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Team:Chiba/jk/γ/week 2
From 2008.igem.org
Contents |
WEEK 2
24,August 2008
- [http://partsregistry.org/Part:BBa_J63001 BBa_J63001](yeast optimized YFP)-->colony
- [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](1)①
- [http://partsregistry.org/Part:BBa_F2620 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
- [http://partsregistry.org/Part:BBa_E2030 BBa_E2030]①
- [http://partsregistry.org/Part:BBa_J33202 BBa_J33202]②
- BL21③
- [http://partsregistry.org/Part:BBa_J52008 BBa_J52008]8④
- [http://partsregistry.org/Part:BBa_I712019 BBa_I712019]⑤
| 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 Check
| 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 |
- Venus
- [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
- Venus
- PUC19
- pGFPUV
- pGEX Venus YFP
- Membren Venus YFP
- [http://partsregistry.org/Part:BBa_J63001 BBa_J63001]①
| 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
- [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
- [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
- [http://partsregistry.org/Part:BBa_J63001 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:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
| Sample No | [http://partsregistry.org/Part:BBa_F2620 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:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] insert:BBa_J52008①
vector:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] insert:BBa_E2030②
Negative control:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620]③
| 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
29,August,2008
30,August,2008
Transformation
mcherry
PCR
BBa_J52008(rLuc)
| Sample No | BBa_J52008 |
| DNA tamplate | 1 |
| rLuc_fwd | 2.5 |
| Rev primer | 2.5 |
| Thermo pol Buffer | 5 |
| dNTPmix | 5 |
| Vent pol | 0.5 |
| dH2O | 34 |
| TOTAL | 50 |
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