What's wrong with my DNA sequencing template?

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Fri Feb 24 04:10:04 EST 1995


In article <binnie_betten-2302950852540001 at dipietro.dept-med.pitt.edu>, binnie_betten at paccm.pitt.edu (Binnie Betten) writes:
>This is my first attempt at sequencing, and although I get beautiful
>sequencing info from the control DNA that comes with my sequencing kit, I
>get absolutely no bands of any kind from my single stranded sample DNA.

It looks like you have the "wrong" strand for your primer ( or vise versa),
since many of the packageable plasmids have an f1 ori position, which results in
single strand opposite to that of M13. Try "reverse" primer on your DNA, if
the gel is blank again, you must have isolated the helper band from your gel
( note: you do not normally need such a complex purification as you did ,
and there are some good protocols for sequencing dsDNA; these days with
Sequenase it is quite straightforward) Cheers,

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
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CH-8092 Zurich
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 are excluded from our circle of comprehension" - Kozma Prutkov



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