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RESEARCH DIVISION Laboratory Manual

 


 

Butanol Precipitation of Deprotected Oligonucleotides

Crude oligonucleotides can be precipitated after deprotection directly from the ammonia solution without the need for a separate step to remove the ammonia. This procedure removes small molecular weight impurities such as the ammonia itself and the by products of deprotection (benzamide, isobutyramide). It is taken from a published method (M. Sawadogo and M.W. Van Dyke, NAR 19(3), 674 (1991)).

  1. The ammonia solution of the deprotected oligonucleotide (100l) is mixed with 1 mL of n-butanol (ACS reagent grade) in an Ependorf tube, is vortexed for 15 sec, then centrifuged for 1 min at 12K.
  2. The H2O-containing butanol is removed and discarded. The pellet is redissolved in H2O (100l) and re-extracted with butanol as above.
  3. The final pellet (which may or may not be visible) is then dried under vacuum (desiccator or lyophilizer) and resuspended in TE
  4. The amount of oligonucleotide present should be determined at this stage - you will need to do a 1 in 30 to 1 in 50 dilution for this (from a 0.2 mmole scale synthesis). Typical yield (for a 25mer) is about 50 g per 100l of ammonia solution.
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