Christopher Jackson: Yeast minipreps

Christopher Jackson — Sat, 01 Dec 2012 23:53:15 GMT

Yeast minipreps

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|+ '''''Quick Yeast Plasmid Miniprep'''''
|valign="top"|1. Grow yeast cells in 10mL of culture overnight.
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|valign="top"|2. Pellet culture in 15mL conical. Aspirate supernatant and resuspend pellet in 100μL [[Yu:STET|STET]]. Transfer to a microfuge tube.
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|valign="top"|3. Add ~200mg glass beads and vortex for 5 minutes.
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|valign="top"|4. Heat to 100°C for 3 minutes.
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|valign="top"|5. Transfer 100μL of supernatant to a clean microfuge tube.
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|valign="top"|6. Add 50μL of 7.5M NH4OAc; mix by inversion. Place tube at -20°C for 1 hour.
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|valign="top"|7. Thaw tube and spin at 17,000g for 10 minutes at 4°C.
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|valign="top"|8. Transfer supernatant to a clean microfuge tube. Add 2 volumes of ice-cold 100% ethanol.
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|valign="top"|9. Pellet DNA by spinning at 17,000g for 10 minutes at 4°C
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|valign="top"|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
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|valign="top"|11. Aspirate supernatant and dry pellet. Resuspend in 20μL nuclease-free water. Transform into DH5α.
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|+ '''''Yeast Smash & Grab Plasmid Prep'''''
|valign="top"|1. Grow yeast cells in 10mL of culture overnight.
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|valign="top"|2. Pellet yeast culture in a 15mL conical. Aspirate supernatant, resuspend in 500μL H2O, and transfer to a clean microfuge tube.
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|valign="top"|3. Pellet cells and aspirate supernatant. Resuspend in 200μL [[Yu:TESSX|TESSX]] buffer. Add ~300mg glass beads. Add 200μL Phenol/Chloroform/Isoamyl alcohol (PCI) in the fume hood.
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|valign="top"|4. Vortex for 2 minutes. Add 200μL TE8, vortex, and spin down at 17,000g for 5 minutes at 4°C.
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|valign="top"|5. Transfer aqueous layer to a clean microfuge tube. Add 2 volumes of ice-cold 100% ethanol.
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|valign="top"|6. Pellet DNA by spinning at 17,000g for 10 minutes at 4°C.
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|valign="top"|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
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|valign="top"|8. Aspirate supernatant and dry pellet. Resuspend in 20μL nuclease-free water. Transform into DH5α.
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Christopher Jackson: Yeast minipreps