Induction protocols for GAL1 CUP1 MET17 promoters

Induction protocols for the GAL1, CUP1 and MET17 promoters

Yeast cells containing the relevant transformants were inoculated into 5ml SD medium cultures. Cultures contained 2% raffinose to provide the sugar source and a combination of amino acids depending on the plasmid and selection marker (Ura, His, Met, Trp at 0.2% and Leu at 0.6%).

Cultures were incubated on a shaker overnight at 30oC.

Samples were taken the following day and the OD600 was measured. Specific volumes of the yeast cultures were then taken and inoculated into new 5ml SD raffinose cultures that additionally contained the relevant concentration of inducer/repressor (galactose or copper sulphate or methionine).

The volumes used in these inoculations were calculated so that the OD600 reading obtained prior to testing was 0.6.

After cultures had reached an OD600 of 0.6 samples were washed and re-suspended in PBS

The induction of GAL1, CUP1 and MET17 was then tested by observing the expression of associated fluorescent proteins (GFP and CFP)

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