Difference between revisions of "Team:Freiburg/InterLab Study/Protocol"

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Rabea Jesser, Julia Donauer (Plate reader measurements)
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Ramona Emig, Lara Stühn, Julika Neumann (cloning)
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<li>Rabea Jesser, Julia Donauer (Plate reader measurements)</li>
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Julian Bender (Plate reader measurements, data processing)
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<li>Julian Bender (Plate reader measurements, data processing)</li>
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Revision as of 11:10, 1 September 2015

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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