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Related Techniques
Subprotocol 6
Colony Formation in Soft Agar
Materials
Equipment - Colony counter or dissecting microscope
- Centrifuge and centrifuge tubes
- Petri dish (60 xIS mm)
- Pasteur pipettes
Solutions - Culture medium
Chemicals - Agar (Special Agar-Noble; Difco Laboratories)
- Heart infusion broth (Difco Laboratories)
Miscellaneous - Vinyl tape
Base layer
formation
Seeding
Reseeding
Procedure
1. Prepare base layers by mixing equal volumes of heart infusion broth,
containing 2% agar, with twice-concentrated culture medium, and dispensing 7 ml of this solution in petri dishes.
1. Select a culture containing the cells to be cloned, and suspend the cells
at the logarithmic growth phase in the culture medium.
2. Centrifuge the harvested cells at 150 g for 5 min.
3. Resuspend the cells in culture medium so as to make a very low cell
density (e.g., 10 4 cells/ml).
4. Mix 1 ml cell suspension and melted 0.5% agar at 50°C, and quickly
pour the mixture onto the surface of the base layer.
5. Seal the petri dishes with vinyl tape, and incubate the dishes at 30°C.
6. Examine the colonies formed under a colony counter or dissecting
microscope after 10-14 days.
7. Mark the position of the desired colony on the outside of the dish, and
isolate the colony using a Pasteur pipette.
1. Seed the isolated colony into culture medium, and propagate the cells.
Results
Mcintosh and Rectoris (1974) cloned cells of the cabbage looper
Trichoplusia ni in TC 199-MK medium (see Appendix 1) using this
method with a cloning efficiency of 1 %.