Double stranded DNA sequencing

[Susan L Forsburg]

>RICHARD P. PHIPPS (PHIP at BPHVAX.BIOPHYSICS.ROCHESTER.EDU) wrote:
>: I am currently attempting to sequence double stranded plasmid DNA
from
>: minipreps using the USB sequenase 2.0 Kit.  Unfortunatly after
trying several
>: DNA isolation methods that I could think of or find I am not
having good
>: results in that > 70% of my reactions produce either no bands or
bands in all
>: four lanes all the way up the gel.  I am interested in a miniprep
DNA isolation
>: technique (Kit or otherwise) that reliably yields 50% or greater
readable
>: sequencing reactions.
>
>: Bob Burns

I have always had difficulaty with "standard" ds sequencing. 
Recently I had excellent luck using a PCR based sequencing kit from
USB called Delta-Taq cycle sequencing, with cycled labelling
protocol.  The sequence looked like single stranded, completely and
gorgeously readable.

Usual disclaimers,

susan

------------------------------
Susan L Forsburg
Molecular Biology and Virology Laboratory
The Salk Institute for Biological Studies
email:  susan_forsburg at qm.salk.edu
        formerly forsburg at molbiol.ox.ac.uk