Isolating Plasmid from Cells (Miniprep)

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Revision as of 11:45, 11 September 2008 by Riachalder (Talk | contribs)
  • Pellet overnight culture by centrifuging 13,000g for 10 minutes.
  • Pipette out supernatent and discard.
  • Add 250μl resuspension buffer R3 and mix by pipetting up and down.
  • Transfer to capped tubes.
  • Add 250μl lysis buffer L7 and mix by inverting tube 5 times.
  • Incubate for 5 minutes at room temperature.
  • Add 350μl precipitation buffer. Invert until mixture is homogenous.
  • Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube.
One of the centrifuges in our lab.
  • Load supernatent into spin column and discard capped tube.
  • Centrifuge 13,000g for 1 minute. Discard supernatent.
  • Add 700μl wash buffer W9 (with ethanol).
  • Centrifuge 13,000g for 1 minute. Discard supernatent.
  • Centrifuge 13,000g for 1 minute to remove all liquid. Discard any remaining supernatent and recovery tube.
  • Place spin column in new recovery tube.
  • Add 100μl TE buffer or MilliQ H2O.
  • Incubate for 1 minute at room temperature.
  • Centrifuge 13,000g for 2 minutes. The supernatent in the recovery tube should contain isolated plasmid.
  • Discard spin column and store plasmid at -20°C.
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