Response to ALF Discussion group

Jackson Shea sheaj at ohsu.edu
Wed Oct 19 11:48:35 EST 1994


  
<From: !igloi at oligo.biologie.uni-freiburg.de>

Contribution to ALF discussion group

In order to fill the gap created by Jackson Shea's call for contributions,
I would like to take up the two points he suggested.

1. Mini-Preps. I believe that the best mini-prep method is the one that
gives the best sequence; meaning, that the quality of DNA is not necessarily
method-dependent but has more to do with the person preparing the DNA. In 
our core facility, I have had samples from ordinary good old alkaline-lysis
giving super sequences, while some people can't even get sequenceable DNA
from CsCl gradients. I still recommend PEG pptn.(when asked) because 
relatively little can go wrong, but I have no control about what is finally
submitted for sequencing. I know many people are successful with Qiagen or
similar resins while others manage to get residual resin (e.g. StrataClean)
in their samples, which knocks out the DNA polymerase. We have had fewer
quality problems since changing to cycle sequencing as our routine 
sequencing method.

2. Other ALF applications. A recent issue of BioTechniques contained an
article from the Ansorge group abou the use of ALF for DNA footprinting.
Another application  that I know has been documented is primer extension
analysis.
I am, at the moment, adding the finishing touches to a method for
fluorescence labelling of natural RNA (as opposed to in vitro transcripts)
and have used this for looking at RNA/protein interactions by band shift
gels on ALF. Unfortunately, Alf is not designed to enable cooling of the 
gels so that such gel-retardation experiments at aprox 30 C may miss weak
interactions. For this purpose, our EMBL-ALF prototype is proving more
flexible.

Finally, may I blow my own trumpet by reminding ALF users of their favourable
position (compared to ABI users, without wishing to start a major conflict)
in being more readily able to use labelled primers in primer walking
projects. We do not have to rely on the use of dye terminators, which, I
have been told are expensive and give relatively short reads - (by the way,
has anyone used dye terminators on ALF; and if so where does one get the
fluorescein-labelled terminators from ?) - but have the option of using
fluorescent phosphoramidites in primer synthesis or internal labelling with
FdA. Alternatively, and paricularly for cycle sequencers, I would like point
out the possibilty of the enzymatic addition of the fluorescent label to 
pre-existing primers (BioTechniques 15, 486-497, 1993).



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