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.

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:

Tube A B C D E F G Strain Wild 1, YOR387C 2, YKL121W 3, YBR225W 4, YNL018C 5, YLR426W 6, YGL138C

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.