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RESEARCH DIVISION Laboratory Manual

 


 

CTAB Mini Plasmid Preparation

  1. Inoculate 5ml medium. Grow O/N
  2. Spin 15 min at 13K
  3. Decant supernatant and resuspend pellet in 500 ul STET buffer containing 1mg/ml lysozyme (fresh) and transfer into ependorf.
  4. Incubate 5 min at RT
  5. Place on 95 oC for 1 min
  6. Centrifuge for 15 min
  7. Remove slimy pellet with toothpick
  8. Add 20ul 5% CTAB and mix gently
  9. Centrifuge for 15 min at 13K
  10. Dissolve pellet in 300 ul 1.2 M NaCl
  11. Precipitate DNA by adding 750 ul Ethanol and spinning for 20 min at 13K.
  12. Wash pellet with 70% ethanol and dry
  13. Dissolve pellet in 50 ul TE/RNase (1/20 of 10 mg/ml lab stock)
  14. Use 1/10 volume for analytic restriction cleavages
STET:
- 8%w/v sucrose
- 0.1 % Triton X-100,
- 50mM EDTA
- 50 mM Tris-HCl pH 8,0
CTAB:
- Hexadecyltrimethylammoniumbromide (Sigma: H5882)

 

Reference: Del Sal, G., Manfioletti, G. and Schneider, C. (1988): A one-tube plasmid DNA mini-preparation suitable for sequencing, NAR 16, 9878.

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