Bologna University/Transformation
From 2007.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
::1. Thaw the competent cells in ice (do not refreeze). | ::1. Thaw the competent cells in ice (do not refreeze). | ||
::2. Dispense 100 μl of cells into microfuge tubes on ice. | ::2. Dispense 100 μl of cells into microfuge tubes on ice. | ||
| - | ::3. Add 0.1-0.3 μg of | + | ::3. Add 0.1-0.3 μg of plasmidic DNA or the respective amount of the ligation reaction. |
::4. Keep on ice for 30 min. | ::4. Keep on ice for 30 min. | ||
::5. Heat at 42 °C for 60 seconds without agitation. | ::5. Heat at 42 °C for 60 seconds without agitation. | ||
| Line 7: | Line 7: | ||
::7. Add 0.9 ml of LB medium at room temperature. | ::7. Add 0.9 ml of LB medium at room temperature. | ||
::8. Incubate at 37 °C for 1 hr with agitation. | ::8. Incubate at 37 °C for 1 hr with agitation. | ||
| - | ::9. Pellet the cells and discard supernatant | + | ::9. Pellet the cells and discard most of supernatant, leaving about 100 μl. |
::10. Streak on plates containing appropriate antibiotics. | ::10. Streak on plates containing appropriate antibiotics. | ||
::11. Incubate the plates overnight at 37 °C. | ::11. Incubate the plates overnight at 37 °C. | ||
Revision as of 09:30, 25 October 2007
- 1. Thaw the competent cells in ice (do not refreeze).
- 2. Dispense 100 μl of cells into microfuge tubes on ice.
- 3. Add 0.1-0.3 μg of plasmidic DNA or the respective amount of the ligation reaction.
- 4. Keep on ice for 30 min.
- 5. Heat at 42 °C for 60 seconds without agitation.
- 6. Keep on ice for 2 minutes.
- 7. Add 0.9 ml of LB medium at room temperature.
- 8. Incubate at 37 °C for 1 hr with agitation.
- 9. Pellet the cells and discard most of supernatant, leaving about 100 μl.
- 10. Streak on plates containing appropriate antibiotics.
- 11. Incubate the plates overnight at 37 °C.
