215-Lab3B-Yeast_ONs

215-Lab3B-Yeast_ONs - Lab 3B Setting up Yeast ON Cultures...

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Lab 3B – Setting up Yeast ON Cultures 3B-1 Lab #3B Yeast ONs Background: Next week you will set up spotting assays to quantitatively measure the level of silencing of the TRP1 reporter in your mutant strains. To set up those assays you will need to have cultures of the yeast so that you can make serial dilutions of each of the strains. As part of this week’s lab you will set up those cultures. One usually maintains an interesting fragment of DNA (one that contains a gene, gene regulatory sequence, etc.) in a plasmid vector. A plasmid, in turn, is carried in a bacterial or yeast host strain under conditions that maintain a selection for the plasmid. In bacteria this usually means growing cells containing a plasmid in a medium containing an appropriate antibiotic. In yeast this means selecting for an auxotrophic marker on the plasmid. In our case, the plasmid that we will work with contains LEU2 marker gene that will complement a leu2 mutant strain. Therefore, to select for the presence of this plasmid in yeast, we must grow the cells harboring this plasmid in media lacking the amino acid leucine. Without positive selection for the plasmid, the yeast may lose the plasmid containing the Hst1:Sir2 chimera and we will not be able to tell if it has a mutant or wild type phenotype. When comparing the growth rate between strains, it important to take roughly the same quantity of cells at the same stage of growth for each of the samples you are going to test. The first step in this process is to generate a fresh liquid culture of the yeast strain containing the vector. After a growth for a day, all the cultures will reach a saturation point and will therefore contain roughly the same quantity of cells. They will also be at the same stage of growth. This
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215-Lab3B-Yeast_ONs - Lab 3B Setting up Yeast ON Cultures...

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