Miniprep-Alkaline Lysis

1. Grow bacterial (E. coli) culture in LB medium with appropriate antibiotics at 37 °C overnight (O/N) with shaking. For >10 copies plasmid, 3 ml cell culture is usually enough.
2. Spin down cell culture.
3. Discard the supernatant.
4. Add 200 μl of resuspension solution (P1 buffer) into each tube, and vortex to completely resuspend cell pellet
5. Add 200 μl of lysis solution (P2 buffer) and mix by gently inverting the tube 5-6 times. The solution should quickly turn transparent and become more viscous indicating bacterial lysis has taken place.
6. Add 200 μl of neutralizing solution (P3 buffer) and mix by inverting the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
7. Transfer to a microcentrifuge tube
8. Centrifuge at high speed for 2X2.5 min.
9. Carefully transfer the supernatant (try to not disturb the white precipitate) to a new labeled 1.5-ml eppendorf tube
10. Add 2.5-3 volume of 200-proof cold ethanol (stores at -20 °C) to each tube and mix by inverting the tubes a few times.
11. Spin down plasmid DNA precipitate (transparency pellet) at high speed for 10 min.
12. Discard the supernatant and remove the remaining liquid as much as possible by leaving the tube upside-down on a piece of paper towel, then keep the tubes in a tube holder and air dry for 10-20 min. To dry faster, keep tubes at 37 °C heat blocker. DNA precipitate turns white when dry.
13. Resuspend the DNA pellet with 50 μl TE. Completely dissolve the pellet by pipetting solution several times.