This is a cached page for the URL (http://www.pmci.unimelb.edu.au/core_facilities/manual/mb150.asp). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
RESEARCH DIVISION Laboratory Manual

 


 

Alkaline Lysis Mini Plasmid Preps

  1. Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75g/ml Amp
  2. Pour into ependorf tube and spin down cells at 7-8K for 2 min
  3. Aspirate s/n and resuspend in 50l 25mM Tris pH 8, 10 mM EDTA; leave lids open
  4. Add 100l of freshly prepared 1% SDS, 0.2M NaOH (5ml = 100ul 10M NaOH added to 4.4ml DDW then 500l 10% SDS). Add it forcefully and you don't need to vortex
  5. Add 75l KoAc solution and vortex
  6. Add 100l of phenol/CHI3, close lids, vortex
  7. Spin 13K for 2 mins
  8. Remove supernatant, add to 500l ethanol. Vortex and spin at 13 K for 5 min
  9. Aspirate s/n, removing all ethanol
  10. Resuspend in 50l TE
  11. Digest 2-5ul, adding 1l preboiled 10mg/ml RNAse A (see Reagents for preparation).
KOAc solution:
- 60 ml 5M potassium acetate
- 11.5 ml glacial acetic acid
- 28.5 ml DDW

NOTE: Large scale plasmid preps can be made quickly by scaling all reagents of the "mini prep" procedure 100-fold. Once the phenol step has been completed treat the supernatant with 50ul 10mg/ml RNA'se for 1hr, isopropanol ppte (0.6volumes) and resuspend DNA in 400l of TE. reprecipitate. If the ppte is very cloudy (residual RNA) hook the DNA out (fluffy ball) to separate it from RNA.

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