ALF 'Blue' sequencing

Jan Hoolwerf hoolwerf at solair.inter.nl.net
Thu Aug 13 03:36:31 EST 1998


Hallo Dr.!

Op 12 Augustus 1998 schreef Dr. Duncan Clark (2:283/211.99) aan Dr. Duncan
Clark:

 DDC> We have just started running an ALF with the blue laser with
 DDC> fluorescein labelled primers.

We used it until we bought a ALFexpress and had the "old" ALF updated to an
ALFred because we didn't want to use two different labels.

 DDC> As this is an older type of sequencer there doesn't appear to be
 DDC> anything out there on a web site about optimum electrophoresis
 DDC> conditions for best resolution and best read lengths etc.

We always used the standard instrument settings and i must admit that we hardly
experimented with them. Takes to much time and the standard setting were good
enough for us.

 DDC> Our current conditions are 6% (30:1) acrylamide, 1x TBE, 7M Urea

We got much better results when we started using ReadyMix gels from Pharmacia
although it's rather expensive compared with home made gel-mixes. A comparable
product from an other manufacturer works the same.

 DDC> and 1600V, 38mA, 45W and 50C. Run time 580 mins at 1.75 secs
 DDC> sampling time.

We used 45C and 2 secs.

 DDC> Using ThermoSequenase, dITP or deaza terminators and labelled
 DDC> primer. We can base call to 500bp at say 98% but never really get
 DDC> good base calling thereafter.

As far as i know it will be rather difficult to read further. If i remember
well we never really got beyond 600 bp apart from lucky runs.

 DDC> Currently running ALF manager v3.01 on an ancient OS2 v2.0. Does
 DDC> anyone know if this version of AM will run on OS2 Warp v3 or v4?

It does on Warp 3, at least it did over here. Keep the installation as simple
as possible imho. I must say that OS/2 v2.0 was much more stable that Warp in
my hands and i've really done my (the) best.

 DDC> I just want access to printer support and don't want to pay
 DDC> UKP2000 to upgrade to the Win95 version of AM just for that!

I can imagine that. On the other hand, the ALFwin software is not 100% equal to
the older OS/2 version. I've tried both on several data files generated with
the "old" ALF and imho the ALFwin software reads more bp's that the older
software when you have a nice chromatogram and if it's not a nice chromatogram
it often has more problems to read a long sequence.

 DDC> that some ALF Express users I know, get much better read lengths
 DDC> than I do, even if I use their run conditions/acrylamide recipes
 DDC> etc.

The signal with the new dye is better: less noise, less background, etc. So
normally you can read longer sequences.


M.v.g., Jan Hoolwerf

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