Yu:Yeast miniprep
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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. |
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. |