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SDS-Polyacrylamide solutions and buffers

 

 

 

 

 


Preparation of Yeast Protein Extracts

 

(see Clontech YPH, p12)

 

Cracking buffer stock solution (see p54 Clontech YPH)

8M Urea; 5% w/v SDS; 40mM Tris-HCl pH6.8; 0.1mM EDTA; 0.4 mg/ml BMB in H2O

for a final volume of 20ml 9.6g Urea

1g SDS

0.8ml 1M TrisHCl pH6.8

4l 0.5M EDTA

8mg Bromophenol Blue

 

Protocol

1. Set up 5ml O/N cultures in correct SD selection medium (SD-Leu or SD-Trp).

2. Also set up untransformed yeast as a control. Leave at 30C, 230-250rpm, for 18-24h.

3. For each clone to be assayed, separately inoculate 50ml of YPDA with the entire O/N culture. Take an OD600 reading.

4. Incubate at 30C, 230-250rpm, until the OD600 reaches 0.4-0.6 (this can take between 4-8h).

5. Use OD600 Units to determine the volume of each yeast culture to be taken; i.e. decide to take 15 OD600 Units of each culture 15 / OD600= volume to be taken, so if OD600=0.5, take 30ml

6. For each culture, transfer the volume calculated above into a 50ml Falcon half filled with ice. Spin at 2500rpm (MSE) for 5 min.

7. Pour off supernatant and resuspend pellet into 50ml of ice cold H2O and spin again.

8. Remove supernatant and freeze the pellet in liquid N2 (cells can be stored at -80C)

9. Just before use, make up complete cracking buffer