Bologna University/Fluorescence issue
From 2007.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
| - | :: | + | ::1. Growth O/N for 15h in LB (5ml) of: |
| - | - | + | |
| - | - | + | -trasformed E.Coli with appropriate antibiotic; |
| - | 2. All misure has to be done in M9 at OD=1. | + | |
| - | 3.The day after in the morning OD measure. | + | -E. Coli. |
| - | 4. To obtain OD=1: | + | |
| + | ::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; | -centrifugate 4ml of bacteria culture at 4400rpm for 3min at 25°C; | ||
-eliminate the supernatant; | -eliminate the supernatant; | ||
-risuspende the pellet with 6ml in M9 medium with glucose with appropriate antibiotic | -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. |
| - | 6. Adjust PMT offset with untrasformed E.Coli. | + | ::6. Adjust PMT offset with untrasformed E.Coli. |
| - | 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; | -centrifugate bacteria culture at 4400rpm for 3min at 25°C; | ||
-eliminate the supernatant; | -eliminate the supernatant; | ||
-risuspende the pellet in M9 medium and 2mM IPTG with appropiate antibiotic (1000X); | -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. | 10. Fluorescence test after10min, 20min, 30min, 1h, 2h. | ||
Revision as of 10:17, 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.
