The 48cm. Debate/MitochondrialDNASeq.

Patricia Thomas thomas at pharmdec.wustl.edu
Tue Oct 14 09:57:40 EST 1997


The 48cm.Debate

I've been following the 48 cm. Long Ranger discussion and it's news to me
that it doesn't work right!  I don't routinely use 48cm. plates, but after
optimizing my 34cm plates w/Long Ranger I decided to see how far one could
really read out to on a 373 Stretch.  These are the conditions I used with
my 48cm plates:

2500V, 40mAmp, 40watts, 5%LongRanger gel for 12 hours

Except for the run length (use 14-16 hrs!) this was ABI's recipe for the
373. It's from their R&D lab (I visited in April) and is not in any of
their "official" literature to my knowledge.  You may note that ABI's watts
differ from FMC's recommended 31 watts.

The run was the best that I'd ever seen...unfortunately, I forgot to
instruct my collection computer to collect for 14 hours so I don't know how
far things would have gone before I experienced a loss of resolution.  But
every base was represented by a peak.  At base 560 I had a run of 5 A's and
each one had a representative peak--not the usual "hump" that you see at
this point on a 373 run.  Data collection truncated around base 600 and
every P.I. that I showed the data to felt that the data until that point
was unassailable.  So....what's the catch?  I used Promega-made PGEM and
-21 M13 Rev. primer on this run.  So, I had the advantage of no interfering
background noise.  ABI warns that Gumby users will not see this kind of
read.  In other words, garbage-in-garbage-out.  Unless you have a
total-control Core you may have problems getting this length of read for
your users on a 373.

Finally (phew) I assume that you've gone to FMC's website and/or called to
complain.  As a last resort make them send you a 48 cm.Long Ranger Singel
Pack so that you can r/o reagent problems!
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Mitochondrial DNA Sequencing

I don't know if you have this already or not, but ABI has this interesting
little manual on mitochondrial DNA sequencing.  It gives 5 different
sequencing strategies and discusses some of the common problems encountered
when sequencing through this stuff.  Emphasis is on getting through the
"D-loop" control region, but there are references given that might help.
Call your application specialist for a copy. Good luck!!
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...And a question of my own...

Has anyone tried running MacOs 8 with Sequence Analysis version 2.1.2?  I
called tech support and they told me that they hadn't tried it yet
either...looks like it would be a good thing for us sequencers to upgrade
to if it's compatible with our software...

                                                                --Patricia
Thomas




Patricia Thomas
Washington Univ. School of Medicine
Department of Molecular Biology & Pharmacology
660 South Euclid
Box 8103
St. Louis, MO  63110
thomas at pharmdec.wustl.edu
Lab address:
        Dr. Kerry Kornfeld's lab
        Cancer Research Building, 3rd floor
        (314)747-2998






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