ssDNA preparation

Shiao Y. Wang sywang at whale.st.usm.edu
Thu Mar 17 00:07:39 EST 1994


Rick Wilson (rick at TAQ.WUSTL.EDU) wrote:

: 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
: ****************************************
 
Would the following be a good alternative approach?

1. Amplify insert in phage w/ PCR. Perhaps asymetric amplification to get
ssDNA.

2. Gel purify and then sequence PCR product.

I'm thinking of using this approach. Wondering if it's a commonly accepted
approach.



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