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RESEARCH DIVISION Laboratory Manual

 


 

Labeling of Oligonucleotides

  1. Combine:
    1ul (50ng) of oligo in TE
    0.5ul Denaturing buffer
    3.5ul DDW
  2. 2. Incubate for 5min 70C
  3. Quench on ice and pulse spin to remove condensate from lid.
  4. In this order add:
    1.25ul 10X kinase buffer
    0.5ul 100mM DTT
    5ul gATP32 (10uCi/ul)
    0.75ul polynucleotide kinase.
  5. Incubate for 60min 37C.
  6. Add:
    36ul TE
    50ul 4M NH4 acetate
    1ul 10mg/ml tRNA
    200ul ethanol
  7. Vortex and spin 10min.
  8. No 70% rinse. Resuspend in 100ul TE and count 1ul.

Denaturing buffer:

100ul 2M tris HCl pH 9.5
100ul 100mM spermadine
2ul 0.5M EDTA pH 8.0
798ul DDW

10X kinase buffer:

250ul 2M Tris HCl pH 9.5
100ul 1M MgCl2
150ul DDW
500ul glycerol
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