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.