IGEM:British Columbia/Protocols/Miniprep

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Contents

No Kit Miniprep

Materials

  • Overnight culture
  • Isopropanol
  • P1 buffer
  • P2 buffer
  • 3M sodium/potassiuma acetate
  • 70% ethanol
  • sdH2O

Equipment

  • Tabletop Centrifuge
  • Vortex
  • Water bath

Method

  • Spin down up to 5mL of overnight culture at max speed
  • Pipette out the broth; minimize broth as much as possible
  • Add 200uL of buffer P1 (50mM Tris-Cl pH 8.0, 10mM EDTA, 100ug/mL RNAse A), resuspend pellet in this buffer
  • Add 200uL of buffer P2 (200mM NaOH, 1%SDS), invert the tube 6-10 times and wait for 5 minutes at room temperature.
  • Add 300uL of 3M sodium/potassium acetate (pH 5.5), immediately invert the tube 6-10 times, and centrifuge for 10 minutes at 13,000 RPM.
  • Pour or pipette the supernatant into a microfuge tube with 600uL isopropanol, make sure to avoid the white precipitate as much as possible
  • Vortex the tube for 20 seconds and centrifuge for one minute at 13,000 RPM
  • Discard the supernatant and add 750uL of 70% ethanol to the tube, vortex briefly, and centrifuge for another 1 minute at 13,000 RPM
  • Discard the supernatant again and centrifuge for 10 seconds at 13,000 RPM to collect the remaining ethanol.
  • If you can see a pellet, pipette out the remaining ethanol, if not, heat the tube over a hot water bath (50C or more) for as long as it takes to evaporate the ethanol. A P10 tip works best because it won't freak out the ethanol as much when it comes in contact with the tip.
  • Resuspend the plasmid DNA in the 50┬ÁL of sterile distilled water.
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