InterLab
Interlab studies is a great initiative taken by the iGEM headquaters. Reproducibilty is a very big problem in biology. Repeating a result is not always a simple task.Repeating the research can both correct inaccuracies and deepen our understanding of our work.
Here is the protocol that we followed and our results:
Materials:
- 1ml LUDOX (provided in kit)
- H20 (provided by team)
- 96 well plate, black with flat, transparent/clear bottom preferred (provided by team)
Method
- Add 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
- Add 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
- Measure absorbance 600 nm of all samples in all standard measurement modes in
instrument
- Record the data in the table below or in your notebook
- Import data into Excel (OD600 reference point tab) Sheet_1 provided
Materials:
- fluorescein (provided in kit)
- 10ml 1xPBS (phosphate buffered saline; provided by team)
- Corning 96 well plate, black,flat bottom,half-well
Method:
Prepare the fluorescein stock solution:
- Spin down fluorescein stock tube to make sure pellet is at the bottom of tube
- Prepare 2x fluorescein stock solution (100 µM) by resuspending fluorescein in 1
mL of 1xPBS. [Note: it is important that the fluorescein is properly dissolved. To
check this, after the resuspension you should pipette up and down and examine the
solution in the pipette tip – if any particulates are visible in the pipette tip continue
to mix the solution until they disappear.
- Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein
solution and resulting concentration of fluorescein stock solution 50 µM (500µL of
2x fluorescein in 500 µL 1x PBS will make 1 mL of 50 µM (1x) fluorescein solution.)
Prepare the serial dilutions of fluorescein:
- 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
- Measure fluorescence of all samples in all standard measurement modes in instrumen
- Record the data in your noteboo
- Import data into Excel (fluorescein standard curve tab) Sheet_1 provided
- Plate reader settings:
- Light source: Xenon flash lamp
- Excitation wavelength-480nm
- Emission wavelength-510nm
- Measurement time-100ms
- Excitation bandwidth-5nm
- Emission bandwidth-12nm
- 1ml LUDOX (provided in kit)
- H20 (provided by team)
- 96 well plate, black with flat, transparent/clear bottom preferred (provided by team)
Method
- Add 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
- Add 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
- Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
- Record the data in the table below or in your notebook
- Import data into Excel (OD600 reference point tab) Sheet_1 provided
Materials:
- fluorescein (provided in kit)
- 10ml 1xPBS (phosphate buffered saline; provided by team)
- Corning 96 well plate, black,flat bottom,half-well
Method:
Prepare the fluorescein stock solution:
- Spin down fluorescein stock tube to make sure pellet is at the bottom of tube
- Prepare 2x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. [Note: it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.
- Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 µM (500µL of 2x fluorescein in 500 µL 1x PBS will make 1 mL of 50 µM (1x) fluorescein solution.)
Prepare the serial dilutions of fluorescein:
- 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
- Measure fluorescence of all samples in all standard measurement modes in instrumen
- Record the data in your noteboo
- Import data into Excel (fluorescein standard curve tab) Sheet_1 provided
- Plate reader settings:
- Light source: Xenon flash lamp
- Excitation wavelength-480nm
- Emission wavelength-510nm
- Measurement time-100ms
- Excitation bandwidth-5nm
- Emission bandwidth-12nm
Materials:
- Competent cells (Escherichia coli strain DH5α)
- LB (Luria Bertani) media
- Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/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
- Corning 96 well plate,transparent,full well,flat bottom
- Corning 96 well plate,black,half well,flat bottom
- Positive control
- Negative control
- Test Device 1: J23101+I13504
- Test Device 2: J23106+I13504
- Test Device 3: J23117+I13504
- Test Device 4: J23101.BCD2.E0040.B0015
- Test Device 5: J23106.BCD2.E0040.B0015
- Test Device 6: J23117.BCD2.E0040.B0015
Devices (from InterLab Measurement Kit):
Method
Day 1: transform Escherichia coli DH5α with these following plasmids:
- Positive control
- Negative control
- Test Device 1: J23101+I13504
- Test Device 2: J23106+I13504
- Test Device 3: J23117+I13504
- Test Device 4: J23101.BCD2.E0040.B0015
- Test Device 5: J23106.BCD2.E0040.B0015
- Test Device 6: J23117.BCD2.E0040.B0015
Day 2: Pick 2 colonies from each of plate and inoculate it on 5mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
Day 3: Cell growth, sampling, and assay
- Set your instrument to read OD600 (as OD calibration setting)
- Measure OD600 of the overnight cultures
- Record data in your notebook
- Import data into Excel (Dilution Calculation)
- 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).
