Bologna University/Fluorescence issue
From 2007.igem.org
(Difference between revisions)
| Line 5: | Line 5: | ||
::::-E. Coli. | ::::-E. Coli. | ||
| - | ::2. All misure has to be done in M9 at OD=1. | + | ::2. '''All misure has to be done in M9 at OD=1.''' |
::3. The day after in the morning OD measure. | ::3. The day after in the morning OD measure. | ||
::4. To obtain OD=1: | ::4. To obtain OD=1: | ||
| - | + | ::::-centrifugate 4ml of bacteria culture at 4400rpm for 3min at 25°C; | |
| - | + | ::::-eliminate the supernatant; | |
| - | + | ::::-risuspende the pellet with 6ml in M9 medium with glucose with appropriate antibiotic | |
(1000x). | (1000x). | ||
::5. OD measure: adjust OD to 1 through further diluition or growth. | ::5. OD measure: adjust OD to 1 through further diluition or growth. | ||
| Line 16: | Line 16: | ||
::7. Fluorescence test of the culture before IPTG induction. | ::7. Fluorescence test of the culture before IPTG induction. | ||
::8. IPTG induction: | ::8. IPTG induction: | ||
| - | + | ::::-centrifugate bacteria culture at 4400rpm for 3min at 25°C; | |
| - | + | ::::-eliminate the supernatant; | |
| - | + | ::::-risuspende the pellet in M9 medium and 2mM IPTG with appropiate antibiotic (1000X); | |
::9. Incubate at 37°C. | ::9. Incubate at 37°C. | ||
| - | + | ::10. Fluorescence test after10min, 20min, 30min, 1h, 2h. | |
Revision as of 10:20, 4 October 2007
- 1. Growth O/N for 15h in LB (5ml) of:
- -trasformed E.Coli with appropriate antibiotic;
- -E. Coli.
- 2. All misure has to be done in M9 at OD=1.
- 3. The day after in the morning OD measure.
- 4. To obtain OD=1:
- -centrifugate 4ml of bacteria culture at 4400rpm for 3min at 25°C;
- -eliminate the supernatant;
- -risuspende the pellet with 6ml in M9 medium with glucose with appropriate antibiotic
(1000x).
- 5. OD measure: adjust OD to 1 through further diluition or growth.
- 6. Adjust PMT offset with untrasformed E.Coli.
- 7. Fluorescence test of the culture before IPTG induction.
- 8. IPTG induction:
- -centrifugate bacteria culture at 4400rpm for 3min at 25°C;
- -eliminate the supernatant;
- -risuspende the pellet in M9 medium and 2mM IPTG with appropiate antibiotic (1000X);
- 9. Incubate at 37°C.
- 10. Fluorescence test after10min, 20min, 30min, 1h, 2h.
