Plasmid miniprep

Published on Author Giovanni Morelli

Procedure

  1. Shake the culture tube to resuspend the cells.
  2. Label 2mL tubes and pour about 2ml of the cell suspension into each tube.
  3. Close the caps and place the tubes in a centrifuge (remember to balance the centrifuge by putting the tubes opposite one another) and spin at 11.000 rpm for 2 minutes.
  4. Withdraw and discard the supernatant pouring it in a becker, being careful not to lose the cell pellet. Discard the supernatant in a waste container.
  5. Add 100 uL of Buffer  P1 (50 mM Tris-HCl, 10 mM EDTA, 100 ug/mL [optional RNase A], pH 8.0 ) to each tube and resuspend the cells by vortexing. It’s very important that the cell suspension is homogenous and no clumps are visible.
  6. Add 200 uL of Buffer P2 (1% SDS, 0.2 M NaOH ) to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. DO NOT VORTEX as this will break bacterial chromosomes and will contaminate your prep.
  7. Let tubes stand on ice for 5 minutes.
  8. Add 150 uL of ice-cold Buffer P3 (3.0 M Potassium Acetate, pH 5.5 ) to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. A white precipitate will form.
  9. Let tubes stand on ice for 5 minutes.
  10. Place the tubes in a centrifuge (balanced) and spin at 13.200 rpm for 5 minutes. The precipitate will pellet along the side of the tube.
  11. Transfer the supernatants into clean labeled 1.5 mL tubes, being careful not to pick up any of the precipitate. Discard the tubes with the precipitate and KEEP the tubes with the supernatant.
  12. To each tube of supernatant add an equal volume (about 400 uL) of isopropanol to precipitate the nucleic acids. Close the caps and mix vigorously. Let the tubes stand at room temperature for 2 minutes, place them, with their hinges pointing outward from the center, in a centrifuge (balanced) and spin at 13.200 rpm for 5 minutes. This step pellets the nucleic acids but if you leave it around too long, proteins remaining in solution will begin to precipitate as well.
  13. The pellet will be at the bottom and along the hinge side of the tube. Carefully remove and discard the supernatant. The pellet is usually visible at this point. Sometimes it may be too small to see but there is probably enough DNA there.
  14. Add 200 uL of absolute ethanol to each tube and mix by inversion several times.
  15. Spin the tubes at 13.200 rpm in a centrifuge for 5 minutes (hinges out).
  16. Carefully remove and discard the supernatant. Try to get as much out as possible without dislodging the pellet of plasmid DNA.
  17. Place the tubes in the fume hood with the caps open for 15-20 minutes to dry off the last traces of ethanol.
  18. When the ethanol is gone (you can check this by smelling the tube) add 20 uL of H20 to dissolve the pellet. Pipet the 20 uL in and out, up the side of the tube to ensure that all of the plasmid DNA comes into contact with the water.

Solutions.

P1 1 Liter (store at RT)
50mM Glucose 9g Glucose
10mM EDTA 20ml 0.5 M EDTA (pH 8.0)
25mM Tris (8.0) 25ml 1 M (Tris pH 8.0)
P2 1 Liter (store at RT)
1% SDS  5 ml of 20% SDS
 0.2 M NaOH
P3 1 Liter (store at RT but cool down in ice before using)
KOAc 294g
Glacial Acetic Acid 115ml Glacial Acetic Acid

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