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Team:Chiba/jk/γ/week 3
From 2008.igem.org
Contents |
WEEK 3
31,August,2008
- [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]①
- PCR product([http://partsregistry.org/Part:BBa_J52008 BBa_J52008])②
| Sample No. | ① | ② |
| DNA | 10 | 10 |
| SpeⅠ | 1 | 0 |
| PstⅠ | 1 | 1 |
| XbaⅠ | 0 | 1 |
| BSA(x100) | 0.5 | 0.5 |
| Buffer2 | 5 | 0 |
| Buffer3 | 0 | 5 |
| dH2O | 28.5 | 28.5 |
| TOTAL | 50 | 50 |
-->37℃ 3hour
-->①20μl refine
-->②20μl refine
1,September,2008
- vector:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]①
- negative control:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010]②
| Sample No. | ① | ② |
| vector | 2 | 2 |
| insert | 3 | 0 |
| Ligase Buffer | 2 | 2 |
| Ligase | 1 | 1 |
| dH2O | 3 | 5 | TOTAL | 10 | 10 |
-->R/T 2hour
TRANSFORMATION
BBa_R0079(Las promoter)
BBa_R0071(RhlR promoter)
BBa_R0077(cinR promoter+RBS)
BBa_R0078(cnR promoter)
BBa_R0062(LUX promoter)
2,September,2008
COLONY PCR
[http://partsregistry.org/Part:BBa_R0010 BBa_R0010]+[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]
| Sample No. | ① |
| culture | 1 | Fwd primer | 1.5 |
| Rev primer | 1.5 |
| Thermo pol Buffer | 3 |
| dNTP mix | 3 |
| Taq DNA pol (NEB) | 0.2 (1 unit) |
| dH2O | 19.8 | TOTAL | 30 |
MINI Prep
BBa_R0079
BBa_R0071
BBa_R0077
BBa_R0078
BBa_R0062
3,September,2008
Digestion test
BBa_R0079①
BBa_R0071②
BBa_R0077③
BBa_R0078④
BBa_R0062⑤
| Sample No. | ①~⑤ |
| DNA tamplate | 5 |
| Buffer3 | 1 |
| dH2O | 4 |
| EcoRⅠ | 0.1 | TOTAL | 10 |
->37℃30min
->Gel cheack
| Sample No. | ①~⑤ |
| DNA(digestion sample) | 10 |
| loading Dye | 2 |
| TOTAL | 12 |
4,Septempber,2008
TANSFORMATION
LIgation product
rLUciferase([http://partsregistry.org/Part:BBa_J52008 BBa_J52008]+[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] ) x3
Venus YFP([http://partsregistry.org/Part:BBa_E2030 BBa_E2030]+[http://partsregistry.org/Part:BBa_F2620 BBa_F2620]) x3
5,September,2008
TIME RESPONCE(liquid)
Digestion test
[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]+[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] x2
[http://partsregistry.org/Part:BBa_E2030 BBa_E2030]+[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] x2
| DNA(digestion sample) | 2 |
| PstⅠ | 0.1 |
| BSA | 0.8 |
| Buffer3 | 1 |
| dH2O | 7 |
| TOTAL | 10 |
--->37℃ 30in
Gel Cheack
[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]+[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] x2①
[http://partsregistry.org/Part:BBa_E2030 BBa_E2030]+[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] x2②
[http://partsregistry.org/Part:BBa_J52008 BBa_J52008](PCR Product)③
BBa_E2050④
| Sample No. | ①~② | ③,④ |
| DNA(digestion sample) | 10 | 1 |
| loading Dye | 2 | 1 |
| TOTAL | 12 | 6 |
Luciferase cheack->no light
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