Detection of Saccharomyces cerevisiae vectors and cloned gene expression products

Summary

LacZ fusion vectors were constructed to study yeast gene regulation. Because of the simplicity and sensitivity of this assay, yeast genes are often labeled with the functional portion of the LacZ gene to indicate the regulatory function of the yeast gene to be studied. The fusion protein is constructed in such a way that the promoter of the yeast gene plus a few amino acid residues at the N-terminal end of the protein encoded by this gene are fused to the carboxyl-terminal end of the LacZ gene, thus encoding a protein fragment that still maintains β-galactosidase activity. When constructing the LacZ fusion gene, it is critical that the junction of the gene fusion ensures that the protein translation reading frame is correct. Source: Compact Molecular Biology Laboratory Guide, Fifth Edition

Operation method

Screening of yeast colonies expressing beta galactosidase using a filter paper extraction assay

Materials and Instruments

Yeast strains containing the lacZ fusion gene
Selection medium plate Liquid nitrogen Z buffer 20 mg mL x-gal dissolved in dimethylformamide
Incubator 30°C Round nitrocellulose filter membrane Whatman 3MM filter paper

Move

1) Spot or line inoculate yeast clones on a culture plate containing appropriate selection medium and incubate at 30°C

until growth to the desired size (usually takes 48 h).

Setting up a double assay helps to increase the reliability of the results.

2) Place a circular nitrocellulose filter membrane on top of the plate and gently press it against the surface of the plate to transfer all colonies to the membrane. It is recommended to use reinforced cellulose nitrate membranes for this step and to do it gently as they become very brittle when placed in liquid nitrogen.

3) Carefully remove the membranes and place them in a shallow dish with liquid nitrogen, just enough to cover one membrane.

4) Carefully remove the membranes from the liquid nitrogen using a tool such as a wide tongue depressor, place the membranes on dry Whatman filter paper and thaw at room temperature (<5min).

5) Cut another piece of Whatman filter paper into a circle and place it in a petri dish. Add 3 ~ 5 mL of Z buffer containing 1 mg/mL λ-gal so that the liquid just soaks the filter paper but does not flood it. Place the thawed nitrocellulose filter membrane on the Whatman filter paper so that the buffer is slowly absorbed into the membrane.

If the Whatman filter paper is flooded with buffer, it will cause colonies to spread and make the results of the screening uninterpretable.

6) Place the petri dish at 30°C and incubate for a few minutes to overnight until the positive colonies turn blue.

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