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Team:Tokyo Tech/Project/Apple Reporter/Results
From 2010.igem.org
(→The work flow to TLC from culture) |
(→The work flow to TLC from culture) |
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===The work flow to TLC from culture=== | ===The work flow to TLC from culture=== | ||
1. The cells were cultured over night. | 1. The cells were cultured over night. | ||
| + | |||
2. 50ul bacterial sution was taken from culture 1 and moved into 50ml LB culture with appropriate antibiotic. | 2. 50ul bacterial sution was taken from culture 1 and moved into 50ml LB culture with appropriate antibiotic. | ||
Inducer was added,if necessary. | Inducer was added,if necessary. | ||
| - | 3. The cells were incubated at | + | |
| + | 3. The cells were incubated at 37℃24hour. | ||
| + | |||
4. After the O.D reach 2.0, culture was centrifuged for 10minutes at | 4. After the O.D reach 2.0, culture was centrifuged for 10minutes at | ||
4℃, 6000rpm,10min | 4℃, 6000rpm,10min | ||
| - | 5. Supernatant was removed by decanting. The remainder was added with 1 ml | + | |
| - | 6. Moved into 2ml tube by using P1000 pipetter at least twice. | + | 5. Supernatant was removed by decanting. The remainder was added with 1 ml ddH<sub>2</sub>0 and vortexed. |
| + | |||
| + | 6. Moved into 2ml tube by using P1000 pipetter at least twice. | ||
| + | |||
7. Solution is centrifuged for 20minutes at 4℃, 14000rpm | 7. Solution is centrifuged for 20minutes at 4℃, 14000rpm | ||
| + | |||
8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained. | 8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained. | ||
| + | |||
9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment. | 9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment. | ||
| + | |||
10. Solution is centrifuged for 20minutes at 4℃, 14000rpm | 10. Solution is centrifuged for 20minutes at 4℃, 14000rpm | ||
| + | |||
11. Acetone solution was replaced into 2 ml tube. | 11. Acetone solution was replaced into 2 ml tube. | ||
| + | |||
12. Acetone was removed using rotary evaporator. | 12. Acetone was removed using rotary evaporator. | ||
| + | |||
13. 50 ul of Chloroform/Methanol solution(weight ratio of 9:1) was added. | 13. 50 ul of Chloroform/Methanol solution(weight ratio of 9:1) was added. | ||
| + | |||
14. Sample was vortexed and spinned down. | 14. Sample was vortexed and spinned down. | ||
| + | |||
15. The water layer was removed. | 15. The water layer was removed. | ||
| + | |||
16. Spot the sample onto TLC silica gel plate, and developed by acetone/hexane solution.(weight ratio of 1:3) | 16. Spot the sample onto TLC silica gel plate, and developed by acetone/hexane solution.(weight ratio of 1:3) | ||
| + | |||
17. Spot was observed under visible light. | 17. Spot was observed under visible light. | ||
Latest revision as of 14:22, 27 October 2010
Protocols
The work flow to TLC from culture
1. The cells were cultured over night.
2. 50ul bacterial sution was taken from culture 1 and moved into 50ml LB culture with appropriate antibiotic. Inducer was added,if necessary.
3. The cells were incubated at 37℃24hour.
4. After the O.D reach 2.0, culture was centrifuged for 10minutes at 4℃, 6000rpm,10min
5. Supernatant was removed by decanting. The remainder was added with 1 ml ddH20 and vortexed.
6. Moved into 2ml tube by using P1000 pipetter at least twice.
7. Solution is centrifuged for 20minutes at 4℃, 14000rpm
8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained.
9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment.
10. Solution is centrifuged for 20minutes at 4℃, 14000rpm
11. Acetone solution was replaced into 2 ml tube.
12. Acetone was removed using rotary evaporator.
13. 50 ul of Chloroform/Methanol solution(weight ratio of 9:1) was added.
14. Sample was vortexed and spinned down.
15. The water layer was removed.
16. Spot the sample onto TLC silica gel plate, and developed by acetone/hexane solution.(weight ratio of 1:3)
17. Spot was observed under visible light.
