UW Stout/UV Light FA19
Protocol: Exploring DNA Damage and the Effect of Exposure to UV Light on Yeast Colonies
1. 10 Yeast Cultures and 1 Wild Type
a. WT: BY4736; YAL061W; YBL086C; YBL081W; YBR033W; YBR138C; YBR220C; YHR048W; YGL140C; YJR061W; YKR005C
2. Petri Dishes (44) with YPD Media
3. Autoclavable Flask (1L)
4. 1,000 mL Graduated Cylinder
5. Bacto Agar, Bacto Peptone, Sterile 90% Glucose and Yeast Extract
6. Micro Pipets
7. Sterile H2O
8. Glass Beads
9. Small Tubes (11)
11. PPE- gloves, lab coat, glasses, heat gloves, clean room jacket and shoe covers
1. Electronic Pipet-Aid
3. Stir Plate and Magnetic Stir Bar
4. Biosafety Cabinet
5. Model LS-100-3-UV Exposure System in Clean Room @ UW Stout
Pre-Lab Prep: YPD Media in Agar Plates
1. Obtain a large autoclavable flask and add the following:
a. Bacto Agar- 24g
b. Bacto Peptone- 20g
c. Yeast Extract- 10g
d. Water- 950ml
2. Wrap top of autoclavable flask with tinfoil and tape. Place flask in autoclave, close door, and turn it on. When done, use heat gloves to remove flask from autoclave.
3. Add magnetic stir bar to flask, place on stir plate and turn it on.
4. Add 50 ml of sterile 90% Glucose per 1 L of media and let mix.
5. Move the flask of media into a biosafety hood along with the sleeves of petri dishes, and electronic pipet-aid. Make sure you sterilize everything with the 90% Isopropyl Alcohol before placing it in the hood.
6. While media is still warm, use the electronic pipet-aid to pour into the petri dishes. Allow agar to cool and solidify.
7. Tape together, label, and place in freezer room.
a. Make 44 Plates of YPD Media in Agar Plates
b. After all plates are set, each strain and the wild type will get four plates each. Label each groups’ plates 1, 2, 3, and control.
2. Cell Dilution:
a. Aimed for 100 cells in each petri dish from each strand.
b. Start by taking (x) microliters from a specific strain and calculate the amount of sterile H2O you will need to add in order for there to be 100 cells starting out in each dish.
3. Light Exposure Prep:
a. Take the plates labeled “control” from each group, tape them together, label, turn upside down and store in 37*C incubator. Will be placed in freezer after incubation for counting and comparing to the groups that were exposed.
b. Go to UW Stout Clean Room- put on all PPE required for the room.
4. UV Exposure System:
a. Turn on Illumination Controller and press the start switch, then turn on the Time Box. Let the machine warm up.
b. Set timer to 500.0 seconds and the exposure to 400 watts.
c. Gather one strand, take lids off, place all three plates under the lamp, remove hand, and press the TEST button on the timer to line up the plates under the light.
d. Once plates are in correct position, press TIME EXPOSE on the Time Box and wait until the light goes off. Remove plates, place lids back on, tape together, label, place in 37*C incubator.
e. One group goes at a time, but all three plates are exposed at once.
f. After plates have been incubated, they will be moved to the freezer with the control groups to be counted in the future.
5. Collecting Data:
a.Take all 44 plates from the freezer, take a marker and start counting the colonies on the plates. Record the number of colonies from each plate in the appropriate row. Tape groups of plates back up and place back into the freezer. Take the average from each strand using plates labeled 1, 2, and 3 (do NOT include the control).
b.Graph Results on excel