UW-Stout/Heat Shock SP21
Preliminaries: None
Calibration Experiment Materials:
• Distilled H2O
• Wild-type yeast cells
• P20 and P200 micro pipettors
• PCR test tubes (mini)
• Glass beads
• 15 agar filled petri dishes
Calibration Experiment Procedure:
1. Vortex the yeast culture briefly to resuspend the yeast pellet.
2. Set up 5 different PCR test tubes with 50 ul of wild-strain yeast cells and 50 ul of distilled water in each.
3. Label each tube “A, B, C, D, E”.
4. Insert test tube A in the thermocycler for exactly 30 minutes at 48°C.
5. After test tube A is done cycling, place test tube B in the thermal cycler for exactly 15 minutes at 48° C.
6. After test tube B is done cycling, place test tube C in the thermal cycler for exactly 30 minutes at 48° C.
7. After test tube C is done cycling, place test tube D in the thermal cycler for exactly 45 minutes at 48° C.
8. After test tube D is done cycling, place test tube E in the thermal cycler for exactly 60 minutes at 48° C.
9. Carry on and analyze/count the DNA colonies in the wells.
10. Repeat 3 trials
Knockout Experiment Materials:
• Knockout yeast strains
• Wild-type strain
• 21 Agar filled petri dishes
• Distilled water
• Micro pcr tubes
• Glass beads
Knockout Procedure
1. Set thermocycler to 48°C sitting temperature.
2. Vortex the yeast culture briefly to resuspend the yeast cells.
3. Label 7 pcr tubes A through G.
4. Micropipette 50 ul of water into the pcr tubes.
5. Micropipette 50 ul of each strain into the tubes according to this table:
6. Place pcr tubes into thermocycler for 30 minutes.
7. Label each petri dish accordingly.
8. Micropipette the strains onto the coordinated petri dish.
9. Using 4-5 glass beads, spread the mixture around.
10. Place all the petri dishes in the incubator (30°C).
11. Let sit for 48 hours.
12. Take out and analyze the growth.
13. Repeat for 3 trials.
