We exclusively use the ABI BigDye Terminators and are happy with the
results. We have found two methods of cleanup which give no blobs in
your sequence ladders. These are the traditional G50 cleanup. We don't
use expensive Centrisep columns but a G50 microtiter plate cleanup. The
other method we use is EtOH precipitation. The trick we found to not
getting blobs with EtOH is to use a mixture of 95% Ethanol-5%
Isopropanol (Kodak IBI sells molecular biology grade with this
mixture). However, the BDT chemistry always gives blobs that start
before the sequence ladders and this confuses the start point in the
analysis software. On a standard 36 cm gel the actual sequence ladders
should start about scan 1100 NOT 950 or so where the dye blobs start.
Just my two cents.
Robert B. Chadwick
Director, Genotyping/Sequencing Unit
Division of Human Cancer Genetics
Ohio State University
420 West 12th Avenue
Columbus, OH 43210
e-mail chadwick-1 at medctr.osu.edu
"Scientists have odious manners, except when you prop up their theory; then you
borrow money from them." --- Mark Twain
Date: Tue, 24 Mar 1998 12:57:58 +0000
From: Dr Mick Jones <mjones at rpms.ac.uk>
Subject: Rx Clean-up Strategies
Recently we upgraded our 373A sequencer to a stretch and XL instrument.
We have noticed that we have a problem with reading the sequence at the
beginning of a run.
We are using Dye labelled terminators (Amersham kits recently), and
ethanol ppt after cycling before loading the gel.
Before the upgrade to a stretch we found that small amounts of
terminator not removed by pption would mask maybe the first 20 - 30
bases before we got reliable sequence. Now with the stretch we are
finding that the dye blob contamination is masking the first 20 - 60+
bases (even the first 80 bases appear messy).
One naive thiught on this problem is that the un-incorporated dyes move
through the gel as a loose band which spreads out the further it
migrates, so by lowering the detector in the stretch upgrade the dyes
spread out more and in effect cover more underlying real sequence. Is
this a feasible explanation?
The other possibility is that it is a problem with the Amersham
dye-terminator kits (but we have experienced it with ABI kits).
So what are the procedures that people use to clean-up the dye
tyerminator reactions. I should point out that after 1st April we are
switching to ABI Big-Terminator kits.
Do people use ethanol pption (with 3 M sodiume acetate [traditional EtOH
pption], MgCl2 or just EtOH or iPrOH) or should we be investing in
gel-filtration clean-up columns or microtitre plates formats.
Dr Mick Jones Tel: 081-383-3328 (+44-81-383-3328)
Department of Virology FAX: 081-383-2299 (+44-81-383-2299)
RPMS, Du Cane Road Email: mjones at rpms.ac.uk
London, W12 0NN, UK
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"Crackin' toast, Gromit." Wallace (The Wrong Trousers)