TUDelft/19 September 2008

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(New page: {{Template:TUDelftiGEM2008}} {{Template:TUDelftiGEM2008_menu_home}} {{Template:TUDelftiGEM2008_calendar}} =September 19th 2008= ==Luciferase Assay== Today we've done our first luciferase...)
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Today we've done our first luciferase assay. The samples we had in the end were either K115034 (34) or K115012 (12, control).
Today we've done our first luciferase assay. The samples we had in the end were either K115034 (34) or K115012 (12, control).
===Samples===
===Samples===
-
A: 12, Growing overnight with induction at room temperature, lysed by sonification
+
A: 12, Growing overnight with induction at room temperature, lysed by sonification<br>
-
B: 34, Growing overnight with induction at room temperature, lysed by sonification
+
B: 34, Growing overnight with induction at room temperature, lysed by sonification<br>
-
C: 12, Growing overnight with induction at 37oC, lysed by sonification
+
C: 12, Growing overnight with induction at 37oC, lysed by sonification<br>
-
D: 34, Growing overnight with induction at 37oC, lysed by sonification
+
D: 34, Growing overnight with induction at 37oC, lysed by sonification<br>
-
E: 12, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
+
E: 12, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification<br>
-
F: 34, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
+
F: 34, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification<br>
-
G: 12, Growing 2 times overnight with induction at room temperature, lysed by sonification
+
G: 12, Growing 2 times overnight with induction at room temperature, lysed by sonification<br>
-
H: 34, Growing 2 times overnight with induction at room temperature, lysed by sonification
+
H: 34, Growing 2 times overnight with induction at room temperature, lysed by sonification<br>
-
I: 12, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
+
I: 12, Growing 2 times overnight with induction at room temperature, lysed by kit buffer<br>
-
J: 34, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
+
J: 34, Growing 2 times overnight with induction at room temperature, lysed by kit buffer<br>
-
K: 12, Growing o/n at 37oC without induction, to test R0080 for leakiness..
+
K: 12, Growing o/n at 37oC without induction, to test R0080 for leakiness.<br>
===Protocol===
===Protocol===

Revision as of 10:59, 22 September 2008

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Contents

September 19th 2008

Luciferase Assay

Today we've done our first luciferase assay. The samples we had in the end were either K115034 (34) or K115012 (12, control).

Samples

A: 12, Growing overnight with induction at room temperature, lysed by sonification
B: 34, Growing overnight with induction at room temperature, lysed by sonification
C: 12, Growing overnight with induction at 37oC, lysed by sonification
D: 34, Growing overnight with induction at 37oC, lysed by sonification
E: 12, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
F: 34, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
G: 12, Growing 2 times overnight with induction at room temperature, lysed by sonification
H: 34, Growing 2 times overnight with induction at room temperature, lysed by sonification
I: 12, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
J: 34, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
K: 12, Growing o/n at 37oC without induction, to test R0080 for leakiness.

Protocol

The protocol used in the case of most samples, when lysed by sonification was: 1. Grow as indicated above and induce immediately, unless stated otherwise 2. Pellet 3 ml of cells 3. Resuspend in 100 ul 15 mM Tris HCl 4. Destroy by sonification (3 * 15 s) 5. Freeze at -20 6. Add 20 ul of lysed cell sample to a well 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)

If lysis by kit buffer (Promega) was used, the protocol became: 1. Grow as indicated above and induce immediately, unless stated otherwise 2. Pellet 3 ml of cells 3. Resuspend in 100 ul 1X lysis buffer 4. Leave at room temperature for 15-30 minutes 5. Freeze at -20 6. Add 20 ul of lysed cell sample to a well 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)

Results

After a lot of fine tuning of the luminometer, the luminescence values were obtained as shown in graph 1.

Graph 1. Luminescence normalized for cell density (OD=1) at the time of sample preparation.