cycle seq reply to B.L. Cohen (e-mail to U Glasgow failed)

jmv at canctr.mc.duke.edu jmv at canctr.mc.duke.edu
Fri Jun 4 14:54:00 EST 1993


Date: Thu, 27 May 1993 16:28:20 EDT
From: jmv at canctr.mc.duke.edu
To: GBGA13 at UK.AC.GLASGOW.VMS3
Message-ID: <0096D234.66524B40.20680 at canctr.mc.duke.edu>
Subject: Re: cycle sequencing with 35S??

Thanks for the reply, BL, but I didn't originate the question..
Actually, I played with the kits & 33P for weeks to optimize the rx.
for our primers and cosmids. To improve product yield (& decrease exposure
time), try doubling the amount of 33P added to the end-labelling rx; let
the rx incubate at 37C for longer times (45 min.); lessen the stringency
of the seq. rx (eg., decrease the annealing temp. of the PCR-machine cycle,
increase the number of cycles). If individual weak ddNTP lanes are usual,
either your batch of d/ddATP is bad or something is wrong that might be

Press RETURN for more...

MAIL>
    #12         27-MAY-1993 22:34:29.34                                     MAIL
corrected by doubling rx. volumes (pippeting of the larger reagent volumes
may even out inconsistencies). (yes, i've done this too.) I always put the
film cassette containing 33P gels at -70C overnight (peeling the Saran wrap
off the dried gels doesn't seem to improve the signal as it does for 35S).

Our attempt with 35S incorporation using cycle seq. (Promega kit) was so
brilliant that we couldn't read the seq.: a few hrs.' reexposure at RT
was sufficient to seq. these plasmids.

For seq. of PCR products, i find that DNA:primer ratios are sensitive enough
that if you can't measure out the precise amount of fmols, the rx won't work.
There is a window of template concentration (all templates we've tried) out-
side of which the rx just dies. When seq. agarose-purified PCR prods., I try
a range of volumes, & usually get a legible seq.

Good luck!

--DBLoeb
___Duke U Med Ctr//Neurology



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