Team:NTHU Taiwan/InterLab

Interlab


InterLab Study




In the InterLab study of this year, our team followed InterLab_2017_Plate_Reader_Protocol to conduct the experiment. The work can be separated into two parts:(1) Calibration (2) Cell Measurement.




(1) Calibration

a. OD 600 Reference point

Materials:

1ml LUDOX

dd water

96 well plates, clear with a flat bottom

Use LUDOX-S40 as a single point reference to obtain a ratiometric conversion factor to transform the absorbance data into a standard OD 600 measurement.

We will get the data for OD 600 of the H 2 O and LUDOX. The corrected Abs 600 is calculated by subtracting the H 2 O reading. To convert measured Abs 600 to OD 600 is to let Reference OD 600 divided by Abs 600.



b. Protocol fluorescein fluorescence standard curve

Materials:

Fluorescein

10ml 1xPBS

96 well plates, clear with a flat bottom

Prepare a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate in the plate reader. Generate a standard curve of fluorescence for fluorescein concentration and use this to correct the cell-based readings to an equivalent fluorescein concentration. We must (1) Prepare the fluorescein stock solution (2) Prepare the serial dilutions of fluorescein. [*See pdf. for detail]

Get “OD 600 reference point” & “Fluorescein standard curve” sheet. [*See the Excel Sheet in pdf. presented below]




(2) Cell Measurement

Materials:

Competent cells (Escherichia coli strain DH5α)

LB (Luria Bertani) media

Chloramphenicol (working stock 25 ug/mL)

50 ml Falcon tube

Incubator at 37°C

1.5 ml Eppendorf tubes

Ice bucket with ice

Pipettes

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

Day 1: Transform Escherichia coli DH5α with the Devices

Day 2: Pick 2 colonies from each of plate and grow the cells overnight

Day 3: Cell growth, sampling, and assay [*See pdf. for detail]



Layout for Abs600 and Fluorescence measurement (one plate for each time point: 0, 2, 4, and 6 hours). Get “Raw Plate Reader Measurements” & “Fluorescence Measurement” sheet. [See the Excel Sheet in pdf. presented below]



The experiment went quite smoothly; however, some of the “Summary Statistics” in the Fluorescence Measurement sheet cannot be calculated due to the negative value we got from blanks in Fluorescence – Background. We think that it might be the mistake (ex. wrong concentration, mistakes in the volume or different gain number…) when we’re measuring the reference point, causing the background larger than the sample OD value.

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