UW-Stout/Cold Shock
Contents
Protocol: Cold Shock Calibration
Materials
- Bucket filled with ice
- Stop watch
- Wild Type Yeast Strain (BY4735)
- 200 Microliter Tubes(12)
- Pipetter
- 96-Well Assay Plate
Purpose
Using cold shock we will test the wild type yeast, each having been in the ice bath for different times. We can calibrate where the cold shock affected the wild type the most, and use that calibration point to test other yeast strains to see how that cold shocks them compared to the wild type yeast.
Procedure
- Collect tube(s) of yeast and separate out into 11(200 microliter) tubes each containing 100 microliter of yeast.
- With the remaining 100 microliter of yeast, place that into the 1 well of the 96-well assay plate and we will use as our control, wild type yeast without stress.
- With the 11 test tubes each containing 100 microliter of yeast,label them 2-12 and place them in the ice bath. The time increments will increase by 5 minutes. For the first tube with a number 2 on it, it should be in the ice bath for 5 minutes. For the tube with a 3 on it, it should be in the ice bath for 10 minute and so on the pattern continues. The last tube in the ice bath (number 12), should be on the ice for a total of 55 minutes.
- After each one is finished you can begin placing them in the 96-well assay plate along with the yeast without the stress. Place them in the rows from time ranging from smallest to largest.
- The 96-well assay plate is ready to be tested.
Calibration Results
Analyzing our data from the calibration protocol, the yeast in the ice bucket for the longest time(B12) had the most affect on the yeast with out stress(B1). Therefore we will cold shock for 55 minutes in the experiment protocol.
B1 is the beginning of the time, each one from there will go up in five minute increments, the last one will have been on the ice for 55 minutes.
Protocol: Cold Shock Experiment
Materials
- 8 Knockout Yeast Strains
- Wild Type Yeast Strain (BY4735)
- Pipetter
- Bucket filled with ice
- Stop watch
- 96-Well Assay Plate
- 200 Microliter Tubes(9)
Purpose
Using cold we will shock each type of yeast to see if it is more or less sensitive to the stress we did on a wild type of yeast labeled BY4735.
Procedure
- Gather all 9 different yeast strains and place in the hood along with all 200 microliter tubes.
- Pipette out 100 microliter of each yeast strain placing them each into their own 200 microliter tube. *CHANGING TIP OF PIPETTER IN BETWEEN EACH STRAIN*After each strain is in their own tube, they are now ready to be placed on ice.
- Place each tube on ice for 55 minutes.
- After the 55 minutes, transfer each yeast strain into the 96-well assay plate. Each tube gets its own well, and make sure to change tips in between each strain. The 96-well assay plate is ready to be tested.