- Incubate the cultures at 37°C and 220 rpm.
- Take 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time point, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 samples per time point)
- Place samples on ice or 4℃ fridge
- At the end of sampling point you need to measure your samples (OD and Fl measurement)
- Take two different plates(black and transparent), load 100µL of sample on black plate for fluorescence measurement and 120µL in transparent plate for OD measurement
- Record data in your notebook
- Import data into Excel
uM Fluorescein | 50.00 | 25 | 12.5 | 6.25 | 3.125 | 1.5625 | 0.78125 | 0.390625 | 0.195313 | 0.097656 | 0.048828 | 0 |
Replicate 1 | 13414.1 | 11455.1 | 8410.2 | 5255.2 | 2576.53 | 1581.79 | 674.3 | 364.496 | 219.688 | 110.455 | 42.6915 | 0.116654 |
Replicate 2 | 13484.6 | 11463.9 | 8163.66 | 4963.2 | 2863.6 | 1566.24 | 705.598 | 438.018 | 189.537 | 88.2177 | 47.6698 | 0.181143 |
Replicate 3 | 13468.1 | 11541.2 | 8193.38 | 5177.1 | 2939.83 | 1587.29 | 823.477 | 422.591 | 233.03 | 115.595 | 54.3853 | 0.152354 |
Replicate 4 | 13574.4 | 9851.55 | 6476.66 | 5177.48 | 2949.9 | 1585.38 | 828.868 | 426.119 | 221.582 | 117.785 | 58.7254 | 0.159733 |
Arith. Mean | 13485.3 | 11077.94 | 7810.975 | 5143.245 | 2832.465 | 1580.175 | 758.0608 | 412.806 | 215.9593 | 108.0132 | 50.868 | 0.152471 |
Arith. Std.Dev. | 66.59474 | 818.5062 | 896.3049 | 125.5233 | 174.9193 | 9.565697 | 79.71022 | 32.87594 | 18.57476 | 13.54986 | 7.099149 | 0.026818 |
Experimental Values: | Raw Abs600 | Raw Fluorescence | |||||||||||||||||||||||||||
Sample set: | Replicate 1 | Replicate 2 | Replicate 3 | Replicate 4 | Replicate 1 | Replicate 2 | Replicate 3 | Replicate 4 | |||||||||||||||||||||
Blank media | 0.039 | 0.038 | 0.038 | 0.038 | 11.92 | 11.32 | 11.99 | 11.91 | |||||||||||||||||||||
Blank mean: | 0.03825 | 11.785 | OD - Background | Fluorescence - Background | uM Fluorescein / OD600 | Summary Statistics | |||||||||||||||||||||||
Hour 0: | Replicate 1 | Replicate 2 | Replicate 3 | Replicate 4 | Replicate 1 | Replicate 2 | Replicate 3 | Replicate 4 | Replicate 1 | Replicate 2 | Replicate 3 | Replicate 4 | Arith. Mean | Arith. Std.Dev. | Geo. Mean | Geo. Std. Dev. | |||||||||||||
Negative Control (Colony 1) | 0.047 | 0.047 | 0.047 | 0.047 | 12.75 | 12.83 | 13.35 | 12.82 | 0.00875 | 0.00875 | 0.00875 | 0.00875 | 0.965 | 1.045 | 1.565 | 1.035 | 0.10 | 0.10 | 0.16 | 0.10 | 0.12 | 0.03 | 0.11 | 1.246 | |||||
Negative Control (Colony 2) | 0.047 | 0.047 | 0.047 | 0.046 | 10.89 | 9.781 | 7.717 | 12.44 | 0.00875 | 0.00875 | 0.00875 | 0.00775 | -0.895 | -2.004 | -4.068 | 0.655 | -0.09 | -0.20 | -0.41 | 0.07 | -0.16 | 0.20 | #NUM! | #NUM! | |||||
