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Team:Chiba/jk/γ/week 2
From 2008.igem.org
Contents |
WEEK 2
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
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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