answers to Francis' questions

Rick Wilson rick at GENEMAN.WUSTL.EDU
Mon Oct 26 08:36:53 EST 1992


Hello all,

Francis Ouellette (francis at monod.biol.mcgill.ca) had some additional
questions about the Sequenase terminators:

>In the Lee et al., (199*2*) paper that Bruce Roe reffered to
>(NAR 20:2471-2483), you talk about working on the alkaline denaturation
>of dsDNA to use with the sequenase.  As anything come out of that?

You can do it, but the data is ugly; not good enough for de novo sequencing.
We either recover ssDNA from phagemid clones, or treat PCR products with
T7 gene 6 exonuclease to produce ssDNA.

>Do you now do most of your sequencing with ssDNA and sequenase, or do you
>still use the TAQ cycling dsDNA often?

In our project, 90 percent of sequencing is done with M13 universal
primer and Taq cycle sequencing.  We use the Sequenase terminator chemistry
to walk.

>In a slightly different area, I once read a note from
>your lab in some ABI propaganda bulletin, and in it you indicated
>(if I remember correctly) a 2 phase shotgun cloning approach to cosmids.
>5-7 kb inserts used to link the 1-3 kb based-contigs which had been read
>from one end. Do you still do this?  At the yeast genome meeting I was
>just at the "fastest" sequencer were just doing the 1kb shotgun method
>and closing up contigs with TAQ cycling.  What are your comments about
>that?

We shear cosmid DNA using a French Press and 750 psi.  This produces
fragments of 1-9 kb.  We subclone 1-1.6 kb fragments in M13 and 6-9 kb
fragments in pUC118.  Typically, we assemble about 750 clones; this is
a mix of about 600 M13 clones and 150 pUC clones (sometimes more, sometimes
less).  pUC clones may be sequenced with both universal and reverse primers
to provide contig linkage.  Appropriate M13 clones may also be sequenced
with reverse primer (after PCR).  Is this method fast enough?  You tell me:
this month we finished a 43 kb cosmid insert in three weeks.  We now have
1 Mb finished and we're happy with the strategy.

>Excuse those of us that are not as close as you to R&D :), but what
>is QC?  Quality Check?

Quality Control.

Hope this helps!

R. Wilson
St. Louis




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