Miniprep/GET buffer

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[[Category:In vitro]]
[[Category:In vitro]]
[[Category:Escherichia coli]]

Revision as of 21:13, 15 May 2007

Cheap non-kit plasmid miniprep:

  1. take 1.8 ml from an overnight culture into a 2 ml centrifuge tube, spin down 1 minute at full speed, and discard the medium
  2. resuspend the bacterial pellet by vortexing with 100 ul of chilled (4C) GET solution
  3. lyse the bacteria by adding 200 ul of room temperature alkaline SDS, not longer than 5 minutes. Invert to mix: do not vortex. The solution should become clear.
  4. precipitate proteins, membranes, and chromosomal DNA with 150 ul chilled (4C) potassium acetate solution, mix by inverting gently.
  5. centrifuge at full speed 5 to 10 min
  6. transfer 400 ul of the clean supernatant into a new tube preloaded with 900 ul of 100% EtOH to precipitate the plasmid DNA
  7. place the tubes in the -80 freezer for 30 minutes
  8. centrifuge the precipitated plasmid DNA 10 to 15 minutes at full speed, and discard supernatant
  9. optional: wash the pellet with 70% EtOH, respin and discard the supernatant
  10. air dry the pellet for 10-15 minutes at room temperature with the open tube on the bench
  11. resuspend the plasmid DNA in 20 ul of TE. The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.

GET buffer: 50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8

Alkaline SDS: 0.2 N NaOH, 1% SDS

Potassium acetate: 3 M potassium acetate, 1.8 M acetic acid, no pH adjustment

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