sequencing problems w/ ALF II automated sequencer

Alexander Kraev kraev at bc.biol.ethz.ch
Fri Apr 4 08:30:22 EST 1997


In article <19970401022001.VAA18327 at ladder01.news.aol.com>, gizbab at aol.com
(Gizbab) wrote:

> Hi!
> 
> I wonder if anyone has run into this problem.... I have had some problem
> with the ALF II automated sequencing machine. Namely, when I work with
> cell lines, the sequences come up fine, yet when I use tissues extracted
> from paraffin, and sequence the stuff for p53 mutations (homo and
> heterozygous), I get some strange results, if any! It got to the point
> htat I simply took some of the cell line DNA, amplified it for one of the
> p53 exons, and cycle sequenced it all in one reaction tube, and then ran
> the product in all lines of the machine and actually found different
> results in most of the cases! I really have little problem sequencing the
> conventional way (radioactive) and get great results that way. Any help
> for user of the ALF II will be very much appreciated.

Although you are not sufficiently specific here, you problem may be in the
difference in yield you get from your PCR reactions.  Theoretically (and
in practice as well) all non-radioactive methods, and all automated sequencers
are sensitive to the fluctuations in the template amount.  You need to be
within one order of magnitude with your sequencing signal to get the best data.
Quantitating your template by fluorimetry eliminates most, if not all,
surprises.
You should use a defined molar amount of template in sequencing (for the
ALF between 0.1 and 0.3 pmol if you use cycle sequencing, and 5-10 pmol in non-
cycled reactions).  This is tedious, but  with the kind of samples you
have quantitation is a
must. In other cases sets of similar samples, i.e. plasmid minipreps from
one cloning
experiment do not require such an extensive quantitation (one to three
random tests are
enough). 

The cheapest fluorimeter on the market at the moment seems to be the Dynaquant
from Pharmacia. The investment is really worth the money (no affiliation)!
It also helps to ensure reproducibility of many other DNA experiments, and
actually changes your mentality towards being more "quantitative".

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch



More information about the Methods mailing list