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1. Pick up the desired colony from solid nonselective medium
(SD-U + A + 100 mM KCl) and inoculate it in 3 mL of liquid
nonselective medium. Grow cells overnight in 30 °C shaking
incubator in the dark.
2. Centrifuge 2 mL of the overnight cell culture at 2700 × g for
2 min and discard supernatant.
3. Resuspend pellet with 2 mL sterile water to wash cells.
4. Centrifuge cells at 2700 × g for 2 min and discard the
supernatant.
5. Add 1 mL sterile water and resuspend cells.
6. In a different tube, transfer 50 μL resuspended cells and add
950 μL water (1:20 dilution).
7. Measure OD 600 of the 1:20 dilution and calculate the OD 600 of
the resuspended cells.
8. Dilute the resuspended cells to OD 600 = 0.8 with water.
9. Prepare tenfold serial dilutions in 2 mL tubes: transfer 100 μL
cell from OD 600 = 0.8 preparation in a new tube and add
900 μL water (1:10 dilution). After mixing, repeat the same
procedure starting from 1:10 dilution to obtain 1:100 dilution
and from the 1:100 dilution to obtain 1:1000 dilution.
10. Spot 7 μL drops of each dilution onto plates of SEL-U-M
medium, supplemented with KCl at the desired selective concentration. Prepare each plate in duplicate to test simultaneously two functional conditions.
11. Seal plates with parafilm and incubate inverted for 3 days at
30 °C under light or dark conditions in the chamber described
in Subheading 2.8.
12. To purify the plasmid from selected colonies, pick the desired
colony from solid nonselective medium and inoculate in 3 mL
of liquid nonselective medium.
13. Grow cells overnight in a shaking incubator at 30 °C in the
dark.
14. Purify yeast plasmid with any commercially available plasmid
extraction kit (see Note 15).
15. In a new 2 mL tube transform 10 μL DH5α E. coli competent
cells with 1 μL of the yeast extracted plasmid.
16. Plate 90 μL transformed cells on LB agar plate supplemented
with selective antibiotics and incubate overnight at 37 °C.
17. Inoculate colonies in a 50 mL tube containing 7 mL LB broth,
supplemented with selective antibiotics; grow cells overnight
at 37 °C in a shaking incubator.
18. Proceed as per any commercially available DNA plasmid purification kit workflow.
Cristian Cosentino et al.
1. Pick up the desired colony from solid nonselective medium
(SD-U + A + 100 mM KCl) and inoculate it in 3 mL of liquid
nonselective medium. Grow cells overnight in 30 °C shaking
incubator in the dark.
2. Centrifuge 2 mL of the overnight cell culture at 2700 × g for
2 min and discard supernatant.
3. Resuspend pellet with 2 mL sterile water to wash cells.
4. Centrifuge cells at 2700 × g for 2 min and discard the
supernatant.
5. Add 1 mL sterile water and resuspend cells.
6. In a different tube, transfer 50 μL resuspended cells and add
950 μL water (1:20 dilution).
7. Measure OD 600 of the 1:20 dilution and calculate the OD 600 of
the resuspended cells.
8. Dilute the resuspended cells to OD 600 = 0.8 with water.
9. Prepare tenfold serial dilutions in 2 mL tubes: transfer 100 μL
cell from OD 600 = 0.8 preparation in a new tube and add
900 μL water (1:10 dilution). After mixing, repeat the same
procedure starting from 1:10 dilution to obtain 1:100 dilution
and from the 1:100 dilution to obtain 1:1000 dilution.
10. Spot 7 μL drops of each dilution onto plates of SEL-U-M
medium, supplemented with KCl at the desired selective concentration. Prepare each plate in duplicate to test simultaneously two functional conditions.
11. Seal plates with parafilm and incubate inverted for 3 days at
30 °C under light or dark conditions in the chamber described
in Subheading 2.8.
12. To purify the plasmid from selected colonies, pick the desired
colony from solid nonselective medium and inoculate in 3 mL
of liquid nonselective medium.
13. Grow cells overnight in a shaking incubator at 30 °C in the
dark.
14. Purify yeast plasmid with any commercially available plasmid
extraction kit (see Note 15).
15. In a new 2 mL tube transform 10 μL DH5α E. coli competent
cells with 1 μL of the yeast extracted plasmid.
16. Plate 90 μL transformed cells on LB agar plate supplemented
with selective antibiotics and incubate overnight at 37 °C.
17. Inoculate colonies in a 50 mL tube containing 7 mL LB broth,
supplemented with selective antibiotics; grow cells overnight
at 37 °C in a shaking incubator.
18. Proceed as per any commercially available DNA plasmid purification kit workflow.
Cristian Cosentino et al.