215-Lab2-Yeast_Streakouts - Lab 2 Yeast Streakouts Lab #2...

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Lab 2 Yeast Streakouts 2-1 Lab #2 Yeast Streak Outs In last week’s lab you plated the mutagenized yeast cultures on selective media (SD-leu, trp) to select for mutants in the Hst1:Sir2 chimera protein that would fail to silence transcription of the hml::TRP1 reporter. Mutants that fail to silence will grow on media lacking tryptophan while the wild type strain should not be able to grow. However, this selection is somewhat “ leaky ” i.e. wild type colonies can also grow but at a slower rate. This is often because there are a large number of cells on the plate that were not transformed with the LEU2 plasmid. Since they cannot grow on the media, they die. Cells that were transformed with a wild type copy of the LEU2 plasmid can grow because they can scavenge tryptophan released by the dead cells. Therefore, even if the TRP1 gene is repressed, these cells can still grow. We therefore need to perform a second round of the screen to distinguish real mutants among the wild type cells. The best way to do this is to streak out the colonies for single cells and examine the differences in growth of these individual colonies. A streak out is exactly like it sounds. 1. Take a sterile toothpick and gently touch one of the colonies on the plate. A yeast colony typically has around 10 6 cells. Touching the toothpick to the colony transfers a large number of the cells onto the toothpick. 2. Gently scrape the toothpick on a fresh plate. Normally this is just a short line (Streak 1 in Fig L2-1). A large number of the cells will transfer from the toothpick to the plate. Often so many cells will transfer that a patch of cells will appear after the plate is incubated for a few days. This patch may contain a number of cells from different parents. 3. Take a new sterile toothpick and drag it in perpendicular direction across the end of the first streak and then extend it a short distance (Streak 2 in Fig L2-1). The toothpick will pick up some cells from the first streak and then spread them on the media as the toothpick is dragged across
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This note was uploaded on 10/23/2011 for the course BIO 201 taught by Professor Frenkel/cervantes during the Spring '10 term at Rutgers.

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215-Lab2-Yeast_Streakouts - Lab 2 Yeast Streakouts Lab #2...

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