Difference between revisions of "Yu:Yeast miniprep"

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Latest revision as of 16:53, 1 December 2012

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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.