ALF Woes!

Burkhard Hassel bhassel at orion.rz.mdc-berlin.de
Fri Jul 7 11:56:32 EST 1995


In article <DBC97M.MG at news.tcd.ie>
dbarton at acer.gen.tcd.ie (Dr David E Barton) writes:

>  We have a problem with our Pharmacia ALF sequencer that is proving very
>  difficult to resolve: The intensity of the signals falls off from left to
>  right accross the gel AND with increasing fragment size.   (...)

Dear Dr Barton!
The first problem could have something to do with the
focusation or vertical adjustment of the laser, so the left
diodes on the left side ("clone 1") get more radiation than
the ones on the right.
Or --- the gel is just turbid. 
Tried a run without loading probes?

Second, we had also had the problems of decreasing peak
height with increasing fragment length. It turned out, that
it was a problem of the gel chemistry:
The peaks of the larger fragments were not as good resolved
as the small ones. This led to wider but smaller peaks
(with the same area under the peak - sequencing reactions
were ok) and gave the impression of falling lines from
small to large peaks.
In our case, it turned out that it was the APS. We now make
a fresh Eppi full of APS for every new gel.
Our Gel: a 6 percent long-ranger (!) hydrolink Gel
solution. Its from
AT Biochem and distributed by Serva.
We obtain about 300-500 bp from minipreps and 400-650 bp
from maxis.

If you like to have more infos, drop a line and perhaps one
can exchange some examplary runs via data wire.

Yours
Burkhard Hassel (bhassel at orion.rz.mdc-berlin.de)
MDC
Berlin



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