ds DNA sequencing surefire protocol wanted.

[friedman_j at mscf.med.upenn.edu]

In article <D005uz.Kqt at arris.com>, matthews at oyster.arris.com (David Matthews) writes:
> 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
Just thought I'd second the ease and utility of this method.  Also, check your 
HCl and NaOH by mixing equal volumes and checking the pH - even though it 
should, in theory, always be neutral, it isn't.  So get a good matched set.