty A.Doherty at Bris.ac.uk
Tue Dec 3 05:41:41 EST 1996


Martin Offterdinger wrote:
> 
> Hi everyone!
> 
> I have problems with a special restriction enzyme NdeI.
> I try to cut PCR products after RT PCR with restriction enzymes.
> What I do normally is to precipitate the PCR products with
> isopropanol, wash with 80% EtOH air dry and dissolve the pellet
> in the digestion mixture and incubate for 2h . Afterwards I load the
> digest directly onto an agarose gel. This procedure works very well,
> if I use BamH I to digest and I get my products cut very well-
> BUT IT DOES NOT WORK WITH NDE I. Even Phenol extraction of the product
> does not improve the situation, neither is the enzyme itself inactive
> as a control plasmid is still cut well.
> Is there anyone who has experience with this particular enzyme?
> Is it sensitive to impurities that can not be removed by phenol
> extraction?
> Or do you rather think that the PCR product simply does not have the
> Nde I site any longer? ( But the cell RNA which I use was prepared
> from cells at a low passage number)
> Please help me !!! Any hints will be appreciated.
> Martin
Hi Martin, there was a long thread about this very thing a while ago on
this newsgroup - it'll all be in the archives. Basically, NdeI is a
swine of an enzyme - you have to use bucket-loads of enzyme for a long
time to get it to cut. Hunt thru' the archives, it'll open your
eyes!!!!!

Hope it helps
Andy D
-- 
*************************************************************
Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK
BS8 1TD
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