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Related Techniques
Solutions - Culture media
- 0.05% Ethyl methanesulfonate (EMS) solution (Sigma)
- 0.0025% 5-Fluoro-2'-deoxyuridine solution (Sigma)
- 50% w/w PEG 6000 in serum-free culture medium
Chemicals - Thymidine (Sigma)
Cells - Mutant cell lines of Drosophila that cannot grow at temperatures above
30°C
Procedure
Mutation 1. Seed 4 x 10 7 cells in glass dishes, and incubate the cells in a CO 2 incubator at 23°C.
2. Treat the growing cells with 0.05% EMS at 23°C for 24 h. This may
yield a 20% survival rate.
3. Culture the surviving cells in EMS-free medium for a further 72 h.
4. Reseed the cells at a density of 5 x 10 5 cells/dish in heat-treated used
(conditioned) medium.
5. Maintain the culture for several days at 23°C, and then increase the
temperature to 30°C.
6. After 24 h, add 0.0025% 5-fluoro-2'-deoxyuridine, and incubate for
72 h at 30°C.
7. Wash out the drug, and culture the surviving cells at 23°C in medium
containing 10 Ilg/ml thymidine.
8. Maintain the culture for approximately 3 weeks.
9. Disperse the cell colonies by pipetting, and culture the dispersed cells
at 30°C.
10. Repeat steps 6-9 three more times using the same dish.
11. After that, seed the cells at a density of 1,000 cells/dish, and culture
the cells at 30°C.
12. After 2-4 days, decrease the temperature to 23°C, and culture for a
further 10 days.
Selection of 1. Select approximately 10 colonies per dish, and seed them in heatts mutants
treated used medium in culture plates at 23°C for approximately 2
weeks.
2. Divide the cells in each well into corresponding wells of two plates,
and incubate one plate at 23°C and the other at 30°C.
3. Select colonies that grow well at 23°C but not at 30°C.
Related Techniques
Solutions - Culture media
- 0.05% Ethyl methanesulfonate (EMS) solution (Sigma)
- 0.0025% 5-Fluoro-2'-deoxyuridine solution (Sigma)
- 50% w/w PEG 6000 in serum-free culture medium
Chemicals - Thymidine (Sigma)
Cells - Mutant cell lines of Drosophila that cannot grow at temperatures above
30°C
Procedure
Mutation 1. Seed 4 x 10 7 cells in glass dishes, and incubate the cells in a CO 2 incubator at 23°C.
2. Treat the growing cells with 0.05% EMS at 23°C for 24 h. This may
yield a 20% survival rate.
3. Culture the surviving cells in EMS-free medium for a further 72 h.
4. Reseed the cells at a density of 5 x 10 5 cells/dish in heat-treated used
(conditioned) medium.
5. Maintain the culture for several days at 23°C, and then increase the
temperature to 30°C.
6. After 24 h, add 0.0025% 5-fluoro-2'-deoxyuridine, and incubate for
72 h at 30°C.
7. Wash out the drug, and culture the surviving cells at 23°C in medium
containing 10 Ilg/ml thymidine.
8. Maintain the culture for approximately 3 weeks.
9. Disperse the cell colonies by pipetting, and culture the dispersed cells
at 30°C.
10. Repeat steps 6-9 three more times using the same dish.
11. After that, seed the cells at a density of 1,000 cells/dish, and culture
the cells at 30°C.
12. After 2-4 days, decrease the temperature to 23°C, and culture for a
further 10 days.
Selection of 1. Select approximately 10 colonies per dish, and seed them in heatts mutants
treated used medium in culture plates at 23°C for approximately 2
weeks.
2. Divide the cells in each well into corresponding wells of two plates,
and incubate one plate at 23°C and the other at 30°C.
3. Select colonies that grow well at 23°C but not at 30°C.