283
This step allows the replication of the same colony over two plates
with selective medium. The replica plates are then incubated under
different growing conditions and differentially growing colonies
identified and selected for further analysis.
1. With a sterile velvet cloth, replicate colonies grown onto each
nonselective plate onto two different Ø150 mm plates containing SEL-U-M+A supplemented with the desired selective
KCl concentration.
2. Seal and number the two selective plates according to mother
plate number and selection condition and mark each to report
the orientation as for the nonselective plate (see Note 16).
3. Incubate plates at 30 °C in the growth chamber. One subset of
plates must be placed under effective light irradiation, the
second subset must be grown in the dark (see Note 17).
4. After 3 days, light-irradiated and dark grown plates are compared
to find differentially grown yeast colonies (see Note 18).
Colonies that have been identified by differential growth in
replica plating must be validated to eliminate false positives.
5. To validate the phenotype, identify the colonies that show
differential growth between light and dark conditions following replica plating.
6. Inoculate the selected colonies in liquid nonselective medium
(SD-U+A + 100 mM KCl) and purify plasmid from the liquid
growth.
7. Transform S. cerevisiae competent cells and confirm the phenotype (dark/light differential growth) by drop test.
4 Notes
1. Cover plates with aluminum foil to keep LB agar supplemented
with ampicillin in the dark. Ampicillin is light sensitive and
degrades under high temperature and light exposure.
Moreover, storing plates in an inverted position (with their lid
down) allows the condensed water to accumulate on the plastic lid instead of on the medium.
2. Aliquot 1 mL of antibiotics stock solution into 1.5 mL
Eppendorf tubes. When preparing aliquots of Ampicillin, cover
tube with aluminum foil to keep solution in the dark. Stock
solutions are 1000×, when supplemented to LB media add
1 μL antibiotic to each mL of medium.
3. Dissolve 14.9 g KCl in 100 mL ultrapure water to have a 2 M
stock solution. Add 250 μL, 500 μL, or 2 μL of the obtained
stock solution to supplement 1 L medium with 0.5 mM,
3.4.2 High-Throughput
Screening by Replica
Plating
Engineering Light-Regulated K
+
Channels
This step allows the replication of the same colony over two plates
with selective medium. The replica plates are then incubated under
different growing conditions and differentially growing colonies
identified and selected for further analysis.
1. With a sterile velvet cloth, replicate colonies grown onto each
nonselective plate onto two different Ø150 mm plates containing SEL-U-M+A supplemented with the desired selective
KCl concentration.
2. Seal and number the two selective plates according to mother
plate number and selection condition and mark each to report
the orientation as for the nonselective plate (see Note 16).
3. Incubate plates at 30 °C in the growth chamber. One subset of
plates must be placed under effective light irradiation, the
second subset must be grown in the dark (see Note 17).
4. After 3 days, light-irradiated and dark grown plates are compared
to find differentially grown yeast colonies (see Note 18).
Colonies that have been identified by differential growth in
replica plating must be validated to eliminate false positives.
5. To validate the phenotype, identify the colonies that show
differential growth between light and dark conditions following replica plating.
6. Inoculate the selected colonies in liquid nonselective medium
(SD-U+A + 100 mM KCl) and purify plasmid from the liquid
growth.
7. Transform S. cerevisiae competent cells and confirm the phenotype (dark/light differential growth) by drop test.
4 Notes
1. Cover plates with aluminum foil to keep LB agar supplemented
with ampicillin in the dark. Ampicillin is light sensitive and
degrades under high temperature and light exposure.
Moreover, storing plates in an inverted position (with their lid
down) allows the condensed water to accumulate on the plastic lid instead of on the medium.
2. Aliquot 1 mL of antibiotics stock solution into 1.5 mL
Eppendorf tubes. When preparing aliquots of Ampicillin, cover
tube with aluminum foil to keep solution in the dark. Stock
solutions are 1000×, when supplemented to LB media add
1 μL antibiotic to each mL of medium.
3. Dissolve 14.9 g KCl in 100 mL ultrapure water to have a 2 M
stock solution. Add 250 μL, 500 μL, or 2 μL of the obtained
stock solution to supplement 1 L medium with 0.5 mM,
3.4.2 High-Throughput
Screening by Replica
Plating
Engineering Light-Regulated K
+
Channels