Team:NEU-China/InterLab
Calibration Protocols
◻ Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
◻ Add 100 μl of H 2 O into wells A2, B2, C2, D2 (or 1 mL H 2 O 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 the notebook
◻ Import data into Excel ( OD600 reference point tab ) Sheet_1 provided
Protocol fluorescein fluorescence standard curve
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.
◻ 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, D
◻ Measure fluorescence of all samples in all standard measurement modes in instrument
◻ Record the data in the notebook
◻ Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided
Cell measurement protocol
Day 1 :
transform Escherichia coli DH5α with provided plasmids:
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 220 rpm.
Day 3 : Cell growth, sampling, and assay
◻ Set the instrument to read OD600 (as OD calibration setting)
◻ Measure OD600 of the overnight cultures
◻ Record data in the notebook
◻ Import data into Excel ( Dilution Calculation ) Sheet_1 provided
◻ Dilute the cultures to a target OD 600 of 0.02 (see the volume of preloading culture and media in Excel ( Dilution Calculation ) Sheet_1) in 12 m l LB medium + Chloramphenicol in 50 mL falcon tube (amber, or 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.
◻ At the end of sampling point, measure the samples (OD and Fl measurement), see the below for details.
◻ Record data in the notebook
◻ Import data into Excel ( cell measurement tab ) Sheet_1 provided
Fluorescence Timeline
Colony 1
Figure 1a
Colony 2
Figure 1b
Figure 1a&b. The change of fluorescence over time. 5 different devices were employed in duplicate. Samples were collected every hour for 6 total hours and afterwards had their fluorescence measured using a plate reader and 96-well plate. Graph produced by Jiawen Yang.
Abs600 Timeline
Colony 1
Figure 2a
Colony 2
Figure 2b
Figure 2a&b. OD600 nm absorbance over time. Samples were collected every hour for 6 total hours and measured absorbance using a plate reader and 96-well plate. Graph produced by Jiawen Yang.
