ALF Woes!

[Ari-Matti Sar‚n]

In article <DBC97M.MG at news.tcd.ie> dbarton at acer.gen.tcd.ie (Dr David E Barton) writes:
>From: dbarton at acer.gen.tcd.ie (Dr David E Barton)
>Subject: ALF Woes!
>Date: Fri, 7 Jul 1995 09:04:33 GMT

> 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. 
<SNIP>

SOME fall is inevitable in both cases... 

In the left-right case you loose signal, because the intensity of the laser 
drops as it travels through the gel. We commonly have about 5 percent units 
lower signal at clone 10 than clone 1 (i.e. baseline is 25% in clone 1 
and 20% in clone 10). 

If you take same amount of DNA in ug:s, you naturally get fewer 
molecules and thus less label as insert size goes up. We usually use 5 ug of 
DNA for template sizes (total sizes with vector, that is) up to 5-6 kb, and 
then start increasing the amount of template. For example with total size 
of 15 kb you need closer to 10 ug of DNA. 

So how big a drop are we talking about anyway? What's the baseline at the 
start of the run? What is the average diffrence with the baseline and peaks?

Maybe you could mail me personally so we could discuss this in more detail.

Ari-Matti
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Ari-Matti Saren, Institute of Biotechnology, Helsinki, Finland
E-Mail: saren at operoni.helsinki.fi, tel + 358 (9)0 434 6094
s-mail (home): Ohjaajantie 18 A 2, 00400 Helsinki, Finland
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