Team:CCU Taiwan/InterLab

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Fluorescein Fluorescence standard curve

Plate reader

microplate reader FLUOstar Omega
emission filter: 520 nm
excitation filter: 485 nm

Material

Fluorescein sodium salt
1xPBS
Tissue culture testplate (black with flat bottom)

Method

  1. 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 1xPBS.
3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM

  1. 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.)

  1. repeat serial dilute for Row B、D、E
  1. Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook
  1. Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided


Data result







OD600 Reference point

Plate reader

Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer
Filter: 595 nm

Material

1 ml LUDOX
mQH2O
96 well cell culture plate (clear with flat-bottom)

Method

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

Data result



Cell measure

Material

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 light)
Incubator at 37°C
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
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

Method

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 OD 600 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 OD and fluorescence measurements using the setup described above

Data result





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