Last Week: Dissection and Replica Plating

This past week, I’ve been performing dissections with the yeast I have successfully transformed. Before the yeast can be dissected, they must undergo sporulation, forming four haploid spores. In a dissection, I separate the spores, placing them in columns on a YPD plate. The picture below is from one of the dissections I performed.

DissectionPlate

After letting the spores from the dissection grow, I replica plate the dissection plate. To do this, I get an antibiotic plate matching the antibiotic resistance gene the yeast cells contain. I press the dissection plate onto a piece of velvet fabric, leaving the yeast cells on it. Next, I press the matching antibiotic plate onto the fabric, transferring the yeast cells onto the agar.

I let the yeast on the replica plate grow overnight before checking to see if the dissection was successful. In a successful dissection, two of the yeast spores from the dissection should live while the other two should not. Since the spores are haploid cells produced from a diploid cell, two of the haploid spores should have the antibiotic resistance gene and the gene of interest knocked out while the other two should not. The picture below is the replica plate of the dissection plate depicted above. I still need to work on my technique so the cells are not so sloppily placed on the replica plate.

Replica Plate

I’ve successfully reached this step with a few of my strains. I’ll keep repeating the steps I’ve been performing these past few weeks until I reach this step with all of the strains and genes I am working with. I’ll post a final update soon, detailing what I have accomplished this summer.

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