IGEM:MIT/2005/Old Preliminary Dimer Uptake Experiment Protocol
Preliminary Experiment: Uptake of Fluorescein and Fluorescein Dimers by Competent and Non-competent Bacteria
- 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)
- Prepare dimer solution by dissolving in TE buffer
- Prepare fluorescein solutions and dimer solutions (above)
- Dilute competent cells ~1:100. (Add 24.75 mls for one eppendorf tube.) Put on ice.
- Take OD of overnight culture. Dilute to reach a concentration of ~3.16 X 10^8 cells/ml.
- Centrifuge competent cells and overnight culture.
- Resuspend both in fluorescein solution, fluorescein dimer solution, and M9.
- Aliquot each of 3 samples into 2 tubes.
- Centrifuge and resuspend cells for each of the three different treatments (leave 3 for "wash" control).
- 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
- Microscope: take a 5 microliter sample and drop onto slide; look for presence of fluorescence.