Sequencing running buffer problem?
Chia Jin Ngee
mcblab47 at leonis.nus.sg
Wed Nov 24 04:36:51 EST 1993
Hi to all in bionetland!
Some wierd things have been happening to all DNA sequencers in IMCB. Most
of the time, all our sequencing gels work, good autorads, great sequence
data etc. Then like a flash fire, it just hit all of us. Our DNA
reactions gave smears on the autorads. At first I thot it was my problem
then I found out slowly everybody is running into the same problems. Even
templates that used to work and still working gave these problems. People
have been running up to seven gels with new templates, primers even kits
and come up with the same thing. The rxn seems to be stuck on top of the
gel and fails to run in. Afters the thick band of stuck material is a
smear which disappears (also thinning) halfway down the gel.
I realized there was something we have in common otherwise it can't be
coincidence that all of us from different labs have the same problem. The
only thing I could think of during the task force meeting(Yes, it is so
serious we set up our informal task force) was the water. In IMCB we use
water from an in-house purifying system for gel buffers and washing of
glassware. We use this to dilute our 10X TBE while out 10X TBE is made
from MilliQ water. MilliQ water is too good for diluting such buffers and
is reserved for HPLC. I don't know but we just ran a gel today made with
MilliQ water for both running buffers and gel plus rxns from templates we
know works in the past to confirm my guesses. I will know tomorrow and
will post the results of this run.
Even as I type now, all sequencing jobs are frozen till the outcome of
this test. In fact frozen since the past week.
Anyone out there have seen such problems from your autorads? Is it due to
water use to make the running buffers? If so, what kind of contaminants
I welcome all suggestions. At least to satisfy the mass of angry,
fustrated lab workers and to offer some decent explanation to the building
Still with Blessings and regards after 3 weeks of sequencing mayhem.
Jin Ngee, Chia Chemical Carcinogenesis Laboratory
(Genie, the OligoMan) Institute of Molecular and Cell Biology
mcblab47 at leonis.nus.sg National University of Singapore
Tel: (065)772-3797 Kent Ridge Crescent
Fax: (065)779-1117 Singapore 0511
"I'm Mr Chia not Ms Chia and I know Genie is a girl's name"-Jin Ngee, Chia
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