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Whole Cell Extract Prep of Gal-Induced Yeast Cells Inoculate 3 X 5 ml YEP + 2% raffinose with single yeast colony harboring EE-MOT1-
 

Summary: Auble Lab
Whole Cell Extract Prep of Gal-Induced Yeast Cells
Inoculate 3 X 5 ml YEP + 2% raffinose with single yeast colony harboring EE-MOT1-
expression plasmid. Incubate at 30 degrees O/N.
Next day: check growth of yeast. Cultures may take two days to grow to saturation.
Inoculate 1 liter synthetic media + 2% raffinose minus leucine (to select for plasmid) with
10 ml overnight culture at around 5 pm so that starting OD600 is about 0.1. Incubate at
30 degrees O/N with shaking.
Next day: check growth of yeast cells and induce expression by adding 67 ml 30%
galactose per liter culture when OD is about 1.0 (probably between 9 am and noon).
Incubate for 3-4 hours at 30 degrees with shaking.
Pellet cells at 5000 rpm for 5 minutes in GSA bottles.
Wash cells with 10 ml cold Benoit's Extraction Buffer with BME and protease inhibitors.
Resuspend cells in 10 ml Benoit's buffer and freeze at 80 C or proceed to lyse cells in
French Press.
Remove supernatant to eppendorf tubes on ice, spin samples in microfuge for 30 minutes
at 4 C. (Alternatively, clarify extract by spinning in ultracentrifuge; see Arindam).
Carefully remove supernatant to new tubes on ice. Save 10 ul in separate tube for
protein assay (0.5-5 ul) and western blot (20 g per lane).
Auble Lab

  

Source: Auble, David - Department of Biochemistry and Molecular Genetics, University of Virginia

 

Collections: Biology and Medicine