| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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| This protocol is used in assaying multiple yeast strains for β-Galactosidase activity directly from patches grown on solid media and is similar to the β-Galactosidase Filter Assay in Yeast protocol. Some researchers find it more convenient to immobilize the filters in Agarose and allow the color development to occur at room temperature, as described in this protocol. This is useful for qualitative screening purposes and can be adapted for high throughput purposes. |
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1. Either directly after autoclaving the Minimal A Medium (2X) and 1.4% Agar solutions, or after melting the 1.4% Agar solution in a microwave, equilibrate the above solutions into a 50°C waterbath. Keep the X-gal solution and 1 M Magnesium Sulfate ready to add.
2. Transfer yeast cells into patches on a YPD or other yeast medium containing plate and grow cells at desired temperature for the appropriate number of days in an incubator (see Hint #4).
3. Using sterile technique, pick up a circle of Whatman 3M filter paper and carefully overlay the plate containing the patches of cells (see Hint #5). As the filter saturates from the moisture on the plate, ensure that the filter uniformly contacts all the patches.
4. After the filter paper is wet, carefully lift the filter off of the patches, turn the filter over, and transfer it to a container of Liquid Nitrogen (see Hint #2). Allow the filter to float and freeze for approximately 1 to 2 minutes.
5. Carefully transfer the frozen filter, cells side up, to an empty petri dish.
6. Once all the filters have been frozen and placed into petri dishes, mix the following solutions for final concentration (see Hint # 6):
an equal volume of Minimal A Medium,
an equal volume of 1.4% Agar,
1 mM Magnesium Sulfate
120 μg/ml X-gal.
7. Carefully pour approximately 20 ml of the mixture from Step #6 over each filter and allow the plates to solidify at room temperature.
8. Examine the plates over the next few hours. Color development is usually well underway after approximately 6 hours.
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| 2% X-Gal Solution |
| 2% (w/v) X-Gal (5-Bromo-4-Chloro-3-Indolyl-Β-D-Galactosidase)
Prepare in Dimethylformamide and store at -20°C. (CAUTION! see Hint #2)
(see Hint #3)
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| 1 M Magnesium Sulfate (1000X) |
| Filter sterilze.
1 M MgSO4
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| 1.4% Agar (2X) |
| 7 g Bactoagar
Add Agar to 500 ml ddH2O, autoclave in a large flask to sterilize (see Hint #1).
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| Minimal A Medium (2X) |
| 9 g Potassium Phosphate Monobasic (KH2PO4)
1 g Sodium Citrate Dihydrate
21 g Potassium Phosphate Dibasic (K2HPO4)
Add components to 1 liter ddH2O, autoclave to sterilize.
2 g Ammonium Sulfate (NH4)2SO4
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Potassium Phosphate, Monobasic
X-Gal
Bactoagar
Sodium Citrate, Dihydrate
Potassium Phosphate, Dibasic
Ammonium Sulfate
Liquid nitrogen
Magnesium Sulfate
Dimethylformamide (DMF)
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1. CAUTION! When autoclaving solutions containing agar or agarose, be sure to use a flask that is at least twice as large as the volume of the solution. At the end of the autoclave cycle, the agar solution can become superheated and will be prone to boiling over very rapidly with agitation.
2. CAUTION! This substance is a biohazard. Please consult with this agent's MSDS for proper handling instructions.
3. After dissolving X-gal in DMF, wrap the container in aluminum foil to protect the solution from light.
4. More consistent results will be obtained when assaying plates that are relatively "fresh" (i.e. contain cells that have just been growing logarithmically).
5. This is important to ensure that the patches assayed are not contaminated and positives can then be transferred off the assayed plate. In preparation for this step, be sure to have the Whatman filters autoclaved in foil. Forceps can be sterilized in alcohol.
6. If there are multiple plates, process the filters in batches. The solutions can be premixed for added convenience; however, X-gal should always be added last and just before pouring onto the filters.
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