Sma chewing back - suggestions?
Dr S T May
lsrei at csv.warwick.ac.uk
Wed Aug 10 17:24:07 EST 1994
I need to use Sma I to cut a vector that has been partially
digested (by the EtBr 'one-cut' method) with Sca I and then
cleaned up before cutting.
This vector will then be circularized/self-ligated at the
two different blunt ends (no need to regenerate a site).
The Sma I MUST not digest back from the cutting site.
I have done this experiment twice now:
Each time, the partial digest has worked a dream, BUT
although the Sca I half is perfect (by sequencing), the
Sma I half has deleted (cut back) a variable amount of DNA
commonly >70bp (the next testable site (pst I)).
So the question:
ARE there any conditions that reduce Sma I's inclination to
'chew-back' from its site
I have 'anecdotal' evidence from other people that Sma I is
prone to ragged ends - but the catalogues all say 95%
ligation and recutting of site with their enzyme.
I AM using the recommended buffer and
I AM doing the digest at 25 C NOT 37 C
Any help gratefully received - otherwise I'll have to use
adaptors/linkers, the sites are quite 'tight'.
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