elevated baselines in raw data view (fwd)

Mr.D.J.Cain dcain at hgmp.mrc.ac.uk
Thu Mar 13 06:32:10 EST 2003

---------- Forwarded message ----------
Date: 13 Mar 2003 09:27:52 -0000
From: Martin Hirst <hirst at interomex.com>
To: bionet-genome-autosequencing at net.bio.net
Newsgroups: bionet.genome.autosequencing
Subject: Re: elevated baselines in raw data view

We had a similar problem a couple of month ago on a 3100.  We are running:

ABI V3.0 dye
POP4 with 36cm array
Ultrapid sequence (40min runs)
Cleanup with ETOH and dry 15min in flow hood

The phenotype was a high basal level, usually blue/green, which faded as the
run progressed.  Turned out the problem was with drying, for some reason the
ambient conditions had changed in the lab - we increased the dry time in a
flow hood and problem was resolved.

Your high basal level could be due to some other fluorescing contaminant
(since you are not using ETOH) -  I would look at your precipitation/
cleanup steps.

why do heat after Formamide addition?




Martin Hirst Ph.D

Senior Scientist

Interomex Biopharmaceuticals Inc.

On 3/11/03 12:43, "jwnagle at codon.nih.gov" <jwnagle at codon.nih.gov> wrote:

> 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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