[Previous] [Top]

sect29

Yeast lysates for westerns

1. Cells are grown for 2-3 days as 1.5ml prep. under selection for the plasmid of interest.

2. Spin cells down 2.6K for 5min.

2. Resuspend in 1ml 0.25m NaOH/1% 2-mercaptoethanol

3. Incubate 10min on ice.

4. Add 0.16ml 50% trichloroacetic acid

5. Incubate for 10min on ice.

6. Pellet 14K 10min.

7. Resuspend pellet in 1ml ice cold acetone.

8. Pellet 14K 10min.

9. Air dry pellet and resuspend in 200-500ul SDS SB and proceed as described below in section on western transfer.

[Previous] [Top]