MiniPrep Protocol

Materials

Lysis Solution
400mg lye
500mg SDS
50mL distilled water
Note: First, mix the water and lye by shaking. Then add the SDS and stir gently. Shaking SDS will create a bubbly mess.

Neutralization Solution
11.5g NaCl
3.7g KCl
43mL distilled white vinegar

Binding Solution
11.5g NaCl
43mL distilled white vinegar

Diatomaceous Earth (DE) slurry
Diatomaceous earth 10g
Binding Solution 30mL
Note: Add 10g of diatomaceous earth to 100mL of distilled water. Shake, then allow the particles to settle for 3 hours. Pour off the liquid, then add 30mL of Binding Solution and shake.

Wash Solution 1
8g NaCl
35mL distilled water
15mL Isopropanol

Wash Solution 2
10mL Distilled Water
40mL Isopropanol

Method

  1. Inoculate a 2mL LB culture with your transformed E. coli strain. Incubate for 12h at 37°C with shaking.
  2. Pipette 1.5mL of your culture into an eppendorf tube and centrifuge for 3 minutes at 7,500 rcf. The bacteria will form a pellet in the tube.
  3. Pour off the supernatant, leaving just the bacterial pellet in the tube.
  4. Add 200uL of distilled water to the tube. Pump the liquid up and down with the pipette to re-suspend the bacteria. Make sure there are no clumps left. The liquid should be cloudy.
  5. Add 200uL of Lysis Solution. Invert the tube 3 times to mix the solutions.
  6. Wait 4 minutes.
  7. Add 300uL of Neutralization Solution. A white precipitate will appear. Invert the tube 20 times to ensure the solutions are completely mixed.
  8. Centrifuge the tube for 10 minutes at 16,000rcf. The white precipitate will form a pellet on the side wall of the tube.
  9. Without disturbing the pellet, pipette the supernatant into a new tube. Discard the tube with the pellet.
  10. Shake the DE Slurry to mix the particles which have settled on the bottom.
  11. Pipette 500uL of DE Slurry into the tube containing the supernatant from Step 9. Shake the tube vigorously to keep the DE suspended.
  12. Keep the DE suspended in the liquid for 2 minutes by shaking whenever the particles settle.
  13. Centrifuge the tube for 30s at 16,000 rcf to pellet the DE.
  14. Pour off and discard the supernatant.   Note: Depending on your particular tube, the pellet may not stick well. If this is the case, gently pipette the supernatant away instead of pouring
  15. Add 500uL of Wash Solution 1 and use the pipette to re-suspend the DE pellet.
  16. Keep the DE suspended for 2 minutes like in Step 12.
  17. Centrifuge the tube for 30s at 16,000 rcf to pellet the DE.
  18. Pour off and discard the supernatant like in Step 14
  19. Add 500uL of Wash Solution 2 and use the pipette to re-suspend the pellet.
  20. Keep the DE suspended for 2 minutes like in Steps 12 and 16
  21. Centrifuge the tube for 30s at 16,000 rcf to pellet the DE.
  22. Pour off and discard the supernatant.
  23. Leave the cap open and let the tube sit in a warm area for a half hour to let the remaining alcohol evaporate.
  24. Add 50uL of distilled water and use the pipette to re-suspend the DE.
  25. Centrifuge the tube for 30s at 16,000 rcf to pellet the DE.
  26. Pipette the supernatant into a new tube and discard the tube with the DE pellet. This new tube contains your purified plasmid DNA.