Bio421
Expt 4: Luciferase Gene Expression
Day 1Splitting Cells
Preparation of Cells
1. Pour the media off the flask into the waste container in the hood.
2. Rinse the cells with 3 ml of PBS.
3. Place 2 ml of Trypsin/EDTA onto the cells and incubate at 37C until the cells detach from the flask.
4. Add 2 ml of media and place the cell suspension in a sterile 15 ml conical tube.
5. Centrifuge the cells at 1000 rpm for 2 minutes.
6. Remove the supernatant and resuspend the cells in 5 ml of fresh media.
7. Add 5 ml of fresh media to a 60 mm plate
8. Prepare 1 plate with0.5 ml of the cell suspension.
9. Swirl in a figure “8” and place in the incubator.
Day 4 (Wednesday or Thursday)
Transfection of human colorectal carcinoma cells, HCT-116 cells.
1. Prepare 1 mixture of DNA/FuGene according to the table below. Mix the solution by pipetting one time.
Amount of Media (ul) / Amount DNA (ul) / Amount of FuGene (ul)Group 1 / 100 / 5.0 / 10
Group 2 / 100 / 5.0 / 20
Group 3 / 100 / 10.0 / 20
Group 4 / 100 / 10.0 / 30
Group 5 / 100 / 10.0 / 40
Group 6 / 100 / 20.0 / 60
2.Incubate for 10 minutes at room temperature..
3. Add the DNA-lipid complexes dropwise to the cells and carefully swirl the plate in a figure “8”
4. Incubate the cells 1 or 2 days at 37C.
Bio421
Expt 4: Luciferase Gene Expression
Day 3 (Friday)
Collection and Splitting of Cells
1. Remove the old media and wash with 2 ml of PBS
2. Add 2 ml of Trypsin and incubate at 37C until the cells detach
3. Add 3 ml of ml of media
4. Place 4 ml of the cell suspension into a 15 ml conical tube and 1ml of cell suspension into another 15 ml conical tube.
5. Centrifuge at 1000 X g for 2 minutes.
6. Pour off the supernatents
7. Store the 4 ml cell pellet at -80C.
8. Resuspend the 1 ml cell pellet in 5 ml of media.
9. Put the 5 ml cell suspension into the new plate.
10. Place the new plate in the 37C incubator.
Bio421
Expt 4: Luciferase Gene Expression
Day 4 (Monday or Tuesday)
Preparation of Cell Pellets
1. Resuspend cell pellets in 3 ml of PBS. Centrifuge for 2 minutes at 1K rpm
2. Remove the PBS and replace with 1 ml of PLB.
3. Resuspend the cell pellet and transfer to a microfuge tube.
4. Place on ice.
Preparation of Cells from plates
1. Rinse the plate with 3ml of PBS.
2. Remove the PBS and add 3 ml of Trypsin/EDTA.
3. Incubate at 37C until the cells are detaching from the bottom of the plate (5-10min)
4. Add 7 ml of PBS and transfer the cell suspension to a 15ml conical tube.
5. Centrifuge for 2 minutes at 1K rpm
6. Remove the PBS and replace with 1 ml of PLB.
7. Resuspend the cell pellet and transfer to a microfuge tube.
8. Place on ice.
9. Take both sets of cells and place them in the -70C freezer for 10 minutes.
10. Thaw the cells completely and pipette the samples 10 times.
11. Repeat this freeze-thaw procedure.
Lucfierase Assay
1. Place 100 ul of LAR II solution in a new microfuge tube.
2. Add 20 ul of cell lysate to the tube and mix by pipetting.
3. Read the samples in the luminometer and record the results.