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ALf express

Dr Toni Whistler TONIW at niv.ac.za
Sun Mar 17 05:54:19 EST 1996

We have just recently purchased the ALF-express and I am a little at 
sea as to getting things going. The machine is running well using 
cy5-labelled primers  and sequencing off plasmid. The majority of the 
sequencing done in this department is off PCR products, using the 
incorporation of cy5-dATP. 
My main complaint so far is the very poor literature that goes with 
this machine .... protocols are scarce.
I have several questions.
1) Sequencing PCR product, I need to make it ss-ed. Asymmetric PCR or 
ExoI digestion.
2) Clean up of ssDNA, what is the best method.
3) What sort of concentrations of template (450bp to some of 1600bp) 
is used, what are the primer concentrations.

I would appreciate any and all advise. If anyone has protocols they 
use in their lab, these would be most appreciated.


    Toni Whistler, PhD
    Department of Molecular Virology
    National Institute for Virology
    P Bag X4 
    Sandringham, 2131
    South Africa

E-mail: Toniw at niv.ac.za
Tel:    (011) 882 9910
Fax:    (011) 882 0596


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