Biology Protocols

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Rapid Large Scale Isolation of Plasmid DNA (based on Promega protocol)

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  • Pick a single colony from a plate and inculate 250ml of LB broth containing the appropriate antibiotic. Grow the cells overnight in a shaker at 37°C.
  • Centrifuge the cells at 5,000g, 4°C for 15m.
  • Resuspend the cell pellet in 6ml of ice-cold freshly prepared lysis buffer:

                                                                         Lysis buffer

                                                                         25mM Tris.HCl, pH8.0
                                                                         10mM EDTA
                                                                         50mM glucose

  • Thoroughly resuspend the cells by pipetting them up and down with a 10ml pipette. Incubate in ice water for 10m.
  • Add 12ml of freshly prepared 0.2M NaOH/1% SDS. Carefully mix by inversion and incubate in ice water for 10m. Do not vortex.
  • Add 7.5ml of 3M sodium acetate, pH4.6. Carefully mix by inversion or gentle vortexing and incubate in ice water for 20m.
  • Centrifuge at 12,000 x g for 15m. Transfer the supernatant to another tube and discard the precipitate.
    Add DNAase-free RNAase A to a final concentration of 20µg/ml. Incubate at 37°C for 20m.
  • Extract twice with 1 volume of TE-saturated phenol/chloroform. Vortex for 1m and centrifuge at 12,000g for 5m.
  • Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1), vortex for 1, and centrifuge at 12,000g for 5m.
  • Transfer the upper, aqueous phase to a fresh tube and add 2 volumes of ethanol. Leave at -20°C for 30m, then centrifuge at 12,000 x g for 20m.
  • PEG purify.

You can expect about 500μg from this protocol.


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