Attn: DS DNA Sequencers

aroger at ac.dal.ca aroger at ac.dal.ca
Fri Nov 12 10:05:17 EST 1993


Here is a summary of my luck with ds DNA sequencing:

1) I routinely get better sequence from a magic miniprep than from a 
straight alkaline lysis+two phenol/choroform extractions (I cannot
compare QIAGEN because I haven't tried it yet)
2) I use about half of a standard miniprep to sequence with
3) The most important thing in ds sequencing is the strain of E.coli
one uses: endA- strains give reproducibly better sequence (fewer pauses
and less dark smeared background).  But some of these strains are
sicker and the time of growth must be carefully monitored..ie-
XL-1 blue should be grown for less than about 10hours in a two ml
culture from a single fresh colony innoculum in 2yt or LB medium 
(100ug/ml ampicillin).  Otherwise, cell lysis seriously comprimises
DNA purity.
4) If there are problems with pausing on a template, these can often
be cured by doing two things:
a) reduce the extension time to 2mins or less
b) dilute the labelling mix 1:20 rather than 1:5
(I'm talking about the USB sequenase version 2 kit).

Those are the most important things that I have noticed about
double stranded sequencing.  I always get strong bands after
only a single night's exposure on fast film (s35 of course).

Andrew Roger
aroger at ac.dal.ca



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