UW-Stout/Heat Shock FA21

Materials

96 channel well

Yeast cells

PCR test tubes

Media and Plates (all in 475ml H2O)

500 ul of YPD

10g Peptide

5g Yeast Extract

10g Agarose

Equipment

Thermocycler

Incubator at 30°C

P20 and P200 micro pipettes

Multichannel pipette

Protocol Page

Media Creation Procedure

• Measure out 10g of Peptide and put into 500ml jar.

• Measure out 5g of Yeast Extract and put into 500ml jar.

• Measure out 10g of Agarose and put into 500ml jar.

• Then add 475ml of H2O into the 500ml jar.

• Afterwards bring the jar to a laboratory steam sterilizer and put it in for 30 minutes.

• Finally pour the media into plates up to half way.

Heat Shock Procedure

• Calibration Experiment Procedure

• Set thermocycler to 48°C sitting temperature.

• Vortex the yeast culture briefly to resuspend the yeast cells.

• Label 6 pcr tubes A through F.

• Micropipette 90 ul of water into the pcr tubes.

• Micropipette 10 ul of each strain

• Label each tube “A, B, C, D, E, F”.

• Insert all 6 of the tubes into the thermocycler for exactly 42 minutes at 48°C.

• Afterwards remove all of the tubes from the thermocycler.

Dilution Procedure

• In a 96 channel well put in your six yeast strains then put them into the left most column desending from A to F.

• Then in the rest of the rows add 90 ul of H2O into A-F.

• After adding the water take 10 ul of the strains add them to the next column and mix.

Plating Procedure

• Label each petri dish accordingly, first plate "A,B", the Second plate "C,D", and the Third plate "E,F".

• Micropipette 5 ul of the strains onto the coordinated petri dish using the .

• Place all the petri dishes in the incubator (30°C).

• Let sit for 48 hours.

• Take out and analyze the growth.

• Repeat for 3 trials.

Important Remaining Items (Remove Before Publish)

The raw data from the calibration experiment.

Your interpretation of the calibration experiment. Which parameter value did you settle on? Why?