Team:Florida Atlantic/InterLab

Florida_Atlantic

Interlab Report


Member Participations

Calibrations:

- Completed by Douglas + Valentina on September 21st


Transformations:

- Completed by Douglas + Ariania and supervised by Dr. Pavlovic on September 26th


Cell Measurement:

- Completed by Douglas + Rachel S. and assisted by Erick and Daniela (Esiobu lab members) on September 27th, 28th, 29th



Methodology

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
● 96 well plate, 2 different plates used: clear with flat bottom for absorbance; black with flat bottom for fluorescence


Devices (from InterLab Measurement Kit):

● 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



Calibration Procedure

◻ 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


Transformation Procedure

◻ 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


Cell Measurement Procedure

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​:

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.

Day 3​:

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 m​l 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


Data


Results


Fluorescence of GFP engineered E.coli Graphs

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