TUDelft/19 September 2008
From 2008.igem.org
(→Samples) |
(→Protocol) |
||
Line 22: | Line 22: | ||
===Protocol=== | ===Protocol=== | ||
The protocol used in the case of most samples, when lysed by sonification was: | 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 | + | 1. Grow as indicated above and induce immediately, unless stated otherwise<br> |
- | 2. Pellet 3 ml of cells | + | 2. Pellet 3 ml of cells <br> |
- | 3. Resuspend in 100 ul 15 mM Tris HCl | + | 3. Resuspend in 100 ul 15 mM Tris HCl <br> |
- | 4. Destroy by sonification (3 * 15 s) | + | 4. Destroy by sonification (3 * 15 s) <br> |
- | 5. Freeze at -20 | + | 5. Freeze at -20 <br> |
- | 6. Add 20 ul of lysed cell sample to a well | + | 6. Add 20 ul of lysed cell sample to a well <br> |
- | 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega) | + | 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)<br> |
- | If lysis by kit buffer (Promega) was used, the protocol became: | + | |
- | 1. Grow as indicated above and induce immediately, unless stated otherwise | + | If lysis by kit buffer (Promega) was used, the protocol became:<br> |
- | 2. Pellet 3 ml of cells | + | 1. Grow as indicated above and induce immediately, unless stated otherwise <br> |
- | 3. Resuspend in 100 ul 1X lysis buffer | + | 2. Pellet 3 ml of cells <br> |
- | 4. Leave at room temperature for 15-30 minutes | + | 3. Resuspend in 100 ul 1X lysis buffer <br> |
- | 5. Freeze at -20 | + | 4. Leave at room temperature for 15-30 minutes <br> |
- | 6. Add 20 ul of lysed cell sample to a well | + | 5. Freeze at -20<br> |
- | 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega) | + | 6. Add 20 ul of lysed cell sample to a well <br> |
+ | 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)<br> | ||
===Results=== | ===Results=== |
Revision as of 10:59, 22 September 2008
July | ||||||
M | T | W | T | F | S | S |
1 | 2 | 3 | 4 | 5 | 6 | |
7 | 8 | 9 | 10 | 11 | 12 | 13 |
14 | 15 | 16 | 17 | 18 | 19 | 20 |
21 | 22 | 23 | 24 | 25 | 26 | 27 |
28 | 29 | 30 | 31 |
August | ||||||
M | T | W | T | F | S | S |
1 | 2 | 3 | ||||
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
September | ||||||
M | T | W | T | F | S | S |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 |
October | ||||||
M | T | W | T | F | S | S |
1 | 2 | 3 | 4 | 5 | ||
6 | 7 | 8 | 9 | 10 | 11 | 12 |
13 | 14 | 15 | 16 | 17 | 18 | 19 |
20 | 21 | 22 | 23 | 24 | 25 | 26 |
27 | 28 | 29 | 30 | 31 |
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.