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Quick and Easy Yeast Extract for Western Blots
Author: Erin M Green, updated date: , view: 18, Q&A: 0

Procedure

1.     Grow yeast in a 5mL culture overnight to saturation.

 

2.     Measure the OD­600 of the cultures.  If using a standard spectrophotometer, dilute the culture 1:10 in media and measure the OD of the diluted culture.  Calculate the OD of the culture by multiplying by the dilution factor (e.g. 10).

 

3.     Spin down 2.5OD600 units of culture at 10K rpm for 2 minutes. An OD unit = mL x OD600 of cells (e.g. 2.5 OD unit = 1 ml at OD600 2.5. )

                  Sample Calculation:

                  Yeast culture OD600= 3.5

                  2.5 OD unit = 2.5/3.5 = 0.714 mL of culture

 

4.     Remove supernatant and resuspend in 100uL of water.

 

5.     Add 100uL 0.2M NaOH and mix well.  Incubate at room temperature, 5 minutes.

 

6.      Pellet cells by spinning at 10Krpm for 2 minutes. 

 

7.     Remove supernatant and resuspend pellet in 50uL 1x SDS loading buffer. Boil for 3 minutes. 

 

8.     Pellet debris by spinning at 10Krpm for 1 minute.  Load 6uL of supernatant on to an SDS-PAGE gel.

 

 

Required Solutions:

0.2M NaOH

0.4 g NaOH in 50mL H20

 

5X SDS loading buffer

250mM Tris-HCl pH 6.8

10%SDS

30% glycerol

5% ß-mercaptroethanol

0.02% bromphenol blue

 

 


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