Tuesday, November 18, 2003

Description
Yeast Glass Bead Prep

Procedure
Sample Buffer: 0.06M Tris-HCl, pH 6.8
10% (v/v) glycerol
2% (w/v) SDS
5% (v/v) 2-mercaptoethanol
0.0025% (w/v) bromophenol blue

0.1 M PMSF

0.5 M Benzamidine

1. Grow 25 ml of cells to mid-log.

2. Spin down (2500 rpm for 5 minutes). Wash 1X with water and spin again.

3. Resuspend in 1 ml of water and transfer to 1.5 ml microfuge tube. Spin down for 5 seconds and pour off water.

4. Resuspend in 0.5 ml of ice cold Sample Buffer with freshly added PMSF (0.5 mM) and benzamidine (0.5 mM).

5. Add glass beads (~0.5 ml).

6. Vortex on high 4X for 45 seconds with 30 seconds on ice in between each mixing.

7. Spin for 5 minutes in microfuge at 4 deg C.

8. Transfer supernatant to a new tube and boil for 5 minutes.

9. Load 10-15 l on a protein gel.


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