Pharmacia ALF problem

Christoph W. Sensen sensencw at niji.imb.nrc.ca
Fri Jan 27 09:58:07 EST 1995


lwashing at sunflower.bio.indiana.edu (Lawrence Washington) wrote:
>
> In article <t.keen-1901951651410001 at mol9.ioo.bpmf.ac.uk>, t.keen at ucl.ac.uk
> (Jeffrey Keen) wrote:
> 
> > Has anybody had the same problem as me with their Pharmacia ALF sequencer?
> > 
> > What has developed is that I am getting a very high peak of fluorescense
> > (way off scale) passing through the gel just before the primer peak.
> > Pre-running the gel appears to allow the contamination to pass through the
> > gel before loading, but still leaves a residual background of around 45%.
> > The fluorescense isn't noisy so doesn't affect the sequence (as far as I
> > can tell anyway).
> > I've stopped using our own RO water and just use Merck Analar stuff
> > without getting rid of the problem. Swapping detergents doesn't seem to
> > help either.
> > 
> > Any suggestions 


May be that I have a suggestion for you:

You are obviously introducing fluorescein into your sequencing
system somewhere. If it is not in the  chemicalss, then check out
the following potential sources:

- permanet pens (especially red ones) used to write things on
  the glass plates prior to gel-loading
- Mr. Clean and his relatives used to wash the  glass plates
  (the greenish colour is often fluorescein)
- Do you use absolute Ethanol only or is there a contamination
  in your Enthanol sources somewhere (check on UV plate)

these are the three most obvious sources of contaminations.
If you do not find the flourecein there, then check everything
else that you use on the UV plate and see wahat is lightening up....

Christoph Sensen



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