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RESEARCH DIVISION Laboratory Manual

 


 

Phage Purification with DE52

  1. Grow Fresh O/N Of Appropriate Cells (Eg. Kw251).
  2. To 10ml Lmm In A 100ml Flask Add 0.2ml O/N And A Single Plaque.
  3. Let Stand For 10min. Then Shake For 3-4hr Until Lysis Has Occurred. Add 0.5ml Chcl3 and shake for a further 15min.
  4. Spin To Remove Debris And Use 0.5ml Of Lysate For Dna Prep.
    Prepare slurry of DE52:
    12g DE52 plus 36ml 50mM HCL (conc. is 8.7M) make about 28ml final slurry.
    Add conc. NaOH to pH 6.8.
    Let settle and remove s/nAdd several volumes of LMM and let settle.
    Remove s/n and repeat 3X
  5. Add 0.5ml lysate to 0.5ml of well mixed slurry. Shake X20 and spin out DE52.
  6. To S/N Add: 25ul 10% Sds 10ul 0.5m EDTA. Let Stand For 5 Min. At 37C.
  7. Extract 2x With Phenol:ChCl3, 1x With ChCl3
  8. Add 1/10 Vol. 3m Naoac And 2.5 Vol Ethanol.
  9. Spin 15min. Final Yield Should Be About 5ug.
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