elevated baselines in raw data view

Brynn Watson Brynn_Watson at hc-sc.gc.ca
Wed Mar 12 08:34:18 EST 2003


We have also noticed similar baseline issues with our 2 3100's, especially
in the last few months.   Our protocols are similar, except that we do not
use SDS, we denature the samples for 5 minutes instead of 2, and we run
them out of sterile distilled water most of the time (we use formamide when
evaporation may be an issue).  One of the machines is running POP4 & an 80
cm array with 1/2 strength reactions, and the other is running POP6 & a 50
cm array with 1/8th strength reactions. The most recent development is
baseline that gets progressively higher toward the end of the run, but
unlike all the other baseline issues, it has only been observed on the
machine with the 50 cm array.  We have also been unable to track down the
problem, so any input would be very helpful.

_________________
Brynn Anne Watson
DNA Core Facility
Canadian Science Centre for Human and Animal Health
1015 Arlington St., Suite H3390
Winnipeg, MB   Canada R3E 3R2
Phone: (204) 789-6035
Fax: (204) 789-2018
Email: brynn_watson at hc-sc.gc.ca



                      jwnagle at codon.nih
                      .gov                      To:      autoseq at net.bio.net
                      Sent by:                  cc:
                      owner-autoseq at hgm         Subject: elevated baselines in raw data view
                      p.mrc.ac.uk


                      03/11/2003 02:43
                      PM






First-timer in this group, long-time sequencer,

We have 3 ABI 3100s in our Facility. We have recently observed some
problems
with our sequencers. In the raw data view, many samples including our own
pos
controls and ABI standards show elevated baseline levels of signal. The
effect
is highly variable from sample to sample, run to run and machine to
machine. In
some cases the phenomenon is of what has been termed "waterfall" in which
the
initial level is high for a period of perhaps 2500 scans and then drops
precipituously which creates drop-outs and miscalls in the chromatograms.
In other
cases the elevation follows a smoother curve from an initial area of
elevation
before reaching a normal level. In some cases, the G and A baselines
(green/brown)
levels are slightly but significantly elevated over over the red/blue
scans.

Formamide, buffer, arrays, polymer have all been systematically changed
without eliminating the problem.

We run BDT v3 or 3.1 at half strength, denature the samples with 2 ul of
2.2% SDS (95 degrees, 5 minutes) then
use sephadex G50 superfine to purify samples as post-reaction clean-up.
Dry the samples down in a spin-vac, resuspend them in Hi-Di formamide.
Heat at 95 for 2 minutes, put em on ice, load the treys onto the 3100s.

any thoughts or suggestions much appreciated

-jim  nagle
NINDS DNA Sequencing Facility
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