Mini-prep Protocol

1. Grow bacterial colonies overnight in LB with antibody. (I do 5 ml cultures)

2. Take out 1.5 ml bacteria and spin down for 30 sec. Remove supernatant.

3. Add 100 ul Solution 1. Vortex to resuspend pellet.

4. Add 200 ul Solution 2. Vortex and wait 1-2 min.

5. Add 150 ul Solution 3. Vortex.

6. Add 150 ul Chloroform/Isoamyl alcohol (IAA). Vortex 5-10 sec.

7. Spin 1 min at room temperature.

8. Remove the supernatant and put in new tube.

9. Add 3X volume of 95% Ethanol (I like to use cold Ethanol).

10. Invert to mix and spin for 3 min at room temperature or 4C. You should see a white pellet now.

11. Discard supernatant.

12. Add 500 ul 70% Ethanol. Spin 2 min, (RT or 4 C) and remove supernatant

13. Dry pellet, we do this in the hood (in a speedvac dry >10 min).

14. Resuspend in 50-100 ul TE.

Solution 1

50 mM glucose 0.9 g glucose (2.25 ml of 40% glucose)

25 mM Tris-Cl (pH 8.0) 2.5mL 1M Tris-Cl (pH 8.0)

10 mM EDTA (pH 8.0) 2.0 ml 0.5M EDTA (pH 8.0)

Water up to 100 ml

Solution 2

0.2 N NaOH 2 ml 10 N NaOH

1% SDS 5 ml 20% SDS

Water up to 100 ml

Solution 3

3 M potassium acetate 29.44g potassium acetate or 60 ml 5M KoAc

11.5% acetic acid 11.5 ml glacial acetic acid

Water up to 100 ml