Bologna University/Fluorescence issue

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::1. Growth O/N for 15h in LB (5ml) of:
::1. Growth O/N for 15h in LB (5ml) of:
-
::::-trasformed E.Coli with appropriate antibiotic;
+
::::-transformed E. Coli with appropriate antibiotic;
::::-E. Coli.
::::-E. Coli.
-
::2. '''All misure has to be done in M9 at OD=1.'''
+
::2. '''All measures have to be done in M9 with OD=1.'''
-
::3. The day after in the morning OD measure.
+
::3. The day after in the morning measure OD.
::4. To obtain OD=1:
::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;
+
::::-discard the supernatant;
-
::::-risuspende the pellet with 6ml in M9 medium with glucose with appropriate antibiotic(1000x).
+
::::-resuspende cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x).
-
::5. OD measure: 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 untrasformed E.Coli.
+
::6. Adjust PMT offset with untransformed E. Coli.
-
::7. Fluorescence test of the culture before IPTG induction.
+
::7. Test culture fluorescence 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;
+
::::-discard the supernatant;
-
::::-risuspende the pellet in M9 medium and 2mM IPTG with appropiate antibiotic(1000X);
+
::::-resuspende cell pellet in M9 medium and 2mM IPTG with appropriate antibiotic (1000X);
-
::9. Incubate at 37°C.
+
::9. Incubate at 37°C.
-
::10. Fluorescence test after10min, 20min, 30min, 1h, 2h.
+
::10. Test fluorescence after 10min, 20min, 30min, 1h, 2h.
 +
 
 +
[[Bologna University/Protocols | Back]]

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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