Nde I restriction enzyme

lj1 at mrc-lmb.cam.ac.uk lj1 at mrc-lmb.cam.ac.uk
Wed Dec 4 11:07:09 EST 1996


>> :
>> : 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 don't know if anybody mentioned it, but Nde1 has an half-life of 15' at
   RT/37 C. So what works with me is to add the required amount of Nde1 in
   aliquots. After adding the first aliquot, I leave it at 37 C for 1h, and
   then add some more, leave it 30' at 37 C again, and so on, for 4-5 times,
   until it is completely digested (obviously, one has to top up buffer and
   water as well). I tried this with an Nde1/BamH1 double digest in BamH1
   buffer (I use NEB enzymes) and it worked fine.
   Hope this help - Luca.

--------------------------------------------------------------------------------
   Luca Jovine
   Structural Studies Division, MRC Laboratory of Molecular Biology
   Hills Road, Cambridge CB2 2QH, ENGLAND.
   Voice: +44.1223.402443. FAX: +44.1223.213556. Telex: 81532.
   E-Mail: lj1 at mrc-lmb.cam.ac.uk - lj200 at cam.ac.uk.
--------------------------------------------------------------------------------




More information about the Methods mailing list