246
(PABA), 0.4 mg/l pyridoxine HCl, 0.4 mg/l thiamine, 2%
(w/v) glucose, supplemented with appropriate amino acids
and/or other nutrients.
3. Solid synthetic complete medium (SC): 0.15% (w/v) Bacto
yeast nitrogen base without amino acids and without ammoniumsulfate, 0.5% (w/v) ammoniumsulfate, 0.2% (w/v) drop-out
mix lacking the compound used for plasmid selection, 2%
(w/v) glucose, 2% (w/v) agar.
1. SORB buffer: 100 mM lithium acetate, 10 mM Tris–HCl pH
8.0, 1 mM EDTA–NaOH pH 8.0, 1 M sorbitol, adjusted with
acetic acid to pH 8.0 and filter-sterilized.
2. Carrier DNA: 10 mg/ml herring sperm DNA, denatured at
100 °C for 10 min and immediately cooled on ice.
3. Polyethylene glycol (PEG) buffer: 100 mM lithium acetate, 10
mM Tris–HCl pH 8.0, 1 mM EDTA–NaOH pH 8.0, 40%
(w/v) PEG 4000; filter-sterilized.
1. Breaking buffer: 2% (v/v) Triton X-100, 1% (w/v) sodium
dodecylsulfate (SDS), 100 mM NaCl, 10 mM Tris–HCl pH
8.0, 1 mM EDTA–NaOH pH 8.0.
1. Alkaline lysis buffer: 1.85 M NaOH, 7.5% β-mercaptoethanol.
2. High urea buffer: 5% (w/v) SDS, 8 M urea, 200 mM Na 2 HPO 4 /
NaH 2 PO 4 pH 6.8, 0.1 mM EDTA, 0.01% (w/v) bromophenol
blue.
3. Protein-precipitation buffer: 55% trichloroacetic acid (TCA)
(w/v)
4. Cycloheximide stock solution: 20 mg/ml.
5. Sodium azide buffer: 100 μM NaN 3 .
6. Antibodies recognizing tagRFP are available from evrogen
(www.evrogen.com), antibodies against Tub1 (loading control) were obtained from Abcam (www.abcam.com).
1. Light emitting diode (LED) stripes or clusters for illumination
of yeast cells grown on plate or in liquid medium. The LED
setup should include a dimmer to achieve a photon flux of 30
μmol m
−2
s
−1
at the level of the yeast cells. Single wavelength
LEDs for blue light illumination (output wavelength 465 nm)
or RGB LEDs with an appropriate controller can be used.
Homogeneous illumination is advisable, which might be easier
to achieve with many LEDs of lower light intensity output
then with few high-power LEDs.
2. An optometer (e.g., P2000, equipped with light detector
D-9306-2, Gigahertz-Optik, Türkenfeld, Germany) to measure the light-intensity used for illumination of yeast cells.
2.4 Yeast
Transformation
2.5 Plasmid Rescue
from Yeast
2.6 Cell Lysis
and Immunoblotting
2.7 Illumination
of Yeast with Blue
Light
Christof Taxis
(PABA), 0.4 mg/l pyridoxine HCl, 0.4 mg/l thiamine, 2%
(w/v) glucose, supplemented with appropriate amino acids
and/or other nutrients.
3. Solid synthetic complete medium (SC): 0.15% (w/v) Bacto
yeast nitrogen base without amino acids and without ammoniumsulfate, 0.5% (w/v) ammoniumsulfate, 0.2% (w/v) drop-out
mix lacking the compound used for plasmid selection, 2%
(w/v) glucose, 2% (w/v) agar.
1. SORB buffer: 100 mM lithium acetate, 10 mM Tris–HCl pH
8.0, 1 mM EDTA–NaOH pH 8.0, 1 M sorbitol, adjusted with
acetic acid to pH 8.0 and filter-sterilized.
2. Carrier DNA: 10 mg/ml herring sperm DNA, denatured at
100 °C for 10 min and immediately cooled on ice.
3. Polyethylene glycol (PEG) buffer: 100 mM lithium acetate, 10
mM Tris–HCl pH 8.0, 1 mM EDTA–NaOH pH 8.0, 40%
(w/v) PEG 4000; filter-sterilized.
1. Breaking buffer: 2% (v/v) Triton X-100, 1% (w/v) sodium
dodecylsulfate (SDS), 100 mM NaCl, 10 mM Tris–HCl pH
8.0, 1 mM EDTA–NaOH pH 8.0.
1. Alkaline lysis buffer: 1.85 M NaOH, 7.5% β-mercaptoethanol.
2. High urea buffer: 5% (w/v) SDS, 8 M urea, 200 mM Na 2 HPO 4 /
NaH 2 PO 4 pH 6.8, 0.1 mM EDTA, 0.01% (w/v) bromophenol
blue.
3. Protein-precipitation buffer: 55% trichloroacetic acid (TCA)
(w/v)
4. Cycloheximide stock solution: 20 mg/ml.
5. Sodium azide buffer: 100 μM NaN 3 .
6. Antibodies recognizing tagRFP are available from evrogen
(www.evrogen.com), antibodies against Tub1 (loading control) were obtained from Abcam (www.abcam.com).
1. Light emitting diode (LED) stripes or clusters for illumination
of yeast cells grown on plate or in liquid medium. The LED
setup should include a dimmer to achieve a photon flux of 30
μmol m
−2
s
−1
at the level of the yeast cells. Single wavelength
LEDs for blue light illumination (output wavelength 465 nm)
or RGB LEDs with an appropriate controller can be used.
Homogeneous illumination is advisable, which might be easier
to achieve with many LEDs of lower light intensity output
then with few high-power LEDs.
2. An optometer (e.g., P2000, equipped with light detector
D-9306-2, Gigahertz-Optik, Türkenfeld, Germany) to measure the light-intensity used for illumination of yeast cells.
2.4 Yeast
Transformation
2.5 Plasmid Rescue
from Yeast
2.6 Cell Lysis
and Immunoblotting
2.7 Illumination
of Yeast with Blue
Light
Christof Taxis