pol I, Klenow, Terminal Transferase

Robert Horton horton at molbio.cbs.umn.edu
Fri Feb 18 15:10:04 EST 1994


V.T. Forsyth (pha23 at cc.keele.ac.uk) wrote:
: Has anybody got any advice on the optimal use of pol I, Klenow and terminal
: transferase for the purposes of producing long polymeric DNA with simple
: repetitive sequences(they are for diffraction studies)? We have tried a
: number of reactions in which we have varied pH (using either KP or Tris) and
: seem to be able to get reasonably efficient usage of dNTPs but the whole
: thing appears to be a bit unpredictable and we find that we have to vary the
: conditions as soon as we change to a new batch of dNTP or enzyme. We are
: also using DTT and BSA in these reactions but are not at the moment
: convinced of the advantages. What is the best way to maintain sterility in
: the reaction mixture over the long incubation times necessary?
:

Why not try the method of Rudert, W.A., and Trucco, M. (1990) NAR 18 (21):6460?
This is a PCR-based method for generating repetitive sequences. It will
automatically heat sterilize your sample!


Bob Horton (Ph.D.!)   /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
St. Paul, MN 55108    ^^   horton at molbio.cbs.umn.edu/(612) 624-3790



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