215-Lab5-Yeast_plasmidprep

215-Lab5-Yeast_plasmidprep - Lab 5 Yeast plasmid rescue Lab...

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Lab 5 – Yeast plasmid rescue 5-1 Lab #5 Plasmid rescue from yeast Today you will purify plasmid DNA from two of the yeast cultures that you started in the previous lab. This is essentially the same protocol as the one described for plasmid DNA minipreps from bacteria, although most of the explanatory notes have been deleted. While this should make the protocol easier to follow in the lab, it is very important that you understand what each step accomplishes. Remember, you are responsible for understanding each protocol used in this course. 1. Obtain your overnight cultures that were set up in the previous lab. These cultures have been spun in a centrifuge to pellet the cells at the bottom of the tube. 2. Pour off the supernatant, being careful to not lose the pellet. Do not worry if there is additional media remaining in the tube. This step is used to concentrate the cells and remove the spent media and cell debris. 3. Using a P-1000, add 500 μ l of sterile water to the pellet and vortex to resuspend the cells. 4. Using a P-1000, transfer the cell suspension to an appropriately labeled microfuge tube. 5. Cap your microfuge tubes and spin them in the microcentrifuge at full speed (14,000 rpm) for 1 minute. 6. After spinning, remove and discard the clear supernatant (sup) by using a P-1000. Remove as much of the supernatant as possible without disturbing the pellet. Discard the clear supernatants into a waste container so they can be poured down the sink later. 7. Resuspend the yeast cell pellet in 250 μl of Buffer P1. Resuspend the pellet with the
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215-Lab5-Yeast_plasmidprep - Lab 5 Yeast plasmid rescue Lab...

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