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BISC110: Series 4 Lab 11 Enzymology: Difference between revisions
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BISC110: Series 4 Lab 11 Enzymology (view source)
Revision as of 12:48, 3 December 2009
, 3 December 2009→Procedure for Invertase Assays
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6. Tubes 5–8 will be used to assay the invertase that has been produced by the wild type and mutant yeast cells. The cells have been grown for you and can be found on your bench in four 13mm tubes. Each yeast strain has been grown in both YEP+2% glucose and YEP+0.05% glucose. The cells grow almost 10X better in normal glucose than in minimal glucose, as you'll be able to see from the turbidity of the tubes. In order to assay approximately the same number of cells, pipet 0.5mL of 2% glucose-grown wild type cells into tube 5, and 5mL of 0.05% glucose-grown wild type cells into tube 6. Next pipet 0.5mL of 2% glucose-grown mutant cells into tube 7 and 5mL of 0.05% glucose-grown mutant cells into tube 8. | 6. Tubes 5–8 will be used to assay the invertase that has been produced by the wild type and mutant yeast cells. The cells have been grown for you and can be found on your bench in four 13mm tubes. Each yeast strain has been grown in both YEP+2% glucose and YEP+0.05% glucose. The cells grow almost 10X better in normal glucose than in minimal glucose, as you'll be able to see from the turbidity of the tubes. In order to assay approximately the same number of cells, pipet 0.5mL of 2% glucose-grown wild type cells into tube 5, and 5mL of 0.05% glucose-grown wild type cells into tube 6. Next pipet 0.5mL of 2% glucose-grown mutant cells into tube 7 and 5mL of 0.05% glucose-grown mutant cells into tube 8. | ||
7. Harvest the cells by spinning tubes 5–8 in a clinical (benchtop) centrifuge for | 7. Harvest the cells by spinning tubes 5–8 in a clinical (benchtop) centrifuge for 4 min (the tubes do not have to be covered; they won't break). You must turn the timer knob all the way to the right before bringing it back to 4 min. to get the centrifuge to start and to go at full speed. Remember that the centrifuge must be balanced and that the volumes in the four tubes are not identical. Use your timer as the centrifuge timers are not always accurate. | ||
8. After the spin, the yeast cells will be pelleted at the bottom of the tubes. Do not use pipette to remove supernatant. Invert each tube over the sink, in one quick turn of your wrist so the the tube is completely upside down. The pellet will NOT fall out! Without turning the tube back upright, touch the inverted tube lip to a paper towel to remove a few more drops of media. When the pellet is drained, turn the tube upright. Add 2.5mL of deionized water to each tube to wash the cells. Vortex each tube to resuspend the cells in the water, then spin tubes again for 5min in the centrifuge, and discard supernatant. | 8. After the spin, the yeast cells will be pelleted at the bottom of the tubes. Do not use pipette to remove supernatant. Invert each tube over the sink, in one quick turn of your wrist so the the tube is completely upside down. The pellet will NOT fall out! Without turning the tube back upright, touch the inverted tube lip to a paper towel to remove a few more drops of media. When the pellet is drained, turn the tube upright. Add 2.5mL of deionized water to each tube to wash the cells. Vortex each tube to resuspend the cells in the water, then spin tubes again for 5min in the centrifuge, and discard supernatant. | ||