Positive Control (Colony 1) | 0.047 | 0.048 | 0.047 | 0.047 | 12.13 | 12.44 | 13.99 | 13.51 | 0.00875 | 0.00975 | 0.00875 | 0.00875 | 0.345 | 0.655 | 2.205 | 1.725 | 0.03 | 0.06 | 0.22 | 0.17 | 0.12 | 0.09 | 0.09 | 2.410 | |||||
Positive Control (Colony 2) | 0.049 | 0.047 | 0.046 | 0.046 | 13.46 | 12.61 | 13.06 | 13.77 | 0.01075 | 0.00875 | 0.00775 | 0.00775 | 1.675 | 0.825 | 1.275 | 1.985 | 0.14 | 0.08 | 0.14 | 0.22 | 0.15 | 0.06 | 0.14 | 1.505 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 0.049 | 0.05 | 0.048 | 0.049 | 18.19 | 20.69 | 19.15 | 19.37 | 0.01075 | 0.01175 | 0.00975 | 0.01075 | 6.405 | 8.905 | 7.365 | 7.585 | 0.52 | 0.66 | 0.66 | 0.62 | 0.62 | 0.07 | 0.61 | 1.119 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 0.047 | 0.046 | 0.047 | 0.046 | 21.62 | 19.76 | 20.76 | 18.28 | 0.00875 | 0.00775 | 0.00875 | 0.00775 | 9.835 | 7.975 | 8.975 | 6.495 | 0.99 | 0.90 | 0.90 | 0.73 | 0.88 | 0.10 | 0.88 | 1.132 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.047 | 0.046 | 0.047 | 0.047 | 13.95 | 14.37 | 14.62 | 14.54 | 0.00875 | 0.00775 | 0.00875 | 0.00875 | 2.165 | 2.585 | 2.835 | 2.755 | 0.22 | 0.29 | 0.28 | 0.28 | 0.27 | 0.03 | 0.27 | 1.147 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.048 | 0.047 | 0.046 | 0.047 | 14.38 | 16.27 | 13.38 | 15.53 | 0.00975 | 0.00875 | 0.00775 | 0.00875 | 2.595 | 4.485 | 1.595 | 3.745 | 0.23 | 0.45 | 0.18 | 0.38 | 0.31 | 0.12 | 0.29 | 1.523 | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.048 | 0.048 | 0.071 | 0.049 | 11.44 | 12.69 | 14.09 | 11.64 | 0.00975 | 0.00975 | 0.03275 | 0.01075 | -0.345 | 0.905 | 2.305 | -0.145 | -0.03 | 0.08 | 0.06 | -0.01 | 0.03 | 0.05 | #NUM! | #NUM! | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.047 | 0.046 | 0.045 | 0.051 | 13.57 | 13.91 | 12.65 | 13.24 | 0.00875 | 0.00775 | 0.00675 | 0.01275 | 1.785 | 2.125 | 0.865 | 1.455 | 0.18 | 0.24 | 0.11 | 0.10 | 0.16 | 0.06 | 0.15 | 1.504 | |||||
Test Device 4: J23101+I13504 (Colony 1) | 0.045 | 0.046 | 0.047 | 0.047 | 14.09 | 13.1 | 14.81 | 13.72 | 0.00675 | 0.00775 | 0.00875 | 0.00875 | 2.305 | 1.315 | 3.025 | 1.935 | 0.30 | 0.15 | 0.30 | 0.19 | 0.24 | 0.08 | 0.23 | 1.416 | |||||
Test Device 4: J23101+I13504 (Colony 2) | 0.048 | 0.046 | 0.046 | 0.046 | 15.34 | 14.29 | 13.91 | 13.42 | 0.00975 | 0.00775 | 0.00775 | 0.00775 | 3.555 | 2.505 | 2.125 | 1.635 | 0.32 | 0.28 | 0.24 | 0.18 | 0.26 | 0.06 | 0.25 | 1.267 | |||||
Test Device 5: J23106+I13504 (Colony 1) | 0.046 | 0.046 | 0.047 | 0.046 | 13.8 | 11.54 | 12.31 | 10.5 | 0.00775 | 0.00775 | 0.00875 | 0.00775 | 2.015 | -0.245 | 0.525 | -1.285 | 0.23 | -0.03 | 0.05 | -0.15 | 0.03 | 0.16 | #NUM! | #NUM! | |||||
