Difference between revisions of "UW-Stout/Heat Shock SP22"
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===Results=== | ===Results=== | ||
− | [[Image: | + | [[Image:Calibration Experiment Graph.jpg|Calibration Experiment Graph]] |
==Final Protocol Instructions== | ==Final Protocol Instructions== |
Revision as of 21:59, 2 May 2022
Contents
Introduction
A heat shock experiment tests a cell's ability to hold up and survive extreme temperature swings. If one of the 9 genetically modified S. Cerevisiae yeast cells holds up better to a heat shock experiment than the wild type, there are many cases where this is beneficial. The cells are shocked in a thermocycler and then measured using a flow cytometer to so the percent dead and alive.
Materials
- Cultured Genetically Modified Yeast Cells in Solution
- Phosphate buffered saline (PBS) solution.
- Propidium Iodide (100 μg per ml)
Equipment
- Hemocytometer
- Flow cytometer
- Microscope
- 1.7 ml centrifuge tubes
- 200 μl PCR tubes
- P-1000, P-200, P-20, and P-10 Micropipettes and accompanying tips
Calibrartion Experiment
Diluting the Yeast Cell Solution
- Vortex the tube of control yeast cells (BY4735), then immediately pipet 500 μl of the cells into a 1.7 ml centrifuge tube.
- Set up the hemocytometer.
- Count the number of cells in the big square in the top left corner.
- Use a calculator to figure out the volume of the cell solution needed to make a 1 ml solution of 200 cells per μl.
- Vortex the cell solution, then immediately pipet that volume into a 1.7 ml centrifuge tube.
- Pipet the rest of the volume with PBS to make a 1 ml solution.
- Vortex the new cell solution to distribute the cells.
- Label that tube with a checkmark.
Heat Shocking the Yeast Cells
- Set a thermocycler to an incubate cycle with the base at 50 C and the lid at 55 C.
- Set out 6 PCR tubes onto a PCR tube rack.
- Label the first tube 1, second 2, third 5, fourth 10, fifth 20, and sixth 40.
- Vortex the cells in the tube with the checkmark and then immediately pipet 100 μl of the solution into each of the PCR tubes.
- Put the six PCR tubes into the thermocycler and set a timer for 40 minutes.
- Take the PCR tubes out of the thermocycler after each of their times are up.
- 1 tube out after 1 minute, 2 tube out after 2 minutes, etc.
- Allow the cells to cool.
Testing the Cells
- Set out 7 1.7 ml centrifuge tubes.
- Label one with the number 0 and the other six with the times for the PCR tubes.
- Vortex the checkmark tube and immediately pipet 100 μl of the cell solution into the centrifuge tube labeled 0.
- Vortex one of the PCR tubes and immediately pipet 110 μl of cell solution and place it into the centrifuge tube labeled with the corresponding time number.
- The P-200 should be set to 110 μl even though there is only 100 μl of the solution to ensure that all of the liquid is gathered.
- Repeat step 4 for the other PCR tubes.
- Add 6 μl of propidium iodide to each of the centrifuge tubes.
- Vortex all of the tubes to distribute the propidium iodide.
- Follow standard protocol for starting up the flow cytometer.
- Set the machine settings to the following
- FSC, SSC: 15 (low sensitivity)
- GRN: 8, YEL: 8, RED: 8 (low sensitivity)
- 5-decade acquisition
- 5000 events
- Flow rate medium
- Set the graph plots to
- Plot 1: FSC-Log/SSC-Log
- Plot 2: GRN-Log/RED-Log
- Plot 3 (hist): RED-Log
- Vortex the 0 tube and load it into the flow cytometer.
- Run the flow cytometer.
- Run the rest of the tubes in the flow cytometer.
Gathering the Data
- Click on one of the 0 controls.
- Make a polygonal region around the clump of black dots on the graph on the top left.
- Apply that gate to the bottom right graph by clicking and dragging the name from the top-left graph to the bottom right graph.
- Make a new histogram region on the bottom right graph that expands the entire width of the curve.
- Create a new stat for that region.
- Select count and percentage.
- Record the data in a table and dispose of the tube.
Results
Final Protocol Instructions
Diluting the Yeast Cell Solution
- Vortex the tube of cultured yeast cells, then immediately pipet 500 μl of the cells into a 1.7 ml centrifuge tube.
- Set up the hemocytometer.
- Count the number of cells in the big square in the top left corner.
- Use a calculator to figure out the volume of the cell solution needed to make a 1 ml solution of 200 cells per μl.
- Vortex the cell solution, then immediately pipet that volume into a 1.7 ml centrifuge tube.
- Pipet the rest of the volume with PBS to make a 1 ml solution.
- Vortex the new cell solution to distribute the cells.
- Label that tube with a checkmark.
Heat Shocking the Yeast Cells
- Set a thermocycler to an incubate cycle with the base at 50 C and the lid at 55 C.
- Set out 3 PCR tubes onto a PCR tube rack.
- Label the tops of the tubes with the number 5.
- Vortex the cells in the tube with the checkmark and then immediately pipet 100 μl of the solution into each of the PCR tubes.
- Put the three PCR tubes into the thermocycler and set a timer for 5 minutes.
- Take the PCR tubes out of the thermocycler after the 5 minutes are up.
- Allow the cells to cool.
Testing the Cells
- Set out six 1.7 ml centrifuge tubes.
- Label three with the number 0 and the other three with the number 5.
- Vortex the checkmark tube and immediately pipet 100 μl of the cell solution into the centrifuge tubes labeled 0.
- Vortex one of the PCR tubes and immediately pipet 110 μl of cell solution and place it into one of the centrifuge tubes labeled 5.
- The P-200 should be set to 110 μl even though there is only 100 μl of the solution to ensure that all of the liquid is gathered.
- Repeat step 4 for the other two PCR tubes.
- Add 6 μl of propidium iodide to each of the centrifuge tubes.
- Vortex all of the tubes to distribute the propidium iodide.
- Follow standard protocol for starting up the flow cytometer.
- Set the machine settings to the following
- FSC, SSC: 15 (low sensitivity)
- GRN: 8, YEL: 8, RED: 8 (low sensitivity)
- 5-decade acquisition
- 5000 events
- Flow rate medium
- Set the graph plots to
- Plot 1: FSC-Log/SSC-Log
- Plot 2: GRN-Log/RED-Log
- Plot 3 (hist): RED-Log
- Vortex one of the 0 tubes and load it into the flow cytometer.
- Run the flow cytometer.
- Run the rest of the tubes in the flow cytometer.
Gathering the Data
- Click on one of the 0 controls.
- Make a polygonal region around the clump of black dots on the graph on the top left.
- Apply that gate to the bottom right graph by clicking and dragging the name from the top-left graph to the bottom right graph.
- Make a new histogram region on the bottom right graph that expands the entire width of the curve.
- Create a new stat for that region.
- Select count and percentage.
- Record the data in a table and dispose of the tube.