Short Mini-Prep Protocol

Fan's Revised Mini-Prep Protocol

1. Spin down 5mL overnight cultures in Sorval for 5 minutes @ 5,000 rpm.
2. Remove the supernatant via aspiration.
3. Add 500 mL solution I. Re-suspend the pellets via P1000 and transfer the suspensions
to clean 1.5 mL eppendorf microfuge tubes.
4. Centrifuge the tubes for 2 minutes @ 3,000 rpm.
5. Remove the supernatant via aspiration.
6. Add 200 uL of solution I and re-suspend the pellet via P1000.
7. Make solution II: 7mL H2O + 2mL (1M) NaOH + 1 mL (10%) SDS.
8. Add 200 uL of solution II. Use the P1000 to mix
(unless the solution is already clear) and put tubes on ice.
9. Add 150 uL of solution III to each tube. Mix a few times
via inversion. Leave on ice for 10 minutes (Note x).
10. Centrifuge @ 4C for 10-15 minutes.
11. Remove the supernatant to a clean microfuge tube.
12. Add 1 mL of ice-cold 100% EtOH.
13. Leave on ice 30 minutes.
14. Centrifuge @ 15,000 rpm @ 4C for 10-15 minutes.
15. Remove the supernatant via aspiration.
16. Add 200 uL of TE/RNAse. (each sample should have 10-20 ug of total RNAse;
This means add ~10 uL of (10 mg/mL stock) RNAse per mL of TE.
17. Incubate 1 h @ 37C.
18. Add 1 volume of Phenol and 1 volume of CHCl3/isoamyl alcohol
(12:1, v/v), and vortex tubes.
19. Centrifuge 5 minutes @ 15,000 rpm.
20. Remove supernatant to a clean microfuge tube.
21. Add 1 mL 100% EtOH and 60 uL Na Acetate (3M, pH 5.2).
22. Store overnight @ -20C or for 20-30 minutes on dry ice to precipitate the DNA.
23. Centrifuge 15,000 rpm @ 4C for 10-15 minutes to collect precipitate.
24. Remove supernatant via aspiration.
25. Dissolve pellet in 20-50 uL of TE.