3.3.2 Lab Steps
Published on Dec 01, 2015
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PRESENTATION OUTLINE
LAB STEPS
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9. Put on safety gloves and goggles.
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10. obtain a WT Yeast Strain starter plate and a Mutant Yeast Strain starter plate
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11. Obtain two sterile, capped, plastic test tubes. Label one of the tubes WT Yeast Strain (wild type yeast). Label the other tube Mutant Yeast Strain
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12. Pick up a toothpick from the box. Be careful not to touch one end of the toothpick
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13. Using the clean end of the toothpick, collect a mass of yeast from the starter plate labeled WT yeast strain. The mass of yeast on your toothpick should be about 1 mm in diameter
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14. Smear the mass of yeast as far down as you can possibly reach in the test tube labeled WT Yeast Strain
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15. Obtain a clean toothpick and repeat steps 14-16 using the Mutant Yeast Strain
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16. Obtain a sterile 5 mL pipet. Pull out the pipet from the packet from the bulb end.
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17. Pipet 5 mL of sterile water into each test tube. Make sure to not touch the walls of the tube with the pipet. You do not want to cross-contaminate your tubes. If you do accidentally touch the wall of the tube with the pipet, obtain a new pipet
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18. vortex the tubes until the yeast cells are completely resuspended in the water (the water should look slightly cloudy)
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19. Obtain two YED plates.
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20. Label one plate WT Yeast Strain and one plate Mutant Yeast Strain. Also label both plates with your and your partner’s names. label the lids too.
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22. Open each of the plates with the lid-side down. Shake 4 – 5 glass beads onto the lid of the plates and immediately close and flip the plates back over
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23. Use the 1 mL pipet and pipet 250 μL of each yeast suspension onto the appropriately labeled plate. Use the diagram below to help you determine the 250 μL mark. Use a different pipet for each yeast strain. Close the plates as soon as the yeast suspension has been added
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24. Spread the yeast cells onto the plates by shaking the glass beads back and forth across the entire surface of the plate. Do not use a swirling motion, as this will only run the beads along the edge of the plate.
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25. Let the plates sit until the excess liquid has soaked into the agar.
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26. Hold each plate vertically over a disposal container and open the plates slightly to allow the beads to drop into the container. Immediately close the plate.
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27. Remove the lid from one of the plates and quickly cover the plate with plastic wrap. Make sure the plastic wrap is pulled taut and does not touch the surface of the plate.
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28. Repeat step 27 with the other plate
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30. Place the squares of aluminum foil so that they cover half of the yeast plates. The aluminum foil will shield half of the yeast plates from sun exposure. Use a permanent marker to label which half is shielded from the sun.
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31. Expose the plates to the sun according to Bish
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32. After the plates have been exposed, remove the plastic wrap and aluminum foil and quickly replace the corresponding lids.
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33. Incubate the plates overnight at 30 ̊ C. Replace aluminum foil to prevent additional UV exposure.
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