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Yeast Protocols

Yeast Two-Hybrid Protocols

Several different two-hybrid systems have been developed to study protein function. The garden-variety application is to learn about the function of a given protein by isolating proteins that interact with it, usually by screening a cDNA library. To conduct such an interactor hunt, a protein is expressed in yeast as a fusion to the DNA-binding domain of a transcription factor lacking a transcription activation domain. The DNA-binding fusion protein is generally called the bait . The yeast strain also contains one or more reporter genes with binding sites for the DNA-binding domain. To identify proteins that interact with the bait, a plasmid library that expresses cDNA-encoded proteins fused to a transcription activation domain is introduced into the strain. Interaction of a cDNA-encoded protein with the bait results in activation of the reporter genes, allowing cells containing the interactors to be identified...

Full protocol (PDF)

Preparing Yeast Plates

Amino Acid Mix (40x) use 0.6 g/litre
Tyrosine 2 g, Arginine 4 g, Phenylalanine 2 g, Serine 2 g, Valine 2 g, Proline 2 g, Threonine 4 g, Isoleucine 2 g, Aspartic Acid 2 g...

Full protocol (PDF)

Streaking & Growing Yeast Plates

Before working with any yeast strain, begin with a newly isolated colony to avoid the possibility of working with a contaminated culture. Some yeast strains are unstable (e. g., small YAC-bearing strains) and need to be repurified by streaking on an agar plate and then verifying the genetic content of the isolated colony before proceeding. In cases where the strain is unstable, plan to streak the cells onto the selective medium to retain the desired stock, (however, most strains can be streaked onto the complete medium, YPD)...

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Yeast Sporulation

Sporulation Protocol 1
1. Grow up a small (5mL) overnight culture in YPD (rich glucose media), 30°C.
2. Count the cells the next day, and dilute the culture down to 1 x 106 cells/mL, again into a total volume of 5 mL of rich media.
3. Let the new culture grow at 30°C until it reaches the end of log phase, which is 8-9 x 107 cells/mL...

Full protocol (PDF)

Measuring Optical Density of a Liquid Culture

1. Pipette 1ml clean YPD solution to the cuvette as Blank, which sets the basal reference point for future measurements. Note that solutions containing different particles and displaying different colours will produce different basal O.D. readings. Therefore, it is important to use the same solution as blank to get the most accurate measurement. In this case, we should use clear YPD media, instead of water as blank.
2. Place the cuvette into the spectrophotometer. Note that the cuvette has two different sides, the clear sides and the rough sides. Place the cuvette into the spectrophotometer so that the clear sides align with the direction of light passing through...

Full protocol (PDF)