(Ausubel et. Al. 1998. Current Protocols in Molecular Biology. Vol. 2, John Wiley & Sons, Inc.: Boston. 13.11.1 - 13.11.4)
Inoculate 10 mL of culture with yeast colonies and grow overnight at 30oC.
Spin culture for 5 min at 5000 rpm (4000 x g) at room temperature. Aspirate off the supernatant and resuspend cells in 500 ml distilled water.
Transfer the resuspended cells to an eppendorf tube and spin 5 sec at maximum speed in tabletop centrifuge at room temperature. Pour off supernatant and disrupt pellet by vortexing briefly.
Resuspend cells in 200 ml Breaking Buffer. Add 0.3 g glass beads (~200 ml volume) and 200 ml Phenol/Chloroform (50:50) and vortex at highest speed for 3 min.
Add 200 mL TE and vortex briefly.
Spin for 5 min at high speed at room temperature and transfer the aqueous layer to a clean eppendorf tube. Add 1 mL of 100% ethanol and mix by inversion.
Spin for 3 min at high speed at room temperature. Remove supernatant and resuspend pellet in 400 ml TE buffer.
Add 30 ml of 1 mg/mL DNase-free RNase, mix and incubate for 5 min at 37oC.
Add 10 ml of 4 M ammonium acetate and 1 mL of 100% ethanol. Mix by inversion.
Spin 3 min at high speed at room temperature. Discard the supernatant and allow pellet to air dry. Resuspend DNA in 100 ml TE buffer.
Breaking Buffer:
2% Triton X-100
1% SDS
100 mM NaCl
10 mM TrisHCl pH 8
1 mM EDTA pH 8
TE Buffer:
50 mM TrisHCl pH 8
20 mM EDTA pH 8
PCR from Yeast genome
Reaction No DNA negative control
[Mg2+] (mM) 0 2 5 10 2 5 10
PCR Buffer (10X) 10 10 10 10 10 10 10
DNA (1/10) 5 5 5 5 0 0 0
Forward Primer (1/50) 5 5 5 5 5 5 5
Reverse Primer (1/50) 5 5 5 5 5 5 5
dNTPs (5mM) 5 5 5 5 5 5 5
MgCl2 (25mM) 0 8 20 40 8 20 40
Taq-Vent (20:1) 1 1 1 1 1 1 1
dH20 68 60 48 28 65 53 33
Rxn Conditions: 1 cycle 940C 5 min
35 cycles 940C 30 sec
Annealing Temperature 30 sec
720C 2 min
1 cycle 720C 7 min
Transformation of Yeast
(Biotechniques. 1992. "A Simple and Efficient Procedure for Transformation of Yeasts." Vol. 13, No. 1, p. 13.)
Inoculate 2 mL culture with yeast colonies and grow overnight at 30oC.
Take 500 ml of culture and spin 10 sec in microcentrifuge. Decant the supernatant by inverting the tube and shaking once. You may also aspirate the supernatant but USE CAUTION.yeast pellets are easily sucked up by aspiration.
Add 10 ml of salmon sperm DNA and 1 mg of transforming DNA. Vortex the mixture.
Add 500 ml Transformation Buffer.
Incubate overnight and room temperature.
Spread 50 ml of mixture (from the bottom of the tube!!) directly onto selective plates.
Transformation Buffer:
90 mL sterile 45% PEG 4000
10 mL 1 M lithium acetate
1 mL 1 M TrisHCl pH 7.5
200 m L 0.5 M EDTA pH 8
Yeast Cell Extracts for SDS-PAGE (For Tris-Tricene Gels)
(Courtesy of Jeff Brodsky, University of Pittsburgh)
Inoculate 5 mL of culture with yeast colonies and grow to saturation at 30oC.
Pellet 2 O.D.600 units of cells in an eppendorf tube.
Add 20 ml Sample Buffer.
Boil immediately for 2 min (For membrane proteins, heat to 65oC for 10 min).
Add ~0.12 g glass beads. (NOTE: scoops can be made to allow rapid measurement of 0.12 g glass beads by cutting off the bottom of an eppendorf tube.)
Vortex at highest speed for 1 min.
