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Team:Chiba/jk/γ/week 4
From 2008.igem.org
Contents |
Week 4
7,September,2008
Cloning
Digestion
vector:BBa_R0040(ptet)① 75ng/μl
insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
vector:BBa_R0010(pLac)⑤ 100ng/μl
insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl
| Sample No | ① | ② | ③ | ④ | ⑤ | ⑥ | |
| DNA(digestion sample) | 50 | 54 | 54 | 45 | 14 | 54 | |
| SpeⅠ | 1 | 0 | 0 | 0 | 1 | 0 | |
| PstⅠ | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | |
| XbaⅠ | 0 | 0.5 | 0.5 | 0.5 | 0 | 0.5 | |
| BSA(x10) | 0.6 | 7 | 7 | 6 | 2 | 7 | |
| Buffer2 | 6 | - | - | - | 2 | - | |
| Buffer3 | - | 7 | 7 | 6 | - | 7 | |
| dH2O | 1.9 | 1 | 1 | 2 | 0.5 | 1 | |
| TOTAL | 60 | 70 | 70 | 60 | 20 | 70 |
----->37℃ 3hour
----->Gel Extraction
| Sample No | ① | ② | ③ | ④ | ⑤ | ⑥ |
| DNA(digestion sample) | 6 | 10 | 10 | 10 | 4 | 10 |
| Loading Dye | 2 | 2 | 2 | 2 | 1 | 2 |
| dH2O | 4 | - | - | - | 1 | 2 |
| TOTAL | 12 | 12 | 12 | 12 | 6 | 12 |
| well | 10 | 7 | 7 | 6 | 5 | 7 |
->DNA purification->①-④,⑥->7μl ⑤->6μl
8,Septmeber,2008
SAP
BBa_R0040(ptet)①
BBa_R0010(pLac)⑤
| Sample No | ① | ⑤ | |
| DNA(digestion sample) | 7 | 6 | |
| SAP/td> | 2 | 2 | |
| SAP Buffer | 3 | 3 | |
| TOTAL | 30 | 30 | |
| TOTAL | 30 | 30 |
->37℃ 1hour
->65℃ 30min
->DNA purification
->21μl 6μl
->Gel cheack
| Sample No | each |
| DNA(digestion sample) | 1 |
| Loading Dye | 1 |
| dH2O | 4 |
| TOTAL | 6 |
vector:BBa_R0040(ptet)① 75ng/μl
insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
vector:BBa_R0010(pLac)⑤ 100ng/μl
insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl
| Sample No | ①&② | ①&③ | ①&④ | ① | ⑤&⑥ | ⑤ |
| DNA(vector) | 3 | 3 | 3 | 3 | 5 | 2.1 |
| DNA(insert) | 3 | 3 | 3 | - | 3.1 | - |
| Ligase | 1 | 1 | 1 | 1 | 1 | 1 |
| Ligase Buffer(x5) | 2 | 2 | 2 | 2 | 2 | 2 |
| dH2O | 1 | 1 | 1 | 4 | 8.9 | 4 | TOTAL | 10 | 10 | 10 | 10 | 20 | 10 |
9,September,2008
10,September,2008
TIME Responce(liquid)
TIME Responce(solid)
Colony PCR
->Gel cheack
MINI Prep
11,September,2008
12,September,2008
13,September,2008
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