Test Device 5: J23106+I13504 (Colony 2) | 0.047 | 0.047 | 0.046 | 0.046 | 13.06 | 14.03 | 13.71 | 13.77 | 0.00875 | 0.00875 | 0.00775 | 0.00775 | 1.275 | 2.245 | 1.925 | 1.985 | 0.13 | 0.22 | 0.22 | 0.22 | 0.20 | 0.05 | 0.19 | 1.320 | |||||
Test Device 6: J23117+I13504 (Colony 1) | 0.046 | 0.047 | 0.047 | 0.047 | 13.49 | 12.07 | 12.23 | 12.83 | 0.00775 | 0.00875 | 0.00875 | 0.00875 | 1.705 | 0.285 | 0.445 | 1.045 | 0.19 | 0.03 | 0.04 | 0.10 | 0.09 | 0.07 | 0.07 | 2.353 | |||||
Test Device 6: J23117+I13504 (Colony 2) | 0.046 | 0.044 | 0.044 | 0.044 | 13.24 | 12.34 | 13.56 | 13.93 | 0.00775 | 0.00575 | 0.00575 | 0.00575 | 1.455 | 0.555 | 1.775 | 2.145 | 0.16 | 0.08 | 0.27 | 0.33 | 0.21 | 0.11 | 0.19 | 1.829 | |||||
Hour 2: | |||||||||||||||||||||||||||||
Negative Control (Colony 1) | 0.055 | 0.068 | 0.054 | 0.053 | 13.36 | 12.83 | 12.3 | 12.85 | 0.01675 | 0.02975 | 0.01575 | 0.01475 | 1.575 | 1.045 | 0.515 | 1.065 | 0.08 | 0.03 | 0.03 | 0.06 | 0.05 | 0.03 | 0.05 | 1.690 | |||||
Negative Control (Colony 2) | 0.232 | 0.209 | 0.148 | 0.054 | 13.54 | 12.85 | 13.88 | 11.16 | 0.19375 | 0.17075 | 0.10975 | 0.01575 | 1.755 | 1.065 | 2.095 | -0.625 | 0.01 | 0.01 | 0.02 | -0.03 | 0.00 | 0.02 | #NUM! | #NUM! | |||||
Positive Control (Colony 1) | 0.058 | 0.053 | 0.053 | 0.052 | 20.39 | 19.68 | 20.97 | 21 | 0.01975 | 0.01475 | 0.01475 | 0.01375 | 8.605 | 7.895 | 9.185 | 9.215 | 0.38 | 0.47 | 0.55 | 0.59 | 0.50 | 0.09 | 0.49 | 1.210 | |||||
Positive Control (Colony 2) | 0.152 | 0.104 | 0.053 | 0.054 | 21.64 | 20.63 | 19.6 | 20.02 | 0.11375 | 0.06575 | 0.01475 | 0.01575 | 9.855 | 8.845 | 7.815 | 8.235 | 0.08 | 0.12 | 0.46 | 0.46 | 0.28 | 0.21 | 0.21 | 2.540 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 0.051 | 0.054 | 0.049 | 0.049 | 26.75 | 29.96 | 29.37 | 29.77 | 0.01275 | 0.01575 | 0.01075 | 0.01075 | 14.965 | 18.175 | 17.585 | 17.985 | 1.03 | 1.01 | 1.43 | 1.47 | 1.24 | 0.25 | 1.22 | 1.226 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 0.204 | 0.193 | 0.195 | 0.136 | 29.84 | 32.35 | 33.21 | 26.81 | 0.16575 | 0.15475 | 0.15675 | 0.09775 | 18.055 | 20.565 | 21.425 | 15.025 | 0.10 | 0.12 | 0.12 | 0.13 | 0.12 | 0.02 | 0.12 | 1.154 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.052 | 0.05 | 0.05 | 0.05 | 31.21 | 29.42 | 29.86 | 31.01 | 0.01375 | 0.01175 | 0.01175 | 0.01175 | 19.425 | 17.635 | 18.075 | 19.225 | 1.24 | 1.32 | 1.35 | 1.43 | 1.33 | 0.08 | 1.33 | 1.063 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.054 | 0.076 | 0.053 | 0.052 | 30.14 | 35.8 | 34.28 | 31.55 | 0.01575 | 0.03775 | 0.01475 | 0.01375 | 18.355 | 24.015 | 22.495 | 19.765 | 1.02 | 0.56 | 1.34 | 1.26 | 1.04 | 0.35 | 0.99 | 1.491 | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.054 | 0.055 | 0.057 | 0.054 | 14.69 | 14.47 | 16.36 | 16.08 | 0.01575 | 0.01675 | 0.01875 | 0.01575 | 2.905 | 2.685 | 4.575 | 4.295 | 0.16 | 0.14 | 0.21 | 0.24 | 0.19 | 0.05 | 0.18 | 1.278 | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.055 | 0.054 | 0.053 | 0.052 | 17.35 | 16.78 | 18.05 | 15.31 | 0.01675 | 0.01575 | 0.01475 | 0.01375 | 5.565 | 4.995 | 6.265 | 3.525 | 0.29 | 0.28 | 0.37 | 0.22 | 0.29 | 0.06 | 0.29 | 1.230 | |||||
Test Device 4: J23101+I13504 (Colony 1) | 0.054 | 0.053 | 0.052 | 0.052 | 21.9 | 22.44 | 23.34 | 23.84 | 0.01575 | 0.01475 | 0.01375 | 0.01375 | 10.115 | 10.655 | 11.555 | 12.055 | 0.56 | 0.63 | 0.74 | 0.77 | 0.68 | 0.09 | 0.67 | 1.154 | |||||
Test Device 4: J23101+I13504 (Colony 2) | 0.051 | 0.052 | 0.051 | 0.05 | 25.2 | 25.93 | 25.8 | 24.39 | 0.01275 | 0.01375 | 0.01275 | 0.01175 | 13.415 | 14.145 | 14.015 | 12.605 | 0.92 | 0.90 | 0.96 | 0.94 | 0.93 | 0.03 | 0.93 | 1.029 | |||||
Test Device 5: J23106+I13504 (Colony 1) | 0.055 | 0.053 | 0.051 | 0.053 | 22.94 | 22.74 | 21.48 | 21.35 | 0.01675 | 0.01475 | 0.01275 | 0.01475 | 11.155 | 10.955 | 9.695 | 9.565 | 0.58 | 0.65 | 0.67 | 0.57 | 0.62 | 0.05 | 0.62 | 1.082 | |||||
Test Device 5: J23106+I13504 (Colony 2) | 0.057 | 0.056 | 0.055 | 0.055 | 22.02 | 22.25 | 22.51 | 21.09 | 0.01875 | 0.01775 | 0.01675 | 0.01675 | 10.235 | 10.465 | 10.725 | 9.305 | 0.48 | 0.52 | 0.56 | 0.49 | 0.51 | 0.04 | 0.51 | 1.075 | |||||
Test Device 6: J23117+I13504 (Colony 1) | 0.053 | 0.054 | 0.056 | 0.055 | 16.19 | 15.64 | 16.24 | 16.07 | 0.01475 | 0.01575 | 0.01775 | 0.01675 | 4.405 | 3.855 | 4.455 | 4.285 | 0.26 | 0.21 | 0.22 | 0.22 | 0.23 | 0.02 | 0.23 | 1.094 | |||||
Test Device 6: J23117+I13504 (Colony 2) | 0.051 | 0.051 | 0.049 | 0.05 | 16.13 | 16.2 | 16.26 | 17.05 | 0.01275 | 0.01275 | 0.01075 | 0.01175 | 4.345 | 4.415 | 4.475 | 5.265 | 0.30 | 0.30 | 0.36 | 0.39 | 0.34 | 0.05 | 0.34 | 1.145 | |||||
Hour 4: | |||||||||||||||||||||||||||||
Negative Control (Colony 1) | 0.191 | 0.15 | 0.134 | 0.139 | 14.45 | 15.48 | 14.63 | 13.73 | 0.15275 | 0.11175 | 0.09575 | 0.10075 | 2.665 | 3.695 | 2.845 | 1.945 | 0.02 | 0.03 | 0.03 | 0.02 | 0.02 | 0.01 | 0.02 | 1.370 | |||||
Negative Control (Colony 2) | 0.147 | 0.161 | 0.151 | 0.142 | 14.89 | 15.77 | 16.32 | 15.74 | 0.10875 | 0.12275 | 0.11275 | 0.10375 | 3.105 | 3.985 | 4.535 | 3.955 | 0.03 | 0.03 | 0.04 | 0.03 | 0.03 | 0.00 | 0.03 | 1.169 | |||||
Positive Control (Colony 1) | 0.184 | 0.139 | 0.13 | 0.127 | 50.64 | 49.12 | 44.43 | 39.3 | 0.14575 | 0.10075 | 0.09175 | 0.08875 | 38.855 | 37.335 | 32.645 | 27.515 | 0.23 | 0.32 | 0.31 | 0.27 | 0.29 | 0.04 | 0.28 | 1.161 | |||||
