"
Team:Chiba/jk/γ
From 2008.igem.org
17,August 2008
PCR
BBa_E0430①
BBa_E0420②
BBa_J06702③
BBa_S01416④
BBa_I73003⑤
BBa_I73004⑥
BBa_I730026⑦
BBa_I763002⑧
| Sample No | ① | ② | ③ | ④ | ⑤ | ⑥ | ⑦ | ⑧ |
| DNA tamplate | 1 | 1 | 1 | 1 | >1 | 1 | 1 | 1 |
| FW primer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| VR primer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| dNTPmix | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| thermo pol buffer | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| DNA pol(VENT) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| dH2O | 68 | 68 | 68 | 68 | 68 | 68 | 68 | 68 |
| TOTAL | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
20,August 2008
TRANSFORMATION
BBa_J52008(Luciferase Renilla)-->colony
BBa_I712019(Luciferase Fire fly)-->no colony
BBa_J332002(LacZ)-->colony
BBa_E2030(Venus YFP)-->not so good
YFP(P-GEX)-->colony
BBa_F2620-->colony
BBa_T9002-->colony
21,August 2008
TRANSFORMATION
BBa_J32007(Lux CDABE)-->no colony
BBa_B0034(RBS)-->colony
22,August 2008
MINI PREP
BBa_J52008(Luciferase)
BBa_F2620
BBa_T9002
BBa_J33202(LacZ)
PUC19
PGEX VENUS YFP(GST fusion)
TRANSFORMATION
PGEX VENUS YFP(GST fusion)
24,August 2008
TRANSFORMATION
BBa_J63001(yeast optimized YFP)-->colony
Digestion
BBa_F2620(1)①
BBa_F2620(2)②
Menbren Venus YFP③
| Sample No | ① | ② | ③ |
| DNA tamplate | 3 | 3 | 3 |
| Buffer3 | 1 | 1 | 1 |
| dH2O | 6 | 6 | 6 |
| EcoRⅠ | 0.5 | 0.5 | 0.5 |
| TOTAL | 10 | 10 | 10 |
->37℃ 30min
| Sample No | ①(afetr digestion) | ②(after digestion) | ③(afetr digestion) |
| DNA tamplate | 10 | 10 | 10 |
| loading Dye | 2 | 2 | 2 |
| TOTAL | 12 | 12 | 12 |
| Sample No | ① | ② | ③ |
| DNA tamplate | 2 | 2 | 2 |
| loading Dye | 1 | 1 | 1 |
| dH2O | 3 | 3 | 3 |
| TOTAL | 6 | 6 | 6 |
->135V 30min
25,August,2008
PCR
BBa_E2030①
BBa_J33202②
BL21③
BBa_J52008④
BBa_I712019⑤
| Sample No | ① | ② | ③ | ④ | ⑤ |
| DNA tamplate | 1 | 1 | 1 | 1 | >1 |
| FW primer | 5(Venus_YFP_fwd) | 5(LacZ_fwd) | 5(LacZ_fwd) | 5(rLUC_fwd) | 5(fLuc_fwd) |
| VR primer | 5(VR) | 5(LacZ_rev) | 5(LacZ_rev) | 5(VR) | 5(VR) |
| dNTPmix | 10 | 10 | 10 | 10 | 10 |
| thermo pol buffer | 10 | 10 | 10 | 10 | 10 |
| DNA pol(VENT) | 1 | 1 | 1 | 1 | 1 |
| dH2O | 68 | 68 | 68 | 68 | 68 |
| TOTAL | 100 | 100 | 100 | 100 | 100 |
->Gel
| Sample No | ① | ② | ③ | ④ | ⑤ |
| DNA tamplate | 1 | 1 | 1 | 1 | >1 |
| loading Dye | 1 | 1 | 1 | 1 | 1 |
| dH2O | 4 | 4 | 4 | 4 | 4 |
| TOTAL | 6 | 6 | 6 | 6 | 6 |
MINI prep
Venus
BBa_F2620
TRANSFORMATRION
Venus
PUC19
pGFPUV
pGEX Venus YFP
Membren Venus YFP
PCR
BBa_J63001①
| Sample No | ① |
| DNA tamplate | 1 |
| FW primer(Venus) | 5 |
| VR primer | 5 |
| dNTPmix | 10 |
| thermo pol buffer | 10 |
| DNA pol(VENT) | 1 |
| dH2O | 68 |
| TOTAL | 100 |
26,August,2008
Gel check
BBa_F2620
BBa_F2620
BBa_J63001
| Sample No | ① | ② | ③ | ④ | ⑤ |
| DNA tamplate | 1 | 1 | 1 | 1 | >1 |
| Loading Dye | 1 | 1 | 1 | 1 | 1 |
| dH2O | 4 | 4 | 4 | 4 | 4 |
| TOTAL | 6 | 6 | 6 | 6 | 6 |
Digestion
vector:BBa_F2620
| Sample No | BBa_F2620 |
| DNA tamplate | 100 |
| SpeⅠ | 2 |
| PstⅠ | 2 |
| BSA(x10) | 13 |
| Buffer2 | 13 | TOTAL | 130 |
insert:BBa_J52008,BBa_E2030
| Sample No | J52008 | E2030 |
| DNA tamplate | 30 | 30 |
| XbaⅠ | 1 | 1 |
| PstⅠ | 1 | 1 |
| DpnⅠ | 1 | 1 |
| Buffer3 | 4 | 4 |
| BSA(x10) | 4 | 4 | TOTAL | 40 | 40 |
->37℃ 3hour
->SAP
->37℃ 30min
->65℃ 15min
->Zymo Clean
Ligation
vector:BBa_F2620 insert:BBa_J52008①
vector:BBa_F2620 insert:BBa_E2030②
Negative control:BBa_F2620③
| Sample No | ① | ② | ③ |
| vector | 5 | 5 | 5 |
| inser | 10 | 7.5 | 0 |
| Ligase Buffer | 4 | 4 | 4 |
| Ligase | 1 | 1 | 1 |
| dH2O | 0 | 2.5 | 10 |
| TOTAL | 20 | 20 | 20 |
->RT 2hour
->TRANSFORMATION XL10G
->37℃ over night
->colony PCR
->MINI PREP
28,August,2008
TIME RESPONCE(liquid)
29,August,2008
TIME RESPONCE(solid)
30,August,2008
TRANSFORMATION
mcherry
PCR
BBa_J52008(rLuc)
| Sample No | BBa_J52008 |
| DNA tamplate | 1 |
| rLuc_fwd | 2.5 |
| VR | 2.5 |
| thermo pol Buffer | 5 |
| dNTP | 5 |
| Vent pol | 0.5 |
| dH2O | 34 | TOTAL | 50 |
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
TIME RESPONCE (Solid)
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
5,September,2008
TIME RESPONCE(liquid)
Digestion test
BBa_J52008+BBa_F2620
BBa_E2030+BBa_F2620
| Sample No | each |
| DNA(digestion sample) | 10 |
| loading Dye | 2 |
| TOTAL | 12 |
Luciferase cheack->no light
7,September,2008
Digestion
BBa_R0040(ptet)①
BBa_S03158(RBS+RhlR)②
BBa_S03156(RBS+lasR)③
BBa_S03160(RBS+cinR)④
BBa_R0010(pLac)⑤
BBa_J52008(RBS+rLuc)⑥
| 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
| ホーム | メンバー紹介 | プロジェクト紹介 | Parts Submitted to the Registry | モデリング | ノート |
|---|
