UW-Stout/Heat shock SP23

Pilot Protocol

Materials

• Yeast Culture
• PCR Tubes
• Sterile Water
• Microcentrifuge Tubes
• Micropetters

Equipment

• Thermocycler

Procedure

1. Check the thermocycler is programmed and holding at 50 degrees Celsius
2. Vortex the yeast solutions so that the yeast cells are resuspended
3. Dilute the yeast with Sterile Water to 1 cell per 1 microliters, do this by using 0.3 microliters of the yeast culture and 300 microliters of the sterile water into a microcentrifuge tube
4. Use 50 microliters of the solution into 6 different PCR tubes and label them 5 minutes 10 minutes 15 minutes 20 minutes and 25 minutes and also a control that does not go into the thermocyler
5. Place diluted yeast cells into thermocycler at 50 degrees Celsius for varied amounts of time
6. Label YP+sucrose plates for the minutes of the corresponding PCR tube time
7. Add 5-10 glass beads into each of the YP+ sucrose plates
8. Pipette 50 microliters of the solution onto the corresponding YP+sucrose plate
9. Gently move the solution around on the plates with the glass beads
10. Remove the glass beads
11. Place in the incubator for 48 hours or more
12. Count colonies on petri dish
13. If 100 colonies then yeast was not affected at all
14. If 0 colonies then yeast was affected too harshly, and they all died
15. Record Data and determine what time amount is best

Data Media:KDA_Pilot_Experiment_Plates.jpeg

Wild Yeast Colonies at 50 degrees Celsius

Time (min)	Colonies
0	>100
5	2
10	0
15	0
20	0
25	0

We came to the conclusion that both our temperature was too high and our time was too great for this protocol. The parameter value we decided would be easiest to change was the amount of time spent heat shocking in the thermocycler, because it would be the most consistent and efficient to conduct all our experiments under the same temperature. After doing some more research we changed our time to seconds and our temperature to 40 degrees Celsius.

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

1. Check the thermocycler is programmed and holding at 40 degrees Celsius
2. Vortex the 3 different Knockout Yeast Strands until the yeast cells are resuspended
3. 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.
4. 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.
5. Label YP+Sucrose plates for the different yeast strands and amount of time the certain sample will be in the thermocycler.
6. Add 5-10 glass beads into each of the YP+Sucrose plates
7. 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.
8. Pipette 50 microliters of the PCR tube into the corresponding YP+Sucrose plates,
9. Mix the Solution around with the glass beads carefully.
10. Discard of glass beads
11. Repeat steps 8-10 for all of the plates and samples
12. Place the plates in the incubator upside down for 48 hours or more.
13. 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.