ds DNA sequencing surefire protocol wanted.

David Matthews matthews at oyster.arris.com
Mon Nov 28 19:11:22 EST 1994


In article <jr.50.0011BCCE at dna.bio.warwick.ac.uk>
jr at dna.bio.warwick.ac.uk writes:

> Dear all,
> 
>               As the heading says it all, I'm in need of a double stranded DNA 
> sequencing protocol for DNA prepared using one of the diatomaceous earth 
> methods, that gives bands as good as M13 sequencing (or am I asking too much 
> in the quality of sequencing bands).
> 
>              Thanks in advance.
> 
> 
> Jaz

I use a protocol similar to that of Hsaio (NAR 19, 2787 (1991)) - I've
found this to be useful not only for "clean" dsDNA minipreps (e.g.
Promega's Wizard preps) but also for relatively dirty DNA (phenol
extract cells, then EtOH ppt). The sequencing protocol involves
denaturation of template with NaOH, annealing at 37 C and
neutralization with HCl. Then, you just follow your favorite enzymatic
sequencing protocol (I use Sequenase). The main critical factor is the
NaOH and HCl concentrations (each 1M) - even though the final solution
is buffered, it's important to add similar quantities of acid and base
to ensure the final pH is about 7.5

David Matthews
matthews at arris.com



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