I want to screen a collection of 96-well plates of yeast (mostly cerevisiae) for sporulation. I was thinking of growing the cells on YPD ON, then transfer the cells into spo media.
But I would like to avoid looking at individual wells under the microscope to look for the tetrads, any idea how I can increase the throughput? Maybe by finding a strain specific to spores, or selection on the size? I just wnat to screen thousands of strains of spo+ or spo-. Thanks!
Maybe you could use a fluorescent DNA dye and pass your cell through a cytometer. The diploids should be bigger and have twice the DNA content than the haploids.
and so the spores should be 4 times bigger? Maybe I could treat my cells with zymolyase and then add the dye so only the spores will "survived"
In theory, the spores will be smaller. If you were talking about tetrads, I think their size is equal to diploid cells. What you could do is run a sample from your unsporulated cells first. Then you could run samples from your sporulated cells treated with zymolyase and compare the two samples. If there is no shift in size/DNA content, you can infer that there is no sporulation. A shift in size/DNA content would indicate that the cells sporulated.
Yes, I could to that. The zymo treatment is to disrupt the membrane of the diploid cells, correct, so we will enrich in tetrads. So we should see bigger cells and a lot of debris.
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