Alkaline Lysis Miniprep protocol

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  • Applications:
    1. Purification of plasmid DNA from bacterial cultures
  • Time to complete protocol:
    • Lab time: 3h
    • Waiting time: 30min, 15min, 1hour, overnight.
  • Approximate cost of materials: $

Contents

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls
Method including controls
  1. Inoculate 5mL of LB medium with appropriate antibiotic (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.
  2. Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration/decanting.
  3. Resuspend the pellet by vortexing in 100ul of ice-cold Solution A:
    -50mM Glucose
    -10mM EDTA
    -25mM Tris Cl (pH 8.0)
  4. Add 200 microliters of freshly prepared Solution B:
    -0.2M NaOH
    -1% SDS
    invert rapidly 4 times. No vortexing.
  5. Add 150 microliters of ice-cold Solution C:
    -3M potassium
    -5M acetate (pH 4.8)
  6. vortex for 10 seconds and place on ice for 15 minutes.
  7. Transfer the supernatant to a fresh tube.
  8. Add an equal amount (400ul) of phenol/chroloform (200/200ul). Vortex briefly and centrifuge at 10,000g for 2 minutes.
  9. Transfer the supernatant to a fresh tube.
  10. Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 minutes. Centrifuge at 10,000g for 15 minutes. Discard the supernatant.
  11. Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes. Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).
  12. Add 30ul of TE buffer (or water) with a small amount of RNAase.
Equipement Required
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