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.

Pitri Dish