Duke University/27 June 2009

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June 27, 2009

Harvest bacterial culture by centrifuging at 8000 rpm (6800xg) in microcentrifuge for 2 min at RT. Decant supernatant and remove remaining medium.

Purification/Mini prep

  1. Resuspend pelleted cells in 250 ul Resuspension Solution. Vortex or pipet up and down until no cell clumps remain.
  2. Add 250 Lysis Solution and mix thoroughly by inverting tube 4-6 times until solution becomes viscous and slightly clear. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
  3. Add 350 ul Neutralization Solution and mix thoroughly by inverting tube 4-6 times.
  4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
  5. Transfer supernatant to spin column by decanting or pipetting. Avoid disturbing or transferring white precipitate.
  6. Centrifuge for 1 min. Discard flow-through and replace column in collection tube.
  7. Add 500 ul Wash Solution to spin column. Centrifuge for 30-60s and discard flow-through.
  8. Repeat step 7.
  9. Centrifuge for 1 min to remove residual Wash Solution.
  10. Transfer spin column into fresh 1.7 ml microcentrifuge tube. Add 50 ul Elution Buffer to center of spin column membrane. Do not touch pipette tip to membrane. Incubate 2 min at RT and centrifuge for 2 min.

Note: An additional elution step with Elution buffer or water will recover residual DNA from the membrane and increase overall yield by 10-20%.

  1. Discard column and store purified plasmid DNA at -20°C.
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