Nde I restriction enzyme

[Jared Head]

Martin Offterdinger (a8803349 at unet.univie.ac.at) 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. 

I had exactly the same problems trying to clone a PCR product into a 
plasmid using NdeI.  Even in a control experiment where I cut a plasmid 
with a single NdeI then tried to religate it, the NdeI sticky ends would 
never come together.

Eventually I just changed my brand of Nde1 (to Promega, if I remember 
correctly) and it worked first time.  Still don't know why, unless it was 
something to do with exonuclease activity.

Good luck,

Jared

-- 
Jared Head     at the Department of Biochemistry, University of Bristol

  "Happily unaware of the disadvantages of being a terrible writer with 
                              nothing to say..."