Sonication
Sonication is used to get an accurate reading of cell number, because it breaks up clumps of cells that can be hard to count. Take 1 ml of cell culture in an eppendorf tube. Put eppi tube into holder and position microtip 2/3 rds down into tube, and close door. Sonicate sample with microtip for 10-15 seconds at setting ã2.5ä (set dial at ã2.5ä, hit start, count, then hit stop). Clean off tip with kimwipe that has been wetted with ethanol. This level of sonication will break the weak attachments between cells that have undergone cell division, but that have not yet separated from each other. It will not kill the cells, nor will it separate buds from mother cells. If after sonication large clumps of cells are still seen, samples can be resonicated again at the same or a higher setting (ã3.0ä).
Alpha factor is a small peptide mating pheromone that can be used to arrest Mat a cells in the G1 phase of the cell cycle. Mat a cells are grown to early log phase in YPD (pH 4.5) and then alpha factor is added to a final concentration of 5 ug /ml . Cells are then incubated at the permissive temperature for 3 hours, during which time they arrest in the cell cycle and begin to form schmoos (projections that are specialized for mating). Alpha factor arrest is reversible and can be stopped by placing the cells into fresh media.
to make YPD pH 4.5, take 200 ml YPD media (containing 2% dextrose) and
add 700 ul of 6 N HCl, mix
check pH with pH paper
Alpha factor stock should be kept in the freezer at a concentration of 0.5 mg/ml (100X)
Yeast Plasmid DNA prep
This protocol is used for recovering plasmids from yeast cultures.
Materials:
breaking buffer: 2% Triton X-100 (Sigma, X100)
% SDS (BioRad, 161-030)
100 mM NaCl
10 mM Tris-Cl, pH 8.0
1 mM EDTA, pH 8.0
- T.E. buffer (pH 8.0): 10 mM Tris-Cl, pH 8.0
1 mM EDTA, pH 8.0
- chilled phenol:choloroform:isoamyl alcohol (25:24:1)
- chilled 95% ethanol
- acid-washed glass beads (Sigma, G 3753, See CPMB, 13.12.1)
Protocol
- grow up yeast culture to appropriate density (near saturation)
- spin 1.5 mls of culture for 1 min in microfuge and aspirate off supernatant
- resuspend pellet in 200 ul breaking buffer
- wear gloves and add:
200 ml phenol:choloroform:isoamyl alcohol (25:24:1)
200 ml (@200 mg) glass beads
close cap tightly and vortex for 2.5 min.
Be careful when vortexing; label can be dissolved by the phenol.
Hold cap tightly so it doesnât open or spill.
- add 200 ml TE buffer and spin for 5 min, in microfuge
- transfer 350 ml aqueous (top) layer to fresh eppendorf.
- add 1 ml 95% ethanol and mix well, let sit for 10 minutes
- spin for 2 min, take off supernatant, and let dry upside down 10 min.
- resuspend pellet in 50 ml TE buffer.
You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli . Having the plasmid in E. coli rather than yeast will make subsequent analysis easier.
Transformation of E. coli with plasmid DNA
This protocol is used to get plasmids into E. coli
Materials:
- pure plasmid DNA or Îsmash and grab yeast DNA prepâ
- DH5a competent cells
- ice
- heat-block, 42*C
- LB media and LB amp plates
Protocol
- thaw competent cells, and for each transformation, aliquot 100 ul into a sterile eppendorf tube on ice.
- add DNA sample (1 ul) to each tube and mix well but gently.
- incubate on ice, 10 min.
- heat shock at 42°C for 45 sec
- add 0.5 ml LB media and incubate at 37°C, 1 hr.
- spread 300 ul of cells onto LB + amp plates and inc at 37°C overnight.
- next day count colonies and innoculate liquid LB amp cultures as needed