Dear Newsgroupers,
Has anyone out there ever seen anything like what has affected lanes 29-32
of this gel (I've placed two pictures of it in David Cain's FTP site. One
basically shows the beginning of basecalling, while the other is further
into the run)? Here are your clues:
1.The samples were user-prepared; all I got were the pellets.
2.Previously the client had submitted these samples and got very low
signal
(ie., 20-50) for all 4 bases...but I could see a very faint outline of
called bases, so since her stuff looked as though it would have otherwise
worked, I advised her to resubmit after using 2X-3X more template--while
leaving her other reagent concentrations the same (except H2O, of course).
3.The Premix she used is of dubious origin. I sell ABI FS Premix (I have
a
373 Stretch), but the client also frequents another Core on campus that
has
converted to the new Dichlororhodamine dyes, which ABI warns are
incompatible with 373s. The Premix is no longer in the tube that it was
sold in (her lab immediately aliquots theirs out into PCR tubes in 8ul
quantities and freezes them). I don't know what the dichlororhodamines
look like on the gel image of a 373. Do you?
4.According to her, they use the oil-less PE 9600 for cycle sequencing and
she uses ETOH ppt.'n and the speed vac to dry things up. I asked about
this because a green haze can be caused by ETOH.
5.Oddly enough, at least 3 of these lanes actually had surprisingly clean,
readable data to 200-350 or so b.p.'s even though the green haze seems to
cover everything. I had a similar experience with Amersham's ET primers
when they 1st came out, except for that the gel image where they were
loaded was intensely blue and obscured all bases. Like this situation,
there would've been no way I would've known if things tracked correctly or
not...but the data were beautiful and even. The next person who used that
kit had lanes that looked normal. I didn't create a new ET matrix for the
new guy's stuff, so I concluded that it must have been a chemistry issue.
Someone else in this newsgroup had the same problem at the same time that
I
did.
So, the evidence has been presented. Thanks, all, for your help with this
matter.
FTP Site details:
address:venus.icr.ac.uk
login name:anonymous
password:your at email.address
directory:pub/autoseq
filenames:373 Who Done It? and 373 mystery lanes
(The files are screen shots and will open with simpletext. I put them in
bin format.)
--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
