Q: How doing partial RE digests?

Tim Lansing lansing~tj at glaxo.com
Mon Jan 27 11:00:11 EST 1997


In article <5cgdgp$8gd at mserv1.dl.ac.uk>, Wolfgang Schechinger
<wgschech at med.uni-tuebingen.de> wrote:

> Hi folks!
> 
> Any ideas/expericence/PROTOCOLS?? on ho to do a partial digest?
> 
> That is: I want to cut off the stop codon from a 1200 bp sequence.
> Unfortunately, this piece of DNA as two sites for MluNI (the RE I want to
> work with), of course one that I don't want to be cut. My idea on that is
> taking samples from the digest at different times, freezing them and running
> them together and then cutting out the fragment I desire. 
> Any ideas on the time scale of this? Should I chose a buffer that is not
> ideal for the enzyme to make it cut slowlier? Does the sequence pattern
> around the cut site affect the speed of being cut? Are any predictions
possible?
> 
> ANY  comments/ideas are appreciated!!
> 
> See you!
> 
> Wolfgang

What has worked well in our hands is to use the normal buffer for a
restriction enzyme and reduce the number of units to cut the DNA to
0.1-0.5 U/ug DNA.  Collect samples at various times (5, 10, 15, 30, 45, 60
min), stop the reaction with EDTA, and run out on a gel to find the time
giving the most linear DNA.  This can be scaled up to prepare more DNA.

Tim Lansing
Glaxo Wellcome R & D



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