1. Grow cells to OD600=0.5
2. Pellet 5 OD units of cells
3. Discard supernatant
4. To pellet add 1160 uL of 0.255M NaOH, 1% B-me
for 20 ml:
1M NaOH 5.1 ml
B-me 200 uL
H2O 14.7 ml
5. Incubate on ice for 10 min (Do not go over this amount of time!!)
6. Add 160 uL 50% TCA
7. Incubate on ice 10 min
8. Spin 2 min, 12000 rpm, 4¢J
9. Discard Supernatant
10. Wash pellet with 500 uL 1M Tris PH 6.8
11. Spin 15 min, 12000 rpm, 4¢J
12. Discard Supernatant
13. Add 150 uL sample buffer
14. 65 degree C, 5 or 10 min, and resuspend pellets during incubation
15. Spin samples 1 min, 14000rpm, RT
16. Remove supernatant to a new tube and load 5 or 10 uL on SDS-PAGE.
Sample Buffer (Ivanka's recipe):
¡@ for 4ml
30% Glycerol 1334 uL
10% SDS 800 uL
1M Tris PH 6.8 260 uL
1M DTT 400 uL
0.05% Bromophenol Blue 160 uL
H2O 1046 uL