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RESEARCH DIVISION Laboratory Manual

 
 

ALF Sequencing with Fluorescein Labelled Nucleotides

  1. Denature, precipitate and anneal primers to template DNA by standard method.
  2. Add 2µl of labelling mix to the annealing reaction and begin labelling reaction by adding 2µl of diluted T7 polymerase. Incubate 10 minutes at 37°C.
  3. After the labelling reaction is complete, add 1µl extension buffer and 3µl DMSO. Proceed to the termination reactions as per the standard protocol.
Fluoro nucleotides for non-labelled primers.
- fluoro-dATP labelling mix:
- 10µM fluorescein-15-dATP
- 1µM each dCTP, dGTP, dTTP.
- fluoro-dUTP labelling mix:
- 10µM fluorescein-12-dUTP
- 1µM each dCTP, dATP, dGTP
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998