Yeast Protocols
iGEM ETH Zurich 2019
Yeast Plasmid Extraction
Protocol provided by Computational Systems Biology Lab, BSSE
Materials
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100T Zymolyase solution (1mg/ml, prepare fresh)
50 mM Tris pH 8
10 mM EDTA
10 mg/mL RNase
Lysis buffer (1% SDS, 0.2 M NaOH pellets)
Neutralization buffer (3 M KAc, 11.5% acetic acid, store at 4°C)
Isopropanol
70% EtOH
Nuclease free water
Protocol
1. Start a 5mL o/n culture in the appropriate medium.
2. Harvest 2mL of cells by centrifugation at 3000g for 3 minutes. Keep the rest of the
culture for comparison.
3. Remove supernatant with a pipette (yellow tip on blue tip).
4. Resuspend in 250µL of Zymolyase solution. Pipette to resuspend.
5. Incubate for 45 minutes at 37°C, 600 rpm shaking.
6. Check cell wall digestion microscopically (3µL from digest + 3µL water, o/n culture
as control). Digestion is complete when cell fragments can be seen floating
around. If digestion is not complete, check again in 15 minutes or add more
Zymolyase solution.
7. Add 11µL of Tris, 6µL of EDTA and 5µL of RNase.
8. Add 250µL of lysis buffer and mix gently (solution becomes transparent).
9. Add 350µL of neutralization buffer and mix gently (formation of white cloud).
10. Spin down at max speed for 10 minutes at room temperature.
11. Transfer 800µL of supernatant to a new 1.5mL Eppendorf tube.
12. Add 640µL of isopropanol and mix gently to precipitate DNA.
13. Centrifuge again at max speed for 10 minutes at room temperature.
14. Add 500µL EtOH to wash the pellet.
15. Centrifuge a third time at max speed for 5 minutes at room temperature.
16. Remove supernatant and dry pellet from EtOH by air drying or on the heat block.
17. Resuspend DNA pellet it 30µL of nuclease free water. At this point the DNA
solution contains plasmid DNA and RNA. RNase digestion is necessary for clean
DNA.
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