Add 80 ml Sample Buffer. Samples may be stored at -20oC.
Briefly vortex and boil the cell extracts prior to loading the gel (For membrane proteins, heat to 65oC for 10 min)
Sample Buffer:
80 mM Tris.HCl pH 6.8
20% SDS
0.1% bromophenol blue
100 mM DTT
10% glycerol
ANALYSIS OF PROTEIN EXPRESSION IN YEAST
(8 HOUR TIMECOURSE EXPERIMENT)
This assay is designed to determine the expression levels of a protein in yeast, often from a plasmid construct. In this particular protocol, the expression of a protein from a yeast plasmid is analyzed under permissive and non-permissive growth conditions.
Inoculate 2 mL of YPG His- media (permissive) with a yeast colony and grow to saturation at 30oC in a rotator. These cultures can be stored at 4oC for 1 week.
Inoculate 5 mL YPG His- media with 10
m L of saturated culture and grow in rotator at 30oC for 16 hours overnight. Take the OD600 reading of a 1:10 dilution of this culture. Only use cultures with OD600 between 1.3 and 2.0 (mid-log phase).
Pellet 2.6 OD600 units of cells. Resuspend the pellet in 20 mL YPG His- (permissive control if necessary) or 20 mL YPD His- (non-permissive). Ensure that the yeast suspension is homogenous.
Split the mixture into four 5 mL culture tubes for time points 2, 4, 6, and 8 hours. Place the tubes in a rotator and grow at 30oC. Take the OD600 readings of the cultures at 2, 4, 6, and 8 hour time points - use undiluted culture for time points 2, 4, and 6 hours while using a 1:10 dilution for the 8 hour time point. If any readings are above 1.0 they should be diluted appropriately and re-measured.
Prepare protein samples as outlined in "Yeast Cell Extracts for SDS-PAGE"
as described above. For the 0 hour time point, use the 5 mL overnight culture as a source of cells. Protein can be detected by Western blot and visualized by chemi-luminescence.
NOTE: To ensure equal loading of protein, use an internal standard yeast protein such as Bip, Sec61p, VDAC, etc.
QUANTITATIVE COMPARISON OF MUTANT PROTEIN EXPRESSION IN YEAST
This assay is a modified version of ANALYSIS OF PROTEIN EXPRESSION IN YEAST and is designed to quantitatively compare expression levels for a wide variety of yeast proteins. It is most often used in the comparison of mutants of a single yeast protein under non-permissive growth conditions.
Inoculate 2 mL of YPG His- media (permissive) with a yeast colony and grow to saturation at 30oC in a rotator. These cultures can be stored at 4oC for 1 week.
Inoculate 5 mL YPG His- media with 10
m L of saturated culture and grow in rotator at 30oC for 16 hours overnight. Take the OD600 reading of a 1:10 dilution of this culture. Only use cultures with OD600 between 1.3 and 2.0 (mid-log phase).
Pellet 0.7 OD600 units of cells. Resuspend the pellet in 5 mL YPD His- media (non-permissive). Ensure that the yeast suspension is homogenous.
Incubate cultures in a rotator at 30oC for 8 hours.
Measure OD600 of 1:10 dilution of culture.
Pellet 2 OD600 units of cells (Repeat until all the culture is used up). The cells can be snap frozen in liquid nitrogen and stored at -200C.
Prepare protein samples as outlined in "Yeast Cell Extracts for SDS-PAGE"
as described above. Protein can be detected by Western blot, visualized by chemi-luminescence and quantified by a Fluor-Imager if necessary.
NOTE: To ensure equal loading of protein, use an internal standard yeast protein such as Bip, VDAC, etc.
This assay is designed to test the ability of yeast mutants to complement a growth defect under non-permissive growth conditions.
Inoculate 2 mL of YPG His- media with a yeast colony and grow to saturation at 30oC in a rotator for 2 days. These cultures can be stored at 4oC for 1 week.
Using sterile technique, streak a loop of saturated yeast culture on both YPG His- (permissive) and YPD His- (non-permissive) agar plates. (See YEAST RECIPES)
Incubate at 30oC for 2 days to allow growth. Ensure that a positive control and negative control are streaked on the plates as well.
