Team:Freiburg/InterLab Study/Protocol

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InterLab Protocol

Individuals responsible for conducting InterLab study:

Rabea Jesser, Julia Donauer (Plate reader measurements)
Ramona Emig, Lara Stühn, Julika Neumann (cloning)
Julian Bender (Plate reader measurements, data processing)

What chassis did you use?

E.coli TOP10 E.coli ArcticExpress (DE3)RP

Please provide the positive control(s) you used.

BBa_I20270

Please provide the negative control(s) you used.

Untransformed for both strains Transformed with BBa_R0040 (ptetR in pSB1C3)

What type of agar did you use?

LB Agar with 30µg/mL chloramphenicol

Workflow

Streak out 1 plate per device and control
Incubate plates for 17h at 37 C
Inoculate test tube with with dimension (WxH) of 16 x 1.3 cm and 5mL LB-medium 30µg/mL chloramphenicol
Incubate test tubes for 8h in incubator with 220 rpm at 37°C
For the 10°C samples: transfer them to 10°C incubator
For the 37°C samples: shake another 8 h in 37°C incubator

Set your instrument to read OD₆₀₀
Measure each sample in cuvette
Take the measurement and record it
Measure OD₆₀₀ of all samples
Calculate the dilution required for each sample
Dilute each sample
Re-measure your sample on OD₆₀₀
If your OD₆₀₀ is within 5% of 0.5, proceed
If your OD₆₀₀ is outside that range, recalculate your dilution and remeasure until it's within the range of +/- 5%

set up sodium fluoresceine standard curve with 500, 375, 250, 125, 50, 25, 10, 5 and 0 ng/mL concentration

pipette the samples and the standard curve in 96-well plates (triplicates)
measure emission at 530 nm after excitation at 485 nm