IGEM:MIT/2005/Old Preliminary Dimer Uptake Experiment Protocol

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Preliminary Experiment: Uptake of Fluorescein and Fluorescein Dimers by Competent and Non-competent Bacteria

Materials

  • Competent cells (1.975 X 10^10 cells/ml)
  • Overnight culture
  • Fluorescein dimer solution: 50 and 500 micromolar (M9 + dimers)
  • Fluorescein solution: 50 and 500 micromolar (M9 + fluorescein)

Procedure

  1. Prepare dimer solution by dissolving in TE buffer
  2. Prepare fluorescein solutions and dimer solutions (above)
  3. Dilute competent cells ~1:100. (Add 24.75 mls for one eppendorf tube.) Put on ice.
  4. Take OD of overnight culture. Dilute to reach a concentration of ~3.16 X 10^8 cells/ml.
  5. Centrifuge competent cells and overnight culture.
  6. Resuspend both in fluorescein solution, fluorescein dimer solution, and M9.
  7. Aliquot each of 3 samples into 2 tubes.
  8. Centrifuge and resuspend cells for each of the three different treatments (leave 3 for "wash" control).
  9. Plate: dilute a portion of each of these six samples so that there will be 3000 cells/ml, then plate 100 microliters on 6 plates
  10. Microscope: take a 5 microliter sample and drop onto slide; look for presence of fluorescence.
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