PCR templates on Automated DNA sequencers

Elizabeth Boulding boulding at uoguelph.ca
Tue Feb 20 08:58:19 EST 1996


  We have a brand new A.B.I. Prism 370 automated DNA
sequencer and are just beginning to use it for direct
sequencing of many different DNA templates generated by PCR.
We have noticed that some PCR templates sequence much
better than others.  (We are using cycle sequencing and dye terminator
chemistry and are gel isolating all our PCR products before 
sequencing).
  I was wondering to what extent other laboratories use asymmetric
PCR (or other methods) to make single stranded templates before
sequencing on a A.B.I..  Do you find this is only necessary for heterologous
(=universal) PCR primers that do not match the template too closely
or do you find it works better for all PCR primers?  
  Thank you,
  Elizabeth Boulding
  University of Guelph




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