Procedure Spray the table with 70% ethanol and wipe dry. Write your name and the date on the YES plate. Make a reference mark on the bottom, outer edge of the plate. Place the YES plate on a Petri dish reference grid, aligning your mark to arrow on the grid. Organize your replica plates in HU-/HU+ sets with orientation aligned in the 12 o’clock position. Compare one set at a time. Look for corresponding yeast colonies that have died on the HU+ plate but are still alive on the HU- plate. Use the characteristics listed in Table 1 to identify colonies that contain possible mutants. Using a sterile toothpick, gently scoop the mutant colony (but not the media) from the HU- plate and transfer it one of the squares on the YES plate. Tip: check to make sure the YES plate is properly aligned on the reference grid so potential mutant colonies don’t get mixed together. Repeat the process with the remaining sets of plates. If you are unsure about a colony, go ahead and transfer it. Incubate plate upside down at 30C for 2-3days. Plates will then be refrigerated until the next class. Post-Lab Assignment Explain why you transferred the yeast colonies from the HU- plate and not the HU+ plate. UV radiation mutates DNA in random locations throughout the genome. Why don’t we see other types on mutants on the plates? Part 4: Verification of DNA Repair and Checkpoint Mutants In this lab you will: Replica plate the potential mutants from Part 3 Verify that the selected yeast colonies are DNA repair or checkpoint mutants Materials 1 plate of potential mutants from last class 70% ethanol 1 YES + pB media plate (HU-) disposable gloves 1 YES + pB + HU media plate (HU+) Sharpie marker 1 square sterile velvet pail of soapy water replica block & collar Safety Information Caution: hydroxyurea and phloxine B are hazardous chemicals. Wear disposable gloves during the experiment. Dispose of the master plate in the biohazard waste container
Procedure Spray the table with 70% ethanol and wipe dry. Write your name and the date on the YES plate. Make a reference mark on the bottom, outer edge of the plate. Place the YES plate on a Petri dish reference grid, aligning your mark to arrow on the grid. Organize your replica plates in HU-/HU+ sets with orientation aligned in the 12 o’clock position. Compare one set at a time. Look for corresponding yeast colonies that have died on the HU+ plate but are still alive on the HU- plate. Use the characteristics listed in Table 1 to identify colonies that contain possible mutants. Using a sterile toothpick, gently scoop the mutant colony (but not the media) from the HU- plate and transfer it one of the squares on the YES plate. Tip: check to make sure the YES plate is properly aligned on the reference grid so potential mutant colonies don’t get mixed together. Repeat the process with the remaining sets of plates. If you are unsure about a colony, go ahead and transfer it. Incubate plate upside down at 30C for 2-3days. Plates will then be refrigerated until the next class. Post-Lab Assignment Explain why you transferred the yeast colonies from the HU- plate and not the HU+ plate. UV radiation mutates DNA in random locations throughout the genome. Why don’t we see other types on mutants on the plates? Part 4: Verification of DNA Repair and Checkpoint Mutants In this lab you will: Replica plate the potential mutants from Part 3 Verify that the selected yeast colonies are DNA repair or checkpoint mutants Materials 1 plate of potential mutants from last class 70% ethanol 1 YES + pB media plate (HU-) disposable gloves 1 YES + pB + HU media plate (HU+) Sharpie marker 1 square sterile velvet pail of soapy water replica block & collar Safety Information Caution: hydroxyurea and phloxine B are hazardous chemicals. Wear disposable gloves during the experiment. Dispose of the master plate in the biohazard waste container
soakin_up_rays_student.docx