blunting with S1 nuclease

[Chris Boyd]

rlab davis (rdlab at gandalf.bio.uci.edu) wrote:
: I am trying to blunt ligate a Bam HI and an Afl III site both of which have 
: been blunted by using S1 nuclease to remove the protruding 5' end.
: I use 10 units of the Boehringer-Mannheim S1 nuclease in the S1 buffer from 
: Sambrook to digest about 0.1 to 0.3 ug of cut DNA.  I incubate for 30 min at 
: 37 C, and then gel purify the products.  I have not had much success.  Can 
: anyone recommend any changes in my protocol, or an alternate method?  I need 
: to remove  rather than fill in the overhanging ends to get the junction I want.

You could try Mung bean nuclease instead of S1.  The latter is supposed
to be more prone to cleaving at a nick, and in my experience causes a
lot of grief.  I think it also tends to get a bit carried away with
chewing back and leaves ragged ends, something you wouldn't necessarily
notice on a gel.  Look in the NEB catalogue for a good write up on
Mung. (No, they don't pay me.)

Best wishes
--
Chris Boyd          | from,  \MRC Human Genetics Unit / Western General Hospital
chrisb at hgu.mrc.ac.uk| not for \        Crewe Road / Edinburgh EH4 2XU / Scotland