Team:NTU-Taida/Notebook/Protocol/minipreparation

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Tips for 10 min plasmid DNA minipreparation

  1. Grow 2.0 ml overnight culture
  2. Spin down 1.0 ml if E. coli overnight cells in a microcentrifuge (13,000 rpm, room temp., 30-45 sec) and remove medium.
  3. Add 50 ul of TEN (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 100mM NaCl)
  4. Vortex for 2 min. (VWR multivortexer)
  5. Add 70 ul phenol/chloroform/isoamyl alcohol (25/24/1, with 1% β-ME)
  6. Vortex for 2-3 sec, microcentrifuge for 10 min (room temp.)
  7. Transfer upper solution 70 ul (without touching the interface) to new microcentrifuge tube
  8. Add 20 ul of NH4OAc (7.5M) and 100 ul of isopropanol (room temp.)
  9. Vortex for 5 sec, microcentrifuge for 1 min (room temp.)
  10. Wash the pellet with 70 % EtOH (1 ml) and dry it completely.
  11. Resuspend the pellet in 15 ul of dd H2O or TE. and 1 / 50 RNase. Store at -20oC

Note. Use 4 ul of DNA for digestion (30-60 min in 15 ul with RNase or “express” digestion 3 X 10 sec in a microwave oven), use 18 ul for sequencing.