FRET - Preparation of the Yeast Strains
We tag proteins with CFP and YFP using the procedure described elsewhere on the web site. The tagged proteins are expressed under the control of their own promoter. They fully replace the wild-type, untagged protein. There is no wild-type gene. This means that for an essential protein the tagged version is functional.
On the day before image acquisition, fresh cells are plated on YPD supplemented with 3X adenine and grown at 30 C. The addition of extra adenine eliminates the red pigment that would otherwise accumulate in our ade2 strains. The next day colonies the size of pin heads are scraped up together and resuspended in 30 ul of SD media. There is no need for sonication before visualization since cell separation is complete under these conditions. A 3 ul aliquot is placed on a 1% SDC low melt agarose pad on a microsope slide. The preparation of the slide is presented on the next few pages.