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RESEARCH DIVISION Laboratory Manual

 


 

Large Scale Plasmid Preps

  1. Prepare an o/n culture of 100-200ml in Terrific broth with 75g/ml ampicillin.
  2. Spin down cells 5K, 5 min
  3. Resuspend in 10ml of 25 mM Tris, 10 mM EDTA pH8.0 per 200ml
  4. Add 20 ml of freshly prepared 1% SDS, 0.2M NaOH (50ml = 1ml 10M NaOH added to 44ml DDW then 5ml 10% SDS). Swirl to lyse cells completely and then add 15 ml KoAc solution per 200ml
  5. Spin 7 K 10 min
  6. Pour supernatant through cheese cloth into SS34 tube and add 22mls of isopropanol (for a 200ml prep).
  7. Spin 7 K 10 min. Drain off all supernatant, being careful to wipe around the inside neck of the bottle to remove all the isopropanol.
  8. Resuspend pellet in 2.4 ml TE. When dissolved thoroughly, add 4.4g CsCl dissolve 1st (handy hint) then add and 40ul 10mg/ml EtBr. Make sure all CsCl is in solution
  9. Layer plasmid solution under 8.5 mls of CsCl/TE p1.47 in a Ti70 tube and spin O/N at 50 K.

CsCl/TE [1.47] = Dissolve 76.68 gms of CsCl to every 100 mls TE

  1. Pull band in 1.0ml or less. Extract 3 times (ie. add equal volume) against butanol saturated in TE (first extraction into ethidium waste, remainder into butanol waste).
  2. Add 1.0ml DDW, estimate volume and add 2.5 vol. ethanol (no salt). Do not chill. Spin at 3K in benchtop for 10min (provided there was a good band to begin with, otherwise in ultracentrifuge for 20' 20K). Pour off s/n and remove residue with a gilson, resuspend in 350l TE. Read OD 260/280 on 5l and reprecipitate remainder in ependorf with 35l 5M NaCl and 1.0ml ethanol. Resuspend at 1g/l
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