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Team:Chiba/jk/γ/week 2
From 2008.igem.org
Contents |
WEEK 2
24,August 2008
- BBa_J63001(yeast optimized YFP)-->colony
| 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
- 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 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
- BBa_F2620
- Venus
- PUC19
- pGFPUV
- pGEX Venus YFP
- Membren Venus YFP
| 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
| 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 |
- 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. | BBa_J52008 | BBa_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
- 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
29,August,2008
30,August,2008
- mcherry
- 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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