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