Positive Control (Colony 2) | 0.157 | 0.14 | 0.133 | 0.134 | 58.28 | 57.94 | 52.46 | 52.37 | 0.11875 | 0.10175 | 0.09475 | 0.09575 | 46.495 | 46.155 | 40.675 | 40.585 | 0.34 | 0.40 | 0.38 | 0.37 | 0.37 | 0.02 | 0.37 | 1.063 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 0.057 | 0.056 | 0.055 | 0.054 | 48.17 | 41.93 | 44.44 | 43.61 | 0.01875 | 0.01775 | 0.01675 | 0.01575 | 36.385 | 30.145 | 32.655 | 31.825 | 1.70 | 1.49 | 1.71 | 1.77 | 1.67 | 0.12 | 1.66 | 1.079 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 0.056 | 0.054 | 0.054 | 0.052 | 46.69 | 50.03 | 47.27 | 45.78 | 0.01775 | 0.01575 | 0.01575 | 0.01375 | 34.905 | 38.245 | 35.485 | 33.995 | 1.72 | 2.13 | 1.97 | 2.17 | 2.00 | 0.20 | 1.99 | 1.110 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.143 | 0.13 | 0.129 | 0.116 | 76.83 | 75.98 | 72.69 | 70.29 | 0.10475 | 0.09175 | 0.09075 | 0.07775 | 65.045 | 64.195 | 60.905 | 58.505 | 0.54 | 0.61 | 0.59 | 0.66 | 0.60 | 0.05 | 0.60 | 1.084 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.131 | 0.106 | 0.1 | 0.096 | 75.71 | 70.61 | 69.71 | 75.01 | 0.09275 | 0.06775 | 0.06175 | 0.05775 | 63.925 | 58.825 | 57.925 | 63.225 | 0.60 | 0.76 | 0.82 | 0.96 | 0.79 | 0.15 | 0.78 | 1.213 | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.208 | 0.152 | 0.144 | 0.126 | 18.56 | 17.22 | 16.66 | 15.38 | 0.16975 | 0.11375 | 0.10575 | 0.08775 | 6.775 | 5.435 | 4.875 | 3.595 | 0.03 | 0.04 | 0.04 | 0.04 | 0.04 | 0.00 | 0.04 | 1.092 | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.155 | 0.131 | 0.114 | 0.115 | 16.97 | 16.11 | 16.05 | 17 | 0.11675 | 0.09275 | 0.07575 | 0.07675 | 5.185 | 4.325 | 4.265 | 5.215 | 0.04 | 0.04 | 0.05 | 0.06 | 0.05 | 0.01 | 0.05 | 1.214 | |||||
Test Device 4: J23101+I13504 (Colony 1) | 0.115 | 0.118 | 0.099 | 0.1 | 46.38 | 44.29 | 43.31 | 42.04 | 0.07675 | 0.07975 | 0.06075 | 0.06175 | 34.595 | 32.505 | 31.525 | 30.255 | 0.40 | 0.36 | 0.45 | 0.43 | 0.41 | 0.04 | 0.41 | 1.111 | |||||
Test Device 4: J23101+I13504 (Colony 2) | 0.107 | 0.103 | 0.099 | 0.094 | 46.3 | 44.96 | 44.81 | 42.31 | 0.06875 | 0.06475 | 0.06075 | 0.05575 | 34.515 | 33.175 | 33.025 | 30.525 | 0.44 | 0.45 | 0.48 | 0.48 | 0.46 | 0.02 | 0.46 | 1.044 | |||||
Test Device 5: J23106+I13504 (Colony 1) | 0.119 | 0.112 | 0.122 | 0.117 | 24.84 | 25.74 | 27.41 | 27.58 | 0.08075 | 0.07375 | 0.08375 | 0.07875 | 13.055 | 13.955 | 15.625 | 15.795 | 0.14 | 0.17 | 0.16 | 0.18 | 0.16 | 0.01 | 0.16 | 1.096 | |||||
Test Device 5: J23106+I13504 (Colony 2) | 0.139 | 0.125 | 0.147 | 0.135 | 24.09 | 22.96 | 26.29 | 26.06 | 0.10075 | 0.08675 | 0.10875 | 0.09675 | 12.305 | 11.175 | 14.505 | 14.275 | 0.11 | 0.11 | 0.12 | 0.13 | 0.12 | 0.01 | 0.12 | 1.083 | |||||
