Subcloning oligos into expression vectors

David H. Gorski d-gorski at uchicago.edu
Mon Jan 20 12:10:43 EST 1997


In article <32E382DF.2DD9 at sheffield.ac.uk>, Kevin Mulcahy
<K.Mulcahy at sheffield.ac.uk> wrote:

> it is possible that you are getting incomplete digestion of the vector
since the 
> Nhe I and Bgl II sites are very close together. For efficient cutting
some enzymes 
> require quite a bit of flanking DNA sequence so that the enzyme molecule can 
> attach and recognise the actual cut site. Therefore, if for example Nhe I
requires 
> at least 10 bp either side of its actual recognition sequence (i.e. not
including 
> the recognition sequence GCTAGC) but the length of intervening flanking
sequence 
> from the Nhe I cut site to the Bgl II cut site is only 8 bp then any vector 
> molecules previously cut with Bgl II may not be subsequently cut with Nhe
I. You 
> would therefore end up with many vector molecules which were just singly
cut with 
> Bgl II which can therefore self-ligate.
> 
> You may therefore have to use much more of each enzyme and incubate for much 
> longer in order to ensure sufficient digestion by both enzymes. If you
still get 
> self-ligation (indicating incomplete digestion) you could try
CIAP-treating the 
> vector molecules such that self-ligation cannot occur. In this way, vector 
> molecules will only re-ligate if they contain your insert. If this still
doesn't 
> work then you may have to try different restriction enzyme sites which
require 
> much smaller flanking sequences.

Thanks for the tip. Oddly enough, though, the NEB catalog doesn't state
that you need that much flanking sequence for Nhe I to work. I'm going to
try to digest with Nhe I and Bgl II for several hours and see if that makes
a difference.

-- 
David H. Gorski, M.D., Ph.D. |"What is man, when you come to think
Department of Surgery        | upon him, but a minutely set, ingenious
University of Chicago        | machine for turning, with infinite
                             | artfulness, the red wine of Shiraz
E-Mail: d-gorski at uchicago.edu| into urine?"--Isaak Dinesen



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