Bologna University/Fluorescence issue

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< Bologna University(Difference between revisions)
 
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::::-discard the supernatant;
::::-discard the supernatant;
::::-resuspende cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x).
::::-resuspende cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x).
-
::5. measure OD: adjust OD to 1 through further diluition or growth.
+
::5. Measure OD: adjust OD to 1 through further diluition or 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.

Latest revision as of 15:46, 4 October 2007

1. Growth O/N for 15h in LB (5ml) of:
-transformed E. Coli with appropriate antibiotic;
-E. Coli.
2. All measures have to be done in M9 with OD=1.
3. The day after in the morning measure OD.
4. To obtain OD=1:
-centrifugate 4ml of bacteria culture at 4400rpm for 3min at 25°C;
-discard the supernatant;
-resuspende cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x).
5. Measure OD: adjust OD to 1 through further diluition or 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;
-resuspende 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.

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