(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
0.8ml 1M TrisHCl pH6.8
4µl 0.5M EDTA
8mg Bromophenol Blue
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 30°C, 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 30°C, 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 -80°C)
9. Just before use, make up complete cracking buffer