Mini Plasmid Preparation
Mini Plasmid Preparation
  1. Collect colonies from bacterial transformed medium from streaks of bacteria on plates.
  2. Resuspend the cells with flicking the cells rolled on wooden sticks in MFT containing 50 µl: 50 mM EDTA, 50 mM Tris, pH 8.0, 5 % Triton X-100, and 8 % sucrose. Plates are stored on a lab shelf for intermediate scale preparation of DNA islation.
  3. Voltex well.
  4. Add equal volume (50 µl) of the same buffer used in step 2, but containing 2 mg/ml lysozyme and voltex briefly.
  5. Leave at room temperature for 3 min
  6. Place tubes in a 100 C water bath for 3 min (Use the clear plastic racks with a couple of rubber bands tighten outside of the rack).
  7. Cool the tubes for 5 min on ice.
  8. Spin the tubes for 15 min (14 K), and poke out the pellet with a tooth pick.
  9. Add an equal volume of 2-propanol (100 µl). mix well, and placed at -20 C overnight.
  10. Spin for 15 min (14 K)
  11. Decant the alcohol, rinse with 70 % ethanol twice, drain upside down .
  12. Resuspend the DNA in 105 µl Milli-Q water, mix by inversion with 35 µl NH4Ac and 350 µl EtOH.
  13. Centrifuge 14K, 20 min.
  14. Drain and rinse with 70 % alcohol twice, drain upside down for 5 to 10 min.
  15. wipe the inside of the MFT carefully (do not touch pellets), and vacuum dry for 5 to 10 min.
  16. Resuspend the DNA in 21 µl Milli-Q water, voltexing well.
  17. Digest DNA with a restriction enzyme of interest with addition of 0.1 µl RNase A(10 mg/ml RNase A) per MFT.