​Fluorescence Standard Curve Created

Note (Zoey): After speaking with Judy I learned that our original settings and interpretation of the interlab protocols settings for the plate reading where incorrect. We errored in not paying close attention to whether it required ABS (Absorption) or OD (Optical density) for the test. There is a specific tab in the plate reader program for Fluorescence (FL). Where we make our setting: Excitation (485 mn) / Emission (530/30). This error made the calibration for our culture/LB broth ratios to more than likely be off. It is expected we will need to redo this interlab experiment once more. In addition to the culture plate run, we should have used ABS setting of 595 nm allowed by the machine and not 450. This setting should have been the same for the Day 3 Culture hourly sequence plate read experiment.

After Fluorescence standard curve was created with plate reader we began the Day 3 section of interlab protocols. Using the ABS setting of 450nm (Read today’s note for details on our error here)

Entering this data into the excel spreadsheet contained on the team drive “Interlab 2017 Measurements.

We made up an LB broth + Chloramphenicol solution .25ml of CAT in 250 ml of LB Broth as our standard. To create a 25ug/ml stock.

Taking a sample from each of our incubation falcon tubes. Pos, neg, TD1, TD2, TD3, TD4, TD5, TD6 all from the “A” samples in cells A1 - A8 respectively and LB+Chloramphenicol in A9. We ran that 96 well plate through the plate reader again using the same ABS 450 nm setting as before. (See today’s note above for details on our error here)

Imputing the resulting numbers into the excel located on Team drive called, “2017 Interlab Dilution Calculation Sheet.” This was provided by IGEM and ran the required calculation to determine the ratio of LB + Chloramphenicol to how much of each culture was required.

Covering 50 mL falcon tubes in tin foil as per protocols following the ratios set down in the excel spreadsheet to equal 12 mL total. We labeled 16 total 1.5 mL microcentrifuge tubes 8 “A” & 8 “B”, a 500uL sample from each of the 16 new culture falcon tubes was placed into each respective microcentrifuge tube. Tape was wrapped over all 16 tubes at hour 0 and placed in ice. The Falcon tubes of culture were placed in the shake plate incubator and timer was set for 2 hours.

Hour 2. The process was repeated as per interlab protocols. 500 uL of culture solution were placed in the 16 microcentrifuge tube corresponding with the same numbered and lettered falcon tube. Labeled and placed into Ice.

Hour 4. The process was repeated as per interlab protocols. 500 uL of culture solution were placed in the 16 microcentrifuge tube corresponding with the same numbered and lettered falcon tube. Labeled and placed into ice.

Hour 6, repeated previous hours steps.

Once all were finished I then plated a sample of 100 uL from the microcentrifuge tube in a vertical pattern beginning with the negative control. A1, A2, A3, A4. For the B sample of the negative control, t went in the next four wells. A5, A6, A7, A8. This process was continued on down the rows. B row = Positive Control. C row = TD1, D row = TD2, E row = TD3, F row = TD3, G row = TD 4, H row = TD4, I row = TD 5, J row = TD 6, K row = LB broth+CAT. A single plate was prepared for each two-hour increment and sent through the plate reader.

All results were recorded manually into the team drive excel spreadsheet for later analysis.