pol I, Klenow, Terminal Transferase
pha23 at cc.keele.ac.uk
Wed Feb 16 09:22:25 EST 1994
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?
Any advice very gratefully received!
Trevor Forsyth, Physics Department, Keele University, U.K.
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