Quint Lab:yeast plasmid prep

yeast plasmid prep

1. inoculate 2 ml of Glucose medium and let grow over night @ 30°C
2. spin down 1.5 ml of the over night culture and resuspend in0.5 ml S-buffer
3. incubate 30 min, 37°C
4. add 0.1 ml lysis solution, vortex
5. incubate 30 min, 65°C
6. add 0.166 ml potassium acetate and mix, chill on ice for 10 min
7. spin down for 10 min, pour the SN into a new tube (2 ml!)
8. DNA precipitation: add 0.8 ml EtOH, 10 min on ice, spin down for 10 min, pour off the SN
9. wash DNA with 0.5 ml 70% EtOH, dry pellet
10. dissolve DNA in 0.04 ml water

S-buffer:

• 10 mM K2HPO4; pH 7.2
• 10 mM EDTA
• 50 mM 2-Mercaptoethanol
• 50 µg/ml zymolase

lysis solution:

• 25 mM Tris-HCl; pH 7.5
• 25 mM EDTA
• 2.5% SDS