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Rx Clean-up Strategies

Dr Mick Jones mjones at rpms.ac.uk
Wed Mar 25 10:34:12 EST 1998


Hi

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.

Many thanks

Mick
_____________________________________________________________________
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
URL: http://www.rpms.ac.uk/departments/virology/virolhome.html
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