Rnase T1 digests

Matt Thomas Thomas at molbio.uoregon.edu
Wed Apr 23 05:15:07 EST 1997


I need to cut an in vitro prepared transcript with RNase T1.  The goal
with this was to cut to completion and anaylize the products on a high
percentage gel.  The problem I'm having is getting the dang enzyme to
cut completely and doing it in a buffer that doesn't make the high
percentage gels all ugly. The crazy idea is too look for G's which
aren't supposed to be there.  But I doubt to many of you really want to
hear the sob story of why I'm doing this.  

My first attempts at this, I used a buffer recomened by people in my lab
who use T1 for protection assays.  This was less the wonderfull.  Does
anyone out there have a protocol that has the activity of T1 optimized
for complete digestions?  Also, I'm useing BRL Rnase T1, is this the
best one out there or can someone recomend a differenct supplyer.  It
seems to me that this enzyme is fairly dirty (at least my + enzyme
pellets are really ugly compared to -enzyme pellets) and that may be
giving me trouble on my gels also.  

thanks for any advise or help.

______________________________________________________________________
Matthew Thomas,
Institute of Molecular Biology, University of Oregon
Thomas at molbio.uoregon.edu

Science is a cross dressing dominatrix.
--a fellow graduate student
------BEGIN GEEK CODE BLOCK-----
  Version: 3.1
GS $ d-(--) S: a- C+>++ U@ P? L E? W+ N+ o? k? w--- O M++ !V(--) PS+ PE
Y+
PGP? t(++)@ 5+++ X+(++) R tv+() b+++ DI++ D+ G e++$>++++ h- r++ Y+
------END GEEK CODE BLOCK------



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