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Team:Chiba/jk/γ/week 3
From 2008.igem.org
Contents |
WEEK 3
31,August,2008
digestion
BBa_R0010①
PCR product(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 |
| dH20 | 28.5 | 28.5 |
| TOTAL | 50 | 50 |
->37℃ 3hour
->①20μl refine
->②20μl refine
1,September,2008
Ligation
vector:BBa_R0010 insert:BBa_J52008①
negative control:BBa_R0010②
| 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
BBa_R0010+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(BBa_J52008+BBa_F2620 ) x3
Venus YFP(BBa_E2030+BBa_F2620) x3
5,September,2008
TIME RESPONCE(liquid)
Digestion test
BBa_J52008+BBa_F2620 x2
BBa_E2030+BBa_F2620 x2
| DNA(digestion sample) | 2 |
| PstⅠ | 0.1 |
| BSA | 0.8 |
| Buffer3 | 1 |
| dH2O | 7 |
| TOTAL | 10 |
--->37℃ 30in
Gel Cheack
BBa_J52008+BBa_F2620 x2①
BBa_E2030+BBa_F2620 x2②
BBa_J52008(PCR Product)③
BBa_E2050④
| Sample No | ①~② | ③,④ |
| DNA(digestion sample) | 10 | 1 |
| loading Dye | 2 | 1 |
| TOTAL | 12 |
Luciferase cheack->no light
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