Preparation of plasmid DNA on a small scale

Reagents:

1. TE buffer: Tris-HCl 10 mM pH 8.0, 1 mM EDTA pH 8.0.

2. Solution II: 0.2 M NaOH; 1.0% SDS.

3. Solution III: Potassium Acetate 3M; 2M glacial acetic acid, final pH 4.8 to 5.0.

4. R Buffer (RT): Tris-HCl pH 8.0; 0.1 mM EDTA pH 8.0.

Steps:

1. Bacterial cells grow overnight at 37 °C shaking.

2. Deposit 1.5ml of culture in microtube by centrifuging at 16.000g for 30 seconds. Discard supernatant and repeat for 3 ml of culture.

3. Resuspend cells in 200ul of TE buffer and incubate the system at room temperature for 5 minutes.

4. Add 360ul of Solution II, mix thoroughly by inverting the tube several times. Keep at room temperature for 5 minutes.

5. Add 300ul of Solution III, mix well and incubate on ice for 5 minutes.

6. Centrifuge at 12,000 g for 5 minutes.

7. Transfer supernatant to another microtube, add 750ul of Isopropanol and mix by inverting the tube. Leave for 5 min room temperature.

8. Centrifuge at 12,000 g for 5 minutes and discard the supernatant. Remove alcohol excess with a micropipette.

9. Add 1 ml of 70% ethanol at -20 °C (without resuspending the pellet) and centrifuging at 12,000 g for 5 minutes.

10. Discard supernatant, dry pellet under vacuum and resuspend in 50ul of buffer A.