284
1 mM, or 4 mM KCl, respectively. Weigh 7.4 g of KCl powder
to prepare 1 L of 100 mM KCl containing medium.
4. Low Potassium Agar from Sigma-Aldrich (#P9338) allows the
preparation of media supplemented with very low potassium
concentrations (e.g., 0.5 mM).
5. Filtering vitamins and trace elements through 0.2 μm syringe
filters ensure sterilization of the solution. Filter under laminar
airflow hood directly into sterile 50 mL falcon tubes. Cover
the tubes with aluminum foil to protect from light irradiation.
Open tubes only in sterile environment.
6. Nine assemblies of Royal-blue Tri-Star Rebel LED, at
2730 mW each, are able to irradiate 1 sq. m table at an average
light distribution (λ = 447 nm) of 160–230 μW/cm
2
.
7. Inoculate 3 mL of cell culture in a 15 mL tube.
8. This process typically requires about 4 h when applying 1:50
dilution and about 3 h when working with 1:25 dilution.
9. Allow cells to cool down slowly by placing tubes into a polystyrene box, within the −80 °C refrigerator. This procedure
allows gradual freezing of the cells and improves their transformation efficiency.
10. Frozen-EZ Yeast Transformation II kit workflow adaptation:
prepare in a 2 mL tube a mixture of 1 μL (>200 ng/ul) expression vector, 5 μL SGY1528 competent cells, 50 μL EZ3
solution. Incubate cells at 30 °C for 45 min and mix the solution every 15 min.
11. The product of three consecutive EP-PCR reactions is typically
enough to perform a gap repair transformation plated on up to
18 petri dishes (Ø150 mm).
12. Cut the plasmid by restriction digestion leaving sticky and blunt
end opposite each other to avoid plasmid re- circularization.
Lacking restriction sites can be inserted by SDM-PCR.
13. This number of plates occupies roughly 1m
2
of an irradiated
area during functional complementation tests. Depending on
the cell density per plate, it can provide about 20,000 growing
colonies per functional screening.
14. SGY1528 is a K
+
uptake defective yeast strain: Mata Ade2-1
can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 trk1::HIS3
trk2::TRP1 [8].
15. When using Zymoprep Yeast plasmid Miniprep II kit a typical
purification from 2 mL cell culture has an average yield between
20 and 120 ng/μL. However, the purity of this extraction is not
suitable for sequencing, for this reason a further cloning step in
E. coli cells and subsequent plasmid purification are necessary.
Cristian Cosentino et al.
1 mM, or 4 mM KCl, respectively. Weigh 7.4 g of KCl powder
to prepare 1 L of 100 mM KCl containing medium.
4. Low Potassium Agar from Sigma-Aldrich (#P9338) allows the
preparation of media supplemented with very low potassium
concentrations (e.g., 0.5 mM).
5. Filtering vitamins and trace elements through 0.2 μm syringe
filters ensure sterilization of the solution. Filter under laminar
airflow hood directly into sterile 50 mL falcon tubes. Cover
the tubes with aluminum foil to protect from light irradiation.
Open tubes only in sterile environment.
6. Nine assemblies of Royal-blue Tri-Star Rebel LED, at
2730 mW each, are able to irradiate 1 sq. m table at an average
light distribution (λ = 447 nm) of 160–230 μW/cm
2
.
7. Inoculate 3 mL of cell culture in a 15 mL tube.
8. This process typically requires about 4 h when applying 1:50
dilution and about 3 h when working with 1:25 dilution.
9. Allow cells to cool down slowly by placing tubes into a polystyrene box, within the −80 °C refrigerator. This procedure
allows gradual freezing of the cells and improves their transformation efficiency.
10. Frozen-EZ Yeast Transformation II kit workflow adaptation:
prepare in a 2 mL tube a mixture of 1 μL (>200 ng/ul) expression vector, 5 μL SGY1528 competent cells, 50 μL EZ3
solution. Incubate cells at 30 °C for 45 min and mix the solution every 15 min.
11. The product of three consecutive EP-PCR reactions is typically
enough to perform a gap repair transformation plated on up to
18 petri dishes (Ø150 mm).
12. Cut the plasmid by restriction digestion leaving sticky and blunt
end opposite each other to avoid plasmid re- circularization.
Lacking restriction sites can be inserted by SDM-PCR.
13. This number of plates occupies roughly 1m
2
of an irradiated
area during functional complementation tests. Depending on
the cell density per plate, it can provide about 20,000 growing
colonies per functional screening.
14. SGY1528 is a K
+
uptake defective yeast strain: Mata Ade2-1
can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 trk1::HIS3
trk2::TRP1 [8].
15. When using Zymoprep Yeast plasmid Miniprep II kit a typical
purification from 2 mL cell culture has an average yield between
20 and 120 ng/μL. However, the purity of this extraction is not
suitable for sequencing, for this reason a further cloning step in
E. coli cells and subsequent plasmid purification are necessary.
Cristian Cosentino et al.