Test Device 6: J23117+I13504 (Colony 1) | 0.137 | 0.145 | 0.142 | 0.125 | 13.65 | 14.24 | 13.76 | 14.66 | 0.09875 | 0.10675 | 0.10375 | 0.08675 | 1.865 | 2.455 | 1.975 | 2.875 | 0.02 | 0.02 | 0.02 | 0.03 | 0.02 | 0.01 | 0.02 | 1.301 | |||||
Test Device 6: J23117+I13504 (Colony 2) | 0.11 | 0.111 | 0.11 | 0.116 | 14.42 | 14.08 | 13.93 | 14.6 | 0.07175 | 0.07275 | 0.07175 | 0.07775 | 2.635 | 2.295 | 2.145 | 2.815 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 0.00 | 0.03 | 1.107 | |||||
Hour 6: | |||||||||||||||||||||||||||||
Negative Control (Colony 1) | 0.403 | 0.396 | 0.385 | 0.384 | 13.86 | 14.13 | 14.1 | 13.53 | 0.36475 | 0.35775 | 0.34675 | 0.34575 | 2.075 | 2.345 | 2.315 | 1.745 | 0.00 | 0.01 | 0.01 | 0.00 | 0.01 | 0.00 | 0.01 | 1.140 | |||||
Negative Control (Colony 2) | 0.282 | 0.399 | 0.446 | 0.312 | 13.67 | 13.87 | 14.02 | 13.94 | 0.24375 | 0.36075 | 0.40775 | 0.27375 | 1.885 | 2.085 | 2.235 | 2.155 | 0.01 | 0.01 | 0.00 | 0.01 | 0.01 | 0.00 | 0.01 | 1.209 | |||||
Positive Control (Colony 1) | 0.308 | 0.279 | 0.297 | 0.282 | 84.88 | 74.53 | 79.93 | 79.25 | 0.26975 | 0.24075 | 0.25875 | 0.24375 | 73.095 | 62.745 | 68.145 | 67.465 | 0.24 | 0.23 | 0.23 | 0.24 | 0.23 | 0.01 | 0.23 | 1.028 | |||||
Positive Control (Colony 2) | 0.393 | 0.303 | 0.237 | 0.411 | 79.7 | 68.25 | 81.04 | 81.93 | 0.35475 | 0.26475 | 0.19875 | 0.37275 | 67.915 | 56.465 | 69.255 | 70.145 | 0.17 | 0.19 | 0.31 | 0.16 | 0.21 | 0.07 | 0.20 | 1.336 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 0.069 | 0.059 | 0.068 | 0.057 | 53.36 | 48.94 | 53.32 | 52.33 | 0.03075 | 0.02075 | 0.02975 | 0.01875 | 41.575 | 37.155 | 41.535 | 40.545 | 1.19 | 1.57 | 1.22 | 1.90 | 1.47 | 0.33 | 1.44 | 1.248 | |||||
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 0.07 | 0.069 | 0.068 | 0.068 | 54.14 | 55.06 | 56.59 | 55.34 | 0.03175 | 0.03075 | 0.02975 | 0.02975 | 42.355 | 43.275 | 44.805 | 43.555 | 1.17 | 1.23 | 1.32 | 1.28 | 1.25 | 0.07 | 1.25 | 1.054 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.308 | 0.307 | 0.288 | 0.324 | 146.1 | 129 | 142.7 | 146.6 | 0.26975 | 0.26875 | 0.24975 | 0.28575 | 134.315 | 117.215 | 130.915 | 134.815 | 0.44 | 0.38 | 0.46 | 0.41 | 0.42 | 0.03 | 0.42 | 1.082 | |||||
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.403 | 0.427 | 0.217 | 0.287 | 125.9 | 120.6 | 132.6 | 121.6 | 0.36475 | 0.38875 | 0.17875 | 0.24875 | 114.115 | 108.815 | 120.815 | 109.815 | 0.27 | 0.25 | 0.59 | 0.39 | 0.37 | 0.16 | 0.35 | 1.487 | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.398 | 0.399 | 0.392 | 0.343 | 16.24 | 15.33 | 14.79 | 16.13 | 0.35975 | 0.36075 | 0.35375 | 0.30475 | 4.455 | 3.545 | 3.005 | 4.345 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.00 | 0.01 | 1.261 | |||||
