ALFexpress Automated Sequencing

[Bill]

     Hi Stephanie,
                      Firstly, the only way to get high definition 
sequence from 
     PCR products is to use Solid phase  (Pharmacia Combs/ Dynal Beads) 
or Cycle 
     Sequencing with the Tabor-Richardson enzymes such as 
theremosequenase, 
     along with a high definition Termination mix. If youn are getting 
random 
     insertions/deletions etc, then there is a problem with the 
gel/sequencing 
     chemistry. The ALF Express is capable of excellent mutation 
detection. 
     Please can you send me a data file so I can assess the situation. I 
am the 
     Pharmacia Applications Specialist fot ALF/ALF Express. MY e-mail is 
as 
     follows:
     bill.vallins at eu.pnu.com
     Fax Number: 0044 (0)1727 814 001
     Tel No. 0044 (0)1727 814000 X 4069
       Please can you compress the file using ARJ/WinZip etc. 
     
        Bill

______________________________ Reply Separator 
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Subject: ALFexpress Automated Sequencing
Author:  Stephk at niv.ac.za at Internet-Europe
Date:    12/06/97 06:22


Hi there,
     
We are using the ALF automated sequencer from Pharmacia for more then 
a year now and we have encountered various problems with the machine. 
The biggest problem though are the random mutations that we are 
getting frequently which are not supposed to be there. 
Various people at our institute are sequencing different viruses (HIV, 
Flu, measles, HHV8 etc) using different methods (such as different 
cycle sequencing kits from Pharmacia or Amersham, the AutoRead Kits 
from Pharmacia or the AutoLoad Kits from Pharmacia for sequencing of 
PCR products...). 
All of us are getting deletions and insertions and random point 
mutations where we know there shouldn't be any (confirmed by manual 
sequencing etc.).
     
Our question is whether anybody has ever experienced anything like 
that using the Pharmacia ALF sequencer??? 
We would really like to know whether this problem only occurs on our 
individual machine and with our reactions or whether it is a common 
problem.
Thanks....
     
Stephanie Kreis
National Institute for Virology
Private Bag X4
Sandringhma 2131
South Africa
Email: stephk at niv.ac.za