elevated baselines in raw data view

jwnagle at codon.nih.gov jwnagle at codon.nih.gov
Tue Mar 11 15:43:00 EST 2003


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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