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Smash and Grab

David Amberg

  1. Grow cells in 3mls selective media o/n.
  2. Pellet cells by 2 quick spins in a microfuge.
  3. Resuspend cells in 200ul of the following soln:
    • 5ml SCE
    • 60ul 10mg/ml 100T Zymolyase
    • 10ul 2-ME
  4. Vortex and inc at 37¡C for 30-60 min.
  5. Add 400ul 0.2N NaOH/1%SDS, invert to mix and inc. on ice for 5 min.
  6. Add 300ul 3M K/5M OAc pH4.5, invert to mix and inc on ice x 5 min.
  7. Spin 2 min in microfuge, pour super into a fresh tube and repeat spin. Remove 500ul to a fresh tube.
  8. Add 300ul iPrOH, vortex and let stand for 5 min. at RT.
  9. Pellet DNA 5 min in a microfuge, then wash pellet with 70% EtOH and repeat spin.
  10. Dry pellet in speed vac and recon in 25ul TE.
  11. Porate 2ul into electrocompetent coli, recover and plate all.
  • SCE:
  • 1M Sorbitol
  • 0.1M Sodium Citrate pH 7.6
  • 0.06M EDTA