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

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.