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Low cost E. coli plasmid alkaline lysis mini-prep

  1. Grow a culture of E. coli at 37 °C overnight. 

  2. Transfer 1 mL of culture to a new Eppendorf tube. 

  3. Centrifuge at top speed for 30s to recover cells in a pellet. Always spin the tubes with the hinge pointing outwards. 

  4. Remove supernatant with a pipette or by decanting. 

  5. Add 200 µL of Buffer P1 to the pellet. Mix by pipetting up and down pipette or by vortexing briefly. There should be no visible cell clumps. 

  6. Wear goggles for this step and make sure you have a timer ready Add 200 µL of buffer P2, mix by slowly inverting the tube during ~4 min. do not mix by vortexing or else the chromosomal DNA might fragment and contaminate the plasmid DNA. Do not incubate more than 5 min. 

  7. Add 200 µL  buffer P3, mix by slowly inverting the tube at least ten times, do not vortex. 

  8. Centrifuge at top speed for 10 min. While centrifuge is running, prepare a new clean Eppendorf tube with 1 ml 99-100% ethanol. 

  9. Transfer ~500 µL of the supernatant from the centrifugation to the ethanol and mix by inversion. Discard the tube from the centrifugation. 

  10. Centrifuge at top speed for 10 min. Make sure the hinge of the tube is facing outwards. 

  11. Pour away supernatant by opening and inverting the tube. The plasmid DNA should be visible as a small white spot in the bottom of the tube. 

  12. Add 1 mL 70% EtOH. Mix by inverting the tube 2-3 times. 

  13. Centrifuge for 1 min at top speed. 

  14. Pour away supernatant by opening and inverting the tube. Be sure to remove as much of the liquid as possible. If necessary, spin the tubes again and remove the remaining liquid with a pipette. 

  15. Let the ethanol evaporate by opening the lid of the tube and leave it on the bench for 15 min or at 50 °C for 5 min. 

  16. Resuspend DNA pellet in 50 µL 1x TE buffer. 

  17. Store the tube in fridge or freezer (4 °C or -20 °C)