Single Stranded DNA Sequencing

the End jgraham at
Tue Sep 14 22:13:28 EST 1993

A particular primer-plasmid pair may sequence well when sequencing 
double stranded templates, and do so reliably. If you have a 
series of inserts downstream of a primer site, this may be the best 
first try.

In all other cases, where a variety of different primers and templates
are being examined, or if the novel sequence composes a large percentage
of the template sequence, single stranded approaches are best in the 
long run.

Don't forget the latest easy single stranded prep mentioned in 
Biotechniques. All you need to do is bring down the phage(mids) with
amonium acetate-PEG, resuspend in 400 ul of TE, and then add SDS to
0.2% and a couple units of proteinase K. Digest for 15 min at 75 C,
(yes, that's 75 C) and then add sodium acetate while the solution is
warm. Spin out the proteins, and precipitate the DNA from the supertanant
with ETOH.

No organic extractions and more reliable single stranded templates. I will
repost the reference (this summer Biotechniques, July ?) when I'm in 

Best wishes,

Still "kit-free" in 93

J. Graham
Biology and Chemistry Departments
Indiana Unviersity Bloomington

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