Nde I restriction enzyme
bijgh at zeus.bris.ac.uk
Wed Dec 4 06:51:02 EST 1996
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.
Jared Head at the Department of Biochemistry, University of Bristol
"Happily unaware of the disadvantages of being a terrible writer with
nothing to say..."
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