Plasmid Mini Prep (Alkaline Lysis)

Plasmid DNA is isolated from small-scale (1-5 ml) bacterial cultures by treatment with alkali and SDS. This procedure can be scaled up for any size culture. The mini-preps are suitable for cloning and transformations, but are not 100% pure. There is contaminating RNA and RNAse at the end of this procedure and the Nanodrop reading will not be accurate.

Contents

1. Buffers and Materials
2. Protocol
3. References

1. Buffers and Materials

• Alkaline lysis solution I (sterilize and store at 4oC)
  • 50 mM glucose
  • 25 mM Tris-Cl, pH 8
  • 10 mM EDTA, pH 8
• Alkaline lysis solution II (needs to be made fresh)
  • 200 µL of 10N NaOH (Cf = 0.2N NaOH)
  • 1 ml of 10% SDS (Cf = 1% SDS)
  • 8.8 ml MilliQ H2O
• Alkaline lysis solution III (store 4oC)
  • 60 mL of 5M potassium acetate
  • 11.5 mL glacial acetic acid
  • 28.5 mL MilliQ H2O
• Antibiotics for plasmid selection
• Bacterial Media (LB, YT, TB, etc)
• Ethanol
• Phenol:chloroform
• TE (pH 8) containing 20 µg/ml RNase A

2. Protocol

1. Inoculate 2-5 ml of rich medium (LB, YT, Terrific Broth) containing appropriate antibiotic with a single colony of transformed bacteria. Incubate @ 37oC overnight with vigorous shaking.
2. Pour 1.5 ml of culture into a microcentrifuge tube. Centrifuge cultures @ 5k for 5 min.
3. Remove medium by aspiration, leaving the pellet as dry as possible.
4. Resuspend the bacterial pellet in 100 µl of ice-cold Alkaline lysis solution I by vigorous vortexing.
5. Add 200 ul freshly prepared Alkaline lysis solution II to each bacterial suspension. Close tube tightly and mix the contents by inverting the tube rapidly five times. Do not vortex! Store tube on ice for 5 mins.
6. Add 150 ul of ice-cold Alkaline lysis solution III. Close tube and invert several times. Store the tube on ice for 3-5 minutes.
7. Centrifuge the bacterial lysate at maximum speed for 5 mins at 4oC. Transfer supernatant to a fresh tube.
8. Add 50 ul phenol:chloroform. Mix organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 mins at 4oC. Transfer the aqueoues upper layer to a fresh tube.
9. Precipitate nucleic acids from supernatant by adding 2 volumes ethanol at room temperature. Mix the solution by vortexing and allow mixture to stand for 2 mins at room temperature.
10. Centrifuge at max speed for 5 mins at 4oC.
11. Aspirate supernatant. Stand the tube in an inverted position on a paper tower to allow all the fluid to drain away. Use kimwipe or disposable pipette tip to remove any drops of fluid adhering to walls of the tube.
12. Add 1ml of 70% ethanol to the pellet and invert the closed tube several times. Recover DNA by centrifugation at max speed for 2 mins at 4oC.
13. Gently aspirate the supernatant. Take care with this step: the pellet may not adhere to the tube.
14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube upright at room temperature for 15 mins or until the ethanol has evaporated and no fluid is visible in the tube.
15. Dissove the nucleic acid in 25 ul TE (pH 8) containing 20 ug/ml DNAse-free RNAse A. Vortex gently for a few seconds. Store DNA at -20oC.

3. References

Adapted from CSH Protocols.