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.3 | 0.295 | 0.209 | 0.181 | 15.95 | 15.66 | 15.93 | 15.44 | 0.26175 | 0.25675 | 0.17075 | 0.14275 | 4.165 | 3.875 | 4.145 | 3.655 | 0.01 | 0.01 | 0.02 | 0.02 | 0.02 | 0.00 | 0.02 | 1.318 | |||||
Test Device 4: J23101+I13504 (Colony 1) | 0.272 | 0.274 | 0.264 | 0.264 | 87.02 | 86.88 | 85.79 | 83.04 | 0.23375 | 0.23575 | 0.22575 | 0.22575 | 75.235 | 75.095 | 74.005 | 71.255 | 0.28 | 0.28 | 0.29 | 0.28 | 0.28 | 0.00 | 0.28 | 1.016 | |||||
Test Device 4: J23101+I13504 (Colony 2) | 0.296 | 0.236 | 0.225 | 0.228 | 97.47 | 97.03 | 97.78 | 82.43 | 0.25775 | 0.19775 | 0.18675 | 0.18975 | 85.685 | 85.245 | 85.995 | 70.645 | 0.29 | 0.38 | 0.40 | 0.33 | 0.35 | 0.05 | 0.35 | 1.158 | |||||
Test Device 5: J23106+I13504 (Colony 1) | 0.36 | 0.366 | 0.368 | 0.347 | 47.54 | 46.09 | 45.93 | 42.66 | 0.32175 | 0.32775 | 0.32975 | 0.30875 | 35.755 | 34.305 | 34.145 | 30.875 | 0.10 | 0.09 | 0.09 | 0.09 | 0.09 | 0.00 | 0.09 | 1.045 | |||||
Test Device 5: J23106+I13504 (Colony 2) | 0.327 | 0.324 | 0.294 | 0.309 | 36.82 | 41.73 | 41.66 | 37.26 | 0.28875 | 0.28575 | 0.25575 | 0.27075 | 25.035 | 29.945 | 29.875 | 25.475 | 0.08 | 0.09 | 0.10 | 0.08 | 0.09 | 0.01 | 0.09 | 1.138 | |||||
Test Device 6: J23117+I13504 (Colony 1) | 0.387 | 0.402 | 0.38 | 0.343 | 14.5 | 14.72 | 14.73 | 14.63 | 0.34875 | 0.36375 | 0.34175 | 0.30475 | 2.715 | 2.935 | 2.945 | 2.845 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.00 | 0.01 | 1.083 | |||||
Test Device 6: J23117+I13504 (Colony 2) | 0.351 | 0.374 | 0.356 | 0.375 | 14.53 | 14.82 | 14.32 | 14.82 | 0.31275 | 0.33575 | 0.31775 | 0.33675 | 2.745 | 3.035 | 2.535 | 3.035 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.00 | 0.01 | 1.060 |
Requirements
- 1L autoclave bottle or erlenmeyer flask
- 6.25g LB broth powder
- 2.5mM KCl
- 20mM MgSO 4 .7H 2 O
- Distilled or milliQ water
- Magnetic stirrer and bead(optional)
- Measuring cylinder
- Autoclave tape
- Cotton plug(for the flask)
- Aluminium foil
- Autoclaving machine
Procedure
- In a 1L autoclave bottle or flask , add 6.25g LB broth powder.
- Calculate and weigh 2.5mM KCl and 20mM MgSO 4 .7H 2 O.
- Adjust pH to 7.5(using NaOH)
- Add distilled water to make the solution upto 250mL.
- Either Swirl or use magnetic stirrer and a bead to mix(might not dissolve completely) .
- Make a nice cotton plug by rolling and folding a big piece of cotton and put it on the mouth of the flask.(Try pulling it out once, if it makes a noise then, Bravo!)
- Put an Aluminium foil on top of it and a piece of autoclave tape
- Keep the cap slightly open if using a bottle.
- Autoclave at 121°C
- Cool to RT before use. Do not tighten cap until cool.
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