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.