| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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| The ability to make yeast cells competent for transformation in advance allows those laboratories working with few strains to prepare large batches of cells for later transformation. This is convenient for laboratories using the two-hybrid and other similar systems. |
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A. Making Competent Yeast Cells
1. Grow yeast cells in YPD (10 ml per transformation) to an OD600 of 0.6 to 1.0 from an inoculum of a single colony. This represents a cell density of approximately 0.6 to 1 X 107 cells/ml.
2. Centrifuge cells at 1,000 X g for 5 min in a table-top centrifuge to pellet the cells.
3. Resuspend the cells in 0.5 volume of Solution 1.
4. Centrifuge the cells at 1,000 X g for 5 min to pellet the cells.
5. Resuspend the cells in 0.02 volume of Solution 1 and split the solution into 0.2 ml aliquots.
6. Freeze the aliquots slowly (see Hint #2).
7. After freezing, store the aliquots at -70°C until needed.
B. Transforming Yeast Cells
1. Add 0.1 to 5 μg of plasmid DNA and 50 μg of Carrier DNA in a maximum volume of 20 μl on top of the frozen cell suspension.
2. Place the tube in a 37°C water bath and mix every 10 to 15 sec until the solution begins to melt. Remove the tube from the water bath and warm it between gloved hands until melting is complete.
3. Add 1.4 ml of Solution 2 and mix by vortexing for 1 min.
4. Incubate at 30°C for 1 hr (or permissive temperature if the strain is temperature sensitive).
5. Centrifuge cells at 5,000 rpm for 5 sec in a microcentrifuge.
6. Decant the supernatant and resuspend the pellet in 1 ml of Solution 3.
7. Plate an appropriate amount of cells onto selective media (SC plates).
8. Incubate cells at 30°C (or permissive temperature, if using a temperature sensitive strain). Transformants should appear in 2 to 3 days (see Hint #3).
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| 20% Glucose |
| 20% (w/v) Glucose
Autoclave 30 min
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| SC Plates |
| 6.7 g Yeast Nitrogen Base
900 ml ddH2O
When plates are cool, add appropriate supplements.
Pour plates
20 g Agar
Add 100 ml 20% (w/v) Glucose
Autoclave for 30 min
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| Carrier DNA |
| 10 mg/ml Sonicated Salmon Sperm DNA
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| Solution 3 |
| 10 mM Bicine-NaOH, pH 8.3
Autoclave or filter sterilize
0.15 M NaCl
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| Solution 2 |
| 40% (w/v) PEG 1000
0.2 M Bicine-NaOH, pH 8.3
Autoclave or filter sterilize
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| Solution 1 |
| 10 mM Bicine-NaOH, pH 8.3
1 M Sorbitol
5% (v/v) DMSO (CAUTION! see Hint #1)
Autoclave or filter sterilize
3% (v/v) Ethylene Glycol
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| YPD |
| 10 g/liter Yeast Extract
Autoclave 30 min
20 g/liter Glucose (Dextrose)
20 g/liter Peptone
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Glucose
Sorbitol
DMSO
Yeast Extract
Yeast Nitrogen Base
Peptone
DNA, Salmon Sperm
Ethylene Glycol
Bicine
Polyethylene Glycol (PEG) 1,000
Agar
Sodium Chloride
Sodium Hydroxide
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Slow freezing results in good viability.
3. Preparation of frozen competent cells from the two-hybrid strain PJ69-4A using the method above routinely gives results between 0.9 to 1.0 X 104 transformants/μg DNA. This protocol has been reported to produce up to 105 transformants/μg plasmid DNA.
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