Registry/Measurement kit/Protocols/Plate reader experiment

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  1. For each test construct, three 5 ml cultures of pre-warmed (37°C) supplemented M9 medium (w/glycerol, see Endy Lab protocol) and antibiotic (kanamycin, 20 µg/ml) were each inoculated with a single colony (~2mm ø) from a freshly streaked plate of TOP10 containing the construct. As a control for each experiment, three 5 ml cultures were inoculated with a single colony each from a freshly streaked plate of TOP10 cells containing no vector. M9 media-supplemented without kanamycin was used for the empty TOP10 cells.
  2. Cultures were grown in 17 mm test tubes for 20 hrs at 37°C with spinning at 70 rpm.
  3. After 20 hrs, cultures were diluted 1:100 into 5 ml of pre-warmed fresh media and grown for approximately four hours under the same conditions(17mm tubes, warm room, spinning at 70 rpm).
  4. After four hours, a 500ul aliquot from each culture was then used for OD600 measurement. The cultures were diluted to OD 0.07 in 5 ml of pre-warmed fresh media to be grown for one hour under the same conditions(17mm tubes, warm room, spinning at 70 rpm).
  5. For each culture a 200 µl aliquot was transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner). An additional control of blank media was also added in the same aliquot. Three replicates (three aliquots of 200 ul) of each culture were measured.
  6. The plate was incubated in a Wallac Victor3 multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements for each well was 2 min and 21 s.
  7. Data processing was used to calculate the GFP synthesis rates for each well. The data for each construct tested was averaged across the three replicates wells and the three replicate cultures.
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