Team:Florida Atlantic/InterLab

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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 Eric 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

Transformation Procedure

add 5uL DNA
let sit 30 min on ice
heat shock at 42C for 30 sec
place back on ice for 2 min
add 450uL Luria Broth and incubate at 37c for 2 hours
plate 100uL on LB/chloramphenicol plates
grow 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.
Grow 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
◻ Dilute 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).
◻ Incubate the cultures at 37°C and 220 rpm.
◻ Take 1 mL 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 you need to measure your samples (OD and Fl measurement), see the below for details.
◻ Record data in your notebook