Nde I restriction enzyme

Martin Offterdinger a8803349 at unet.univie.ac.at
Tue Dec 3 09:58:29 EST 1996


On Tue, 3 Dec 1996 10:41:41 GMT, Andrew Doherty <A.Doherty at Bris.ac.uk>
wrote:

>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
>*************************************************************
Hi!

Are there impurities in PCR products that CANNOT be removed by phenol
extraction, that inhibit Nde I ????
 

Martin



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