strange cloning result

Ian A. York iayork at panix.com
Tue Mar 28 12:41:08 EST 2000


In article <200003281715.KAA78624 at nestor.NMSU.Edu>,
Hiranya S. Roychowdhury <hroychow at nmsu.edu> wrote:
>
>The other possibility is that one of the enzymes did not cut 100% of the
>vector molecules. This is rare, given sufficient time for digestion
>(especially w/ those two).

It should be rare, but sometimes it isn't.  I agree with others who have
said this is the probable reason for the odd results; the high background
being reduced in the presence of insert really points to that as the
explanation.

I don't know why it is, but for me, anyway. it seems to go in waves; I'll
get partial cutting, even with good enzymes, for a few weeks, and then
I'll get complete cutting again.  It's not batches of enzyme or different
plasmid preps; I wonder if the incubator I'm using isn't holding
temperature properly, so I'm actually digesting at 35 oC or something.

I never used to believe you *could* get partial cutting, ever since I
tried to do a partial digest by stopping the cuts quickly and found
complete digestion within 1 (one) minute, even after diluting down the
enzyme; but there it is.  One of those annoying things.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England




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