- Completed by Douglas + Valentina on September 21st
- Completed by Douglas + Ariania and supervised by Dr. Pavlovic on September 26th
- Completed by Douglas + Rachel S. and assisted by Erick and Daniela (Esiobu lab members) on September 27th, 28th, 29th
● 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 ● 96 well plate, 2 different plates used: clear with flat bottom for absorbance; black with flat bottom for fluorescence
● 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
◻ Added 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette) ◻ Added 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette) ◻ Measured absorbance 600 nm of all samples in all standard measurement modes in instrument ◻ Recorded the data in the table below or in notebook ◻ Imported data into Excel (OD600 reference point tab) Sheet_1 provided
◻ added 5uL DNA ◻ let sit 30 min on ice ◻ heat shocked at 42C for 30 sec ◻ placed back on ice for 2 min ◻ added 450uL Luria Broth and incubated at 37c for 2 hours ◻ plated 100uL on LB/chloramphenicol plates ◻ grew overnight at 37C ◻ this was done for all 8 machines
transform Escherichia coli DH5α with these following plasmids:
Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol. Grew the cells overnight (16-18 hours) at 37°C and 220 rpm.
Cell growth, sampling, and assay ◻ Set instrument to read OD600 (as OD calibration setting) ◻ Measured OD600 of the overnight cultures ◻ Recorded data ◻ Imported data into Excel (Dilution Calculation) Sheet_1 provided ◻ Diluted the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in Excel (Dilution Calculation) Sheet_1) in 15 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light). ◻ Incubated the cultures at 37°C and 220 rpm. ◻ Took 1 mL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time point, sampled from each of the 8 devices, two colonies per device, for a total of 16 samples per time point) ◻ Placed samples on ice. ◻ Measured samples (OD and Fl measurement) ◻ Recorded data in notebook
