jgraham at bronze.ucs.indiana.edu
Fri Nov 13 10:06:31 EST 1992
In <email@example.com> bhjelle at carina.unm.edu () writes:
>I encountered an impressively high level of mutagenesis (14
>errors out of 411bp) in amplifying a segment of DNA from
>a plasmid clone. All mutations involved T or A in some
>manner. 11 were point mutations (transitions and
>transversions), but 3 were 1 bp insertions or deletions.
Very interesting and disturbing. We routinely generate templates for in vitro
transcription via PCR using a protocol (Higuchi et al. NAR 16 (15) p.7351)
recomended by a well-known colleague.
This procedure uses plasmid targets at 200-400 ng/ rxn. with dNTPs at 0.175 mM
and a mere 16 cycles. The low cycle number is suggested in order to
reduce the chance of errors generated by Taq. I obtain about 1 to 5 ug of
500 bp fragments from target plasmids around 3 Kb.
In the NAR paper describing this technique, Krummel and associates report that
they have not seen a single base change in preparing 10 different transcription
templates in the 500 bp size range (p. 7359 line 14).
Are you using a significantly lower dNTP concentration ?
Indiana Univeristy -Bloomington
......................."Just saying NO to kits"................................
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