The standard curve of Fluorescein's fluorescence
- microplate reader FLUOstar Omega
- excitation filter: 485 nm
- emission filter: 520 nm
- Fluorescein sodium salt
- Tissue culture microplate (black with flat bottom)
- Prepare fluorescein stock solution
- 1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
- 2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1x PBS.
- 3. Dilute the 2x fluorescein stock solution with 1x PBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM
- Serial dilutions
- 1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12.
- 2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.
- 3. Transfer 100 μl of fluorescein stock solution from A1 into A2.
- 4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3.
- 5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4.
- 6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5.
- 7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6.
- 8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7.
- 9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8.
- 10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9.
- 11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.
- 12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.
- 13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
- (Caution: Do not to continue serial dilution into column 12.)
- Repeat serial dilute for Row B、D、E.
- Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook.
- Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided.
OD600 Reference point
- Thermo ScientificTM MultiskanTM FC Filter-based Microplate Photometer
- Filter: 595 nm
- 1 ml LUDOX
- 96 well cell culture plate (clear with flat-bottom)
- 1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette).
- 2. Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette).
- 3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument.
- 4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided.
- Competent cells (Escherichia coli strain DH5α)
- LB (Luria Bertani) media
- Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg/mL)
- 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block
- Incubator at 37°C
- 1.5 ml eppendorf tubes for sample storage
- Ice bucket with ice
- 96 well plate(cell culture 96 well plate、tissue culture testplate)
- Devices (from InterLab Measurement Kit):
- 1. Negative control(BBa_R0040)
- 2. Positive control(J23151+B0032+E0040+B0010+B0012)
- 3. Test Device 1: J23101+I13504
- 4. Test Device 2: J23106+I13504
- 5. Test Device 3: J23117+I13504
- 6. Test Device 4: J23101+BCD2+E0040+B0015
- 7. Test Device 5: J23106+BCD2+E0040+B0015
- 8. Test Device 6: J23117+BCD2+E0040+B0015
- 1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.
- (Transformation protocol is from iGEM)
- 2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB
- medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.
- 3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures
- 4. Dilute the cultures to a target OD600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).
- 5. Incubate the cultures at 37°C and 170 rpm.
- 6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice.
- 7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD600 and fluorescence measurements using the setup described above.