strange cloning result

Hiranya S. Roychowdhury hroychow at nmsu.edu
Tue Mar 28 13:24:31 EST 2000


Hi Ian,
        You are probably correct; but some enzymes are notorious in that
they require "too-many" bases to latch on. Thus when the sites are close
together such RE's pose that problem. AFAIK, RI and Xb are can work even
with just one extra base on either side. 
Anyway, that is why I am such a die-hard advocate of v~OH. It eliminates a
lot of "whats" and "hows" from my already over-crowded schedules.

At 05:41 PM 3/28/00 GMT, Ian A. York wrote:
>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
>


Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

---




More information about the Methods mailing list