UW-Stout/UV Light SP22

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Introduction

We will be stressing multiple strains of yeast cells with UV light exposure

Materials

  • Phosphate Buffered Saline (PBS)
  • Growth Media (Agarose)
  • Yeast Strains, Knockouts and Wild Type

Equipment

  • P-1000 Micro-pipette
  • P-20 Micro-pipette
  • Bachur & Associates Santa Clara, CA 95050 Model LS-100-3 UV Light Exposure System (400 Watts)
  • Incubator set to 30°C
  • Fine Tip Sharpie
  • 60mm Culture Dish
  • SHAKER THINGY

Calibration Protocol

 Safety Note: When conducting any experiment its important to wear the proper PPE for yourself as well as to protect your experiments in our lab we wore a lab coat and gloves. While we were in our clean room doing the UV Light Exposure we wore additional equipment including: hair nets, coverall top, boot covers and gloves.

Calibration Experiment 1

In our First calibration experiment we conducted UV light exposer based on time, the intervals we used are as follows: 1 second, 3 seconds 5 seconds, 10 seconds, 20 seconds, 50 seconds, 100 seconds, 200 seconds, 500 seconds and 1000 seconds respectively.

  1. Calculate the amount of pure cell culture and amount of PBS in order to get 1000 cells per mL
  2. Using a P-20 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
  3. Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
  4. Mix the PBS and cell culture either by hand or by using a NAME OF THING HERE
  5. Pipette 100uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
  6. Add Glass beads and shake for 30 seconds, the remove glass beads
  7. Turn on the UV Light, set it to 400 Watts
  8. Label cell culture with interval time (1 Second, 2 Seconds, etc.)
  9. Remove top off cell culture plate
  10. Expose the single cell plate for designated time under the UV Light
  11. Repeat Steps 8-10 until out of intervals.
  12. Place cell culture plate in 30 degree C incubator for 48 hours
  13. See Results

Calibration Experiment 2

Due to the RESULTS OF CALIBRATION 1 the time gaps left use needing to do another experiment to test the time between 500 seconds and 1000 seconds. We did new time intervals of 550 seconds, 600 seconds, 650 seconds, 700 seconds, 750 seconds, 800 seconds, 850 seconds and 950 seconds.

  1. Calculate the amount of pure cell culture and amount of PBS in order to get 1000 cells per mL
  2. Using a P-20 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
  3. Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
  4. Mix the PBS and cell culture either by hand or by using a NAME OF THING HERE
  5. Pipette 100uL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
  6. Add Glass beads and shake for 30 seconds, the remove glass beads
  7. Turn on the UV Light, set it to 400 Watts
  8. Label cell culture with interval time (550 Seconds,600 Seconds, etc.)
  9. Remove top off cell culture plate
  10. Expose the single cell plate for designated time under the UV Light
  11. Repeat Steps 8-10 until out of intervals.
  12. Place cell culture plate in 30 degree C incubator for 48 hours
  13. See Results

Calibration Results

Calibration Experiment 1 Results

Results ADD PHOTOS OF 1secound 500 second and 1000 second photos


Interpretation We found that the experiment we conducted was inconclusive. The 500 Second plate had too many yeast cells for us to count and make an accurate count and the 1000 second plate didn't have any yeast colony's on it, we concluded that the the point we want must be between the 500 second mark and the 1000 second mark.

Calibration Experiment 2 Results

Results

  • 550 seconds = 483 colonies
  • 600 seconds = 279 colonies
  • 650 seconds = 198 colonies
  • 700 seconds = 40 colonies
  • 750 seconds = 54 colonies
  • 800seconds = 13 colonies
  • 850 seconds = 4 colonies
  • 900 seconds = 2 colonies
  • 950 seconds = 1 colony

ASHTONMIKOLOSKIYeast Colonys Over Time.png

Interpretation We found that the point we were looking for was at the 600 second mark, with this in mind this will be the amount of time we will use in the knockout protocol.

Knock-out Protocol

Safety Note: When conducting any experiment its important to wear the proper PPE for yourself as well as to protect your experiments in our lab we wore a lab coat and gloves. While we were in our clean room doing the UV Light Exposure we wore additional equipment including: hair nets, coverall top, boot covers and gloves.
  1. Calculate the amount of pure cell culture and amount of PBS in order to get 100 cells per mL
  2. Using a P-10 Micropipette, pipette the amount of cell culture needed into a 1.5mL micropipette tube.
  3. Using a P-1000 Micropipette, pipette the amount of PBS calculated into the same micropipette tube as step 2
  4. Mix the PBS and cell culture either by hand or by using a NAME OF THING HERE
  5. Pipette the full 1mL of the PBS and cell mixture into each 60 mm cell culture plate with agarose in it.
  6. Add Glass beads and shake for 30 seconds, the remove glass beads
  7. Turn on the UV Light, set it to 400 Watts
  8. Label cell culture with the Gene Number
  9. Remove top off cell culture plate
  10. Expose the single cell plate for 600 seconds under the UV Light
  11. Repeat Steps 8-10 until finished
  12. Place cell culture plate in 30 degree C incubator for 48 hours
  13. See Results

Knock-out Results

AshtonMikoloskiFinal Project Graph.png

Photos

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