Bologna University/Fluorescence Test
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< Bologna University(Difference between revisions)
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::1. Growth O/N for 15h in LB (5ml) of: | ::1. Growth O/N for 15h in LB (5ml) of: | ||
| - | ::::-transformed E. | + | ::::-transformed E. coli with appropriate antibiotic; |
| - | ::::-E. | + | ::::-E. coli. |
| - | ::2. '''All measures | + | ::2. '''All measures must be done in M9 with OD=1.2.''' |
| - | ::3. The day after in the morning measure OD. | + | ::3. The day after, in the morning, measure OD. |
| - | ::4. To obtain OD=1: | + | ::4. To obtain OD=1.2: |
| - | ::::- | + | ::::-centrifuge 4ml of bacterial culture at 4400rpm for 3min at 25°C; |
::::-discard the supernatant; | ::::-discard the supernatant; | ||
| - | ::::- | + | ::::-resuspend the cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x [ ]). |
| - | ::5. Measure OD: adjust OD to 1 through further | + | ::5. Measure OD: adjust OD to 1.2 through further dilution or cell growth. |
::6. Adjust PMT offset with untransformed E. Coli. | ::6. Adjust PMT offset with untransformed E. Coli. | ||
::7. Test culture fluorescence before IPTG induction. | ::7. Test culture fluorescence before IPTG induction. | ||
| Line 17: | Line 17: | ||
::::-centrifugate bacteria culture at 4400rpm for 3min at 25°C; | ::::-centrifugate bacteria culture at 4400rpm for 3min at 25°C; | ||
::::-discard the supernatant; | ::::-discard the supernatant; | ||
| - | ::::- | + | ::::-resuspend cell pellet in M9 medium and 2mM IPTG with appropriate antibiotic (1000x [ ]); |
::9. Incubate at 37°C. | ::9. Incubate at 37°C. | ||
::10. Test fluorescence after 10min, 20min, 30min, 1h, 2h. | ::10. Test fluorescence after 10min, 20min, 30min, 1h, 2h. | ||
[[Bologna University/Protocols | Back]] | [[Bologna University/Protocols | Back]] | ||
Latest revision as of 09:34, 25 October 2007
- 1. Growth O/N for 15h in LB (5ml) of:
- -transformed E. coli with appropriate antibiotic;
- -E. coli.
- 2. All measures must be done in M9 with OD=1.2.
- 3. The day after, in the morning, measure OD.
- 4. To obtain OD=1.2:
- -centrifuge 4ml of bacterial culture at 4400rpm for 3min at 25°C;
- -discard the supernatant;
- -resuspend the cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x [ ]).
- 5. Measure OD: adjust OD to 1.2 through further dilution or cell growth.
- 6. Adjust PMT offset with untransformed E. Coli.
- 7. Test culture fluorescence before IPTG induction.
- 8. IPTG induction:
- -centrifugate bacteria culture at 4400rpm for 3min at 25°C;
- -discard the supernatant;
- -resuspend cell pellet in M9 medium and 2mM IPTG with appropriate antibiotic (1000x [ ]);
- 9. Incubate at 37°C.
- 10. Test fluorescence after 10min, 20min, 30min, 1h, 2h.
