Plasmid Miniprep


Course Home Page | Laboratory Home Page | Laboratory Schedule

This procedure is used to extract plasmid DNA from bacterial cell suspensions and is based on the alkaline lysis procedure developed by Birnboim and Doly (Nucleic Acids Research 7:1513, 1979). The procedure takes advantage of the fact that plasmids are relatively small supercoiled DNA molecules and bacterial chromosomal DNA is much larger and less supercoiled. This difference in topology allows for selective precipitation of the chromosomal DNA and cellular proteins from plasmids and RNA molecules. The cells are lysed under alkaline conditions, which denatures both nucleic acids and proteins, and when the solution is neutralized by the addition of Potassium Acetate, chromosomal DNA and proteins precipitate because it is impossible for them to renature correctly (they are so large). Plasmids renature correctly and stay in solution, effectively separating them from chromosomal DNA and proteins. Cool, no?

Procedure

Note: The procedure below is used to make duplicate minipreps. This provides balanced tubes for the centrifuge as well as twice as much product when you are finished.

1. Gently swirl the contents of the culture tube to resuspend the cells.

2. Label two 1.5 mL tubes and pipet 1000 uL 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 maximum speed for 20 s.

4. Withdraw and discard the supernatant using a pipettor, being careful not to disturb the cell pellet. Discard the supernatant in a waste container.

5. Add 100 uL of Buffer 1 (50 mM Tris-HCl, 10 mM EDTA, 100 ug/mL 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 2 (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 since the chromosomal DNA released from the broken cells could be sheared into small fragments and contaminate your plasmid prep. 7. Let tubes stand on ice for 5 minutes (it's OK if they go longer).

8. Add 150 uL of ice-cold Buffer 3 (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 (it's OK if they go longer).

10. Place the tubes in a centrifuge (balanced) and spin at maximum speed for 5 minutes. Team up with some other folks on this spin. The precipitate will pellet along the side of the tube.

11. Transfer the supernatants into clean 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. Before you do this step, make sure there is a centrifuge available. 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 maximum speed 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 reason the you oriented the tubes with the hinges outward is because the plasmid DNA pellet may be difficult to see. It 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. If not, do not despair. It may be too small to see but there is probably enough DNA there.  Remember that we are dealing with small molecules that we can't see unless there is a whole lot of them together!

14. Add 200 uL of absolute ethanol to each tube and mix by inversion several times.

15. Spin the tubes at maximum speed in a centrifuge for 2-3 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 TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) 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 TE buffer.

19. Pool the two 20 uL solutions into one labeled tube and  store it in the freezer.


Course Home Page | Laboratory Home Page | Laboratory Schedule


Sep 7, 2000  Copyright (C) 1996, Ivor Knight and Jonathan Monroe. All rights reserved.