Nde I restriction enzyme

Martin Offterdinger a8803349 at unet.univie.ac.at
Tue Dec 3 02:29:34 EST 1996


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




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