pDDGFP-2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Approximately 30 ng of pDDGFP-2 and 3 μL of 1 ~ 4 PCR fragments of a GPCR (which have a ~30 bp overlapping region with each other) were transformed. Transformants were selected on Ura- plates at 30 °C.

Protocol tips
Approximately 30 ng of pDDGFP-2 and 3 μL of 1 ~ 4 PCR fragments of a GPCR (which have a ~30 bp overlapping region with each other) were transformed. Transformants were selected on Ura- plates at 30 °C.

Publication protocol

"Approximately 30 ng of pDDGFP-2 and 3 μL of 1 ~ 4 PCR fragments of a GPCR (which have a ~30 bp overlapping region with each other) were transformed. Transformants were selected on Ura- plates at 30 °C.

Colonies of transformants harboring the target GPCR were grown in 5 mL of Ura- medium with 2% glucose in 50 mL aerated capped tubes (TPP, Switzerland) at 30 °C overnight. The cultures were diluted to an OD600 of 0.12 and cultured in 10 mL of Ura- medium with 0.1% glucose at 30 °C. At an OD600 of 0.6, galactose was added to the culture to a final concentration of 2%, DMSO was added as needed, and the temperature was lowered to 20 °C as needed. After shaking for 20–22 h at 30 °C (or 40 h at 20 °C), the cells were harvested, and the cell pellets were resuspended in 700 μL of buffer A (50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 10% glycerol, 0.12 M sorbitol, and complete protease inhibitor cocktail [Roche]). The cell suspensions were diluted 20-fold in buffer A, and whole-cell GFP fluorescence was measured with a SpectraMax M2e microplate reader (Molecular Devices, USA) in a 96-well black plate. Fluorescence at an emission wavelength of 525 nm was measured by using a 515 nm cutoff filter after excitation at 490 nm. Purified yEGFP was used as a standard for estimating overexpression."

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