UW-Stout/Heat shock SP23
Final Protocol
Materials
- 3 different Knockout Yeast strands
- 18 PCR Tubes
- 3 Microcentrifuge Tubes
- Sterile Water
- 18 YP+Sucrose Plates
- Glass beads
- Sharpie
Equipment
- Thermocycler
- Vortex
- Micropipettes
- Fume hood
- Incubator
Procedure
- Check the thermocycler is programmed and holding at 40 degrees Celsius
- Vortex the 3 different Knockout Yeast Strands until the yeast cells are resuspended
- Under the fume hood, dilute the yeast with Sterile Water to 1 cell per 1 microliter. This is done by adding 0.3 microliters of the yeast culture to 300 microliters of sterile water into a microcentrifuge tube. Repeat this step for all three of the different yeast strands into three separate microcentrifuge tubes.
- Under the fume hood, pippete 50 microliters of the yeast and sterile water solution into a PCR tube, repeat this 6 times. Then, label them Control, 15, 45, 1:15, 1:45, and 2:15. Repeat this whole process three times, with a differentiator between the different yeast strands.
- Label YP+Sucrose plates for the different yeast strands and amount of time the certain sample will be in the thermocycler.
- Add 5-10 glass beads into each of the YP+Sucrose plates
- Place the PCR tubes in the thermocycler and remove are varied time amount, 15 seconds, 45 seconds, 1 minute 15 seconds, 1 minute 45 seconds, and 2 minutes 15 seconds.
- Pipette 50 microliters of the PCR tube into the corresponding YP+Sucrose plates,
- Mix the Solution around with the glass beads carefully.
- Discard of glass beads
- Repeat steps 8-10 for all of the plates and samples
- Place the plates in the incubator upside down for 48 hours or more.
- Record data by counting the colonies on the YP+sucrose plates.
- If 100 colonies then yeast was not affected at all by the varied amount of time of heat shock.
- If 0 colonies then the yeast was killed off and was not able to survive the heat shock.
