Yu:Yeast miniprep

Quick Yeast Plasmid Miniprep

1. Grow yeast cells in 10mL of culture overnight.

2. Pellet culture in 15mL conical. Aspirate supernatant and resuspend pellet in 100μL STET. Transfer to a microfuge tube.

3. Add ~200mg glass beads and vortex for 5 minutes.

4. Heat to 100°C for 3 minutes.

5. Transfer 100μL of supernatant to a clean microfuge tube.

6. Add 50μL of 7.5M NH4OAc; mix by inversion. Place tube at -20°C for 1 hour.

7. Thaw tube and spin at 17,000g for 10 minutes at 4°C.

8. Transfer supernatant to a clean microfuge tube. Add 2 volumes of ice-cold 100% ethanol.

9. Pellet DNA by spinning at 17,000g for 10 minutes at 4°C

10. Aspirate supernatant and resuspend pellet in 500μL ice-cold 70% ethanol. Pellet DNA by spinning at 17,000g for 10 minutes at 4°C

11. Aspirate supernatant and dry pellet. Resuspend in 20μL nuclease-free water. Transform into DH5α.

Yeast Smash & Grab Plasmid Prep

1. Grow yeast cells in 10mL of culture overnight.

2. Pellet yeast culture in a 15mL conical. Aspirate supernatant, resuspend in 500μL H2O, and transfer to a clean microfuge tube.

3. Pellet cells and aspirate supernatant. Resuspend in 200μL TESSX buffer. Add ~300mg glass beads. Add 200μL Phenol/Chloroform/Isoamyl alcohol (PCI) in the fume hood.

4. Vortex for 2 minutes. Add 200μL TE8, vortex, and spin down at 17,000g for 5 minutes at 4°C.

5. Transfer aqueous layer to a clean microfuge tube. Add 2 volumes of ice-cold 100% ethanol.

6. Pellet DNA by spinning at 17,000g for 10 minutes at 4°C.

7. Aspirate supernatant and resuspend pellet in 500μL ice-cold 70% ethanol. Pellet DNA by spinning at 17,000g for 10 minutes at 4°C

8. Aspirate supernatant and dry pellet. Resuspend in 20μL nuclease-free water. Transform into DH5α.