UA Biophysics:Protocols:Yeast Miniprep

This protocol is derived from Yu:Yeast Miniprep and was the only protocol that worked for plasmid extraction. GenElute HP Plasmid Miniprep Doesn't work on Yeast

Materials

Make sure you have all of this materials before proceding

• 1 g Sodium acetate trihydrate CH3COONa·3H2O (136.08 g/mol)
• 10 mL Tris 0.1 M pH 8.0
• 10 mL EDTA 0.1 M pH 8.0
• 2 g Sucrose
• 5 mL TWEEN 80 20%
• 100% ethanol
• 70% ethanol
• Sterile flasks and eppendorf tubes

For making the stocks of EDTA, Tris and NaAc, make sure of taking the right molecular weight and solubility in water. You may be doing a supersatured solution. Also, adjusting the pH of Tris and EDTA is a process that takes time.

STET 25 mL

STET stands for Sucrose Tris Edta Tween. For making 25 mL of this solution you will use

• 10 mL Tris 0.1 M pH 8.0
• 10 mL EDTA 0.1 M ph 8.0
• Dilute 2 g Sucrose in either Tris or EDTA solution
• 5 mL Tween 80 20%

Filter everything in a sterile flask or falcon.

NaAC

Dilute 1 g of NaAc in 2.450 mL for making a solution of 3 M

Extraction Procedure

• Grow yeast overnight
• Centrifugate 6 mL of your culture in 15 mL falcon. 5000 rpm for 5 minutes
• Aspirate supernatant and resuspend pellet in 100 μL STET
• Sonicate at 37°C for 5 minutes
• Heat to ~100°C for 3 minutes
• Transfer 100 μL of supernatant to a eppendorf
• Add 125 μL NaAc 3 M. Mix by inversion 6 times. Store at -20°C for 1-2 hours
• Thaw tube and centrifuge 17000 g for 10 minutes
• Transfer 200 μL supernatant to a new eppendorf
• Add 400 μL ice-cold ethanol 100%
• Centrifuge 17000 g for 10 minutes
• Aspirate supernatant and resuspend pellet in 500μL ice-cold 70% ethanol.
• Centrifuge 17000 g for 10 minutes
• Aspirate supernatant and dry pellet
• Resuspend in 20 μL nuclease free water
• Run a agarose gel and/or measure concentration in nanodrop

Notes

• Some strains of yeast grow very slowly, so be sure to know how much time is needed to grow your strain to saturation.
• Yu:STET uses other concentrations for all stocks (EDTA, Tris, NaAc). We used this because we have trihydrate or dihydrate salts, and solubility in those are lower. Anyway, final concentrations for STET are *almost* the same
• Yu:Yeast Miniprep uses microcentrifuge at 4°C. This is not possible in the lab because the small centrifuge reaches 17000 g but doesn't have a temperature control. The big centrifuge does but doesn't reach to 17000 g. Anyway, it works pretty well

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