Team:Tongji China/InterLab
Fluorescence data usually cannot be compared because they have been reported in different units or because different groups process data in different ways. This year, we took part in the InterLab study which uses GFP as the measurement marker aiming to address this issue by providing researchers with a detailed protocol and data analysis form that yields absolute units for measurements of GFP in a plate reader.
We characterized 8 plasmids this time:
Negative control (BBa_R0040)
Positive control (BBa_I20270)
Six tested plasmids with GFP fused with promoter or PBS of different strength (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009).
Firstly, we do the calibration and come out with the OD600 reference point, particle standard curve and fluorescence standard curve. After that, we transform these tested devices into DH5α E.coli with the same culturing condition and finally make them the same concentration to test the expression degree of fluorescence.
Experiment 1: OD600 Reference point
Materials:
1 mL LUDOX CL-X (provided in kit)
ddH2O (provided by team)
96-well plate, black with clear flat bottom preferred (provided by team)
Method:
Add 100 μL LUDOX into wells A1, B1, C1, D1
Add 100 μL of ddH2O into wells A2, B2, C2, D2
Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements
Record the data in the table below or in your notebook
Import data into Excel sheet provided (OD600 reference point tab)
Results:
| LUDOX CL-X | H2O | |
| Replicate 1 | 0.069 | 0.039 |
| Replicate 2 | 0.068 | 0.035 |
| Replicate 3 | 0.069 | 0.035 |
| Replicate 4 | 0.069 | 0.036 |
| Arith. Mean | 0.069 | 0.036 |
| Corrected Abs600 | 0.033 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 1.938 |
Experiment 2: Particle Standard Curve
Materials:
300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10^8 microspheres)
ddH2O (provided by team)
96-well plate, black with clear flat bottom preferred (provided by team)
Method:
Prepare the Microsphere Stock Solution:
Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex 4 vigorously for 30 seconds. NOTE: Microspheres should NOT be stored at 0°C or below, as freezing affects the properties of the microspheres.
Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
Add 904 μL of ddH2O to the microspheres
Vortex well. This is your Microsphere Stock Solution.
Prepare the serial dilution of Microspheres:
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN ddH2O ONLY. Initially you will setup the plate with the microsphere stock solution in column 1 and an equal volume of 1x ddH2O in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μL from column to column with good mixing.
Add 100 μL of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
Immediately add 200 μL of microspheres stock solution into A1
Transfer 100 μL of microsphere stock solution from A1 into A2.
Mix A2 by pipetting up and down 3x and transfer 100 μL into A3…
Mix A3 by pipetting up and down 3x and transfer 100 μL into A4...
Mix A4 by pipetting up and down 3x and transfer 100 μL into A5...
Mix A5 by pipetting up and down 3x and transfer 100 μL into A6...
Mix A6 by pipetting up and down 3x and transfer 100 μL into A7...
Mix A7 by pipetting up and down 3x and transfer 100 μL into A8...
Mix A8 by pipetting up and down 3x and transfer 100 μL into A9...
Mix A9 by pipetting up and down 3x and transfer 100 μL into A10...
Mix A10 by pipetting up and down 3x and transfer 100 μL into A11...
Mix A11 by pipetting up and down 3x and transfer 100 μL into liquid waste
Repeat dilution series for rows B, C, D
Re-Mix (Pipette up and down) each row of your plate immediately before putting in the plate reader
Measure Abs600 of all samples in instrument
Record the data in your notebook
Import data into Excel sheet provided (particle standard curve tab)
Results:
Experiment 3: Fluorescence standard curve
Materials:
Fluorescein (provided in kit)
10 ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
96-well plate, black with clear flat bottom (provided by team)
Method:
Prepare the fluorescein stock solution:
Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS
Prepare the serial dilutions of fluorescein:
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN PBS BUFFER ONLY. Initially you will setup the plate with the fluorescein stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μL from column to column with good mixing.
Add 100 μL of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Add 200 μL of fluorescein 1x stock solution into A1, B1, C1, D1
Transfer 100 μl of fluorescein stock solution from A1 into A2.
Mix A2 by pipetting up and down 3x and transfer 100 μL into A3…
Mix A3 by pipetting up and down 3x and transfer 100 μL into A4...
Mix A4 by pipetting up and down 3x and transfer 100 μL into A5...
Mix A5 by pipetting up and down 3x and transfer 100 μL into A6...
Mix A6 by pipetting up and down 3x and transfer 100 μL into A7...
Mix A7 by pipetting up and down 3x and transfer 100 μL into A8...
Mix A8 by pipetting up and down 3x and transfer 100 μL into A9...
Mix A9 by pipetting up and down 3x and transfer 100 μL into A10...
Mix A10 by pipetting up and down 3x and transfer 100 μL into A11...
Mix A11 by pipetting up and down 3x and transfer 100 μL into liquid waste
Repeat dilution series for rows B, C, D
Measure fluorescence of all samples in instrument
Record the data in your notebook
Import data into Excel sheet provided (fluorescein standard curve tab)
Results:
Experiment 4: Cell measurement
Materials:
Competent cells (Escherichia coli strain DH5α )
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
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
Micropipettes and tips
96-well plate, black with clear flat bottom preferred (provided by team)
Methods:
Results:
LUDOX: one tube with 30% colloidal silica suspended in 1 mL of water
1xPBS (phosphate buffered saline)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
50 mL Falcon tube (or equivalent) or 250 mL shake flask for cell growth
1.5 mL eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
Black 96-well plate