ssDNA preparation

Rick Wilson rick at TAQ.WUSTL.EDU
Tue Mar 15 15:48:55 EST 1994


John Nash (nash at nrcbsa.bio.nrc.ca) writes:

>What the researchers did was to PEG/NaCl precipitate their phage,
>resuspend it in TE (25 or 30 ul from memory), and then boil it for 2
>min. They directly sequenced the boilate.

>Anybody tried this?

Yes, we're doing something similar.  As written, the method described in
BioTechniques wasn't all that reliable.  Seems to work well if you cook in
TTE buffer (0.25% Triton-X100 in TE buffer) at 80 C for 10 min., then spin
out the debris.  At this point, we transfer supn. to microtiter plates, add
an equal volume of water, and use 6 ul total for dye-primer cycle sequencing.
Note that this method works very well with SequiTherm, but AmpliTaq doesn't
seem to like the Triton.

My colleague, Elaine Mardis (emardis at watson.wustl.edu) has submitted a one-pager
to NAR describing the method.

Rick
****************************************
Richard K. Wilson, Ph.D.
Genome Sequencing Center
Washington University School of Medicine
St. Louis, MO   63108   USA
(314) 286-1804   rick at geneman.wustl.edu
****************************************



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