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Team:TMU-Tokyo/Notebook experiment/1st
From 2013.igem.org
Experiment > Parts Synthesis > 1st week
September 2nd
PCR following fragment. (1st PCR)
| Fragment | PtetR1 | PtetR2 | FLP | Sm | araC-P |
| Primer | CmNR-stu FLP-IRR/IRL | CmCR-stu FLP-IRR/IRL | FLP-L FLP-R | FLP-SaApL CmC | acaC-2 BAD-C3 |
| Templete | IRR/IRL-GFP (BBa_K880002) | IRR/IRL-GFP (BBa_K880002) | pFTGT | m(C2-Ap)Sm | pBAD/Myc-HisA |
| Fragment | hbiF | Tc | lacI | FRT-Cm | FRT-Ap |
| Primer | pBAD-HbiF RTcN-hbiF | TcN TcC3 | 141-70 FRT-lacIC | R6Kori-Xho-3R R(FRT)2 | lacI-R(FRT)2 mhpA-SaApR |
| Templete | hbiF (BBa_K880000) | Escherichia coli MG1655 recA⁻ (cell suspention) | Escherichia coli MG1655 genome | pSG76C | pSG76A2 |
1: PterR1, 2: PtetR2, 3: FLP, 4: Sm, 5: araC-P,
6: hbiF, 7: Tc, 8: LacI, 9: FRT-Cm, 10: FRT-Ap
September 3rd
Vector Remodeling with PCR. Insertion Recognition sites (Blunt).
| Fragment | Km-Blunt |
| Primer | Km-Blunt-N Km-Blunt-C |
| Templete | pACYC177 |
DNA Existract following fragment from gel.
| PtetR1, FLP, Sm, araC-P, hbiF, Tc, lacI, FRT-Cm, FRT-Ap, Km-Blunt |
September 4th
PCR following fragment. (2nd PCR)
| Fragment | Fragment 1 | Fragment 2 | Fragment 3 |
| Primer | lacA-IRR/IRL cynR-SaApL | araCHC araA-TcC | R(SaApR)-lacIN mhpA-CmC6 |
| Templete | PtetR FLP Sm | araC-P hbiF Tc | lacI FRT-Cm |
1: Fragment 1, 2: Fragment 2, 3: Fragment 3
September 5th
PCR following fragment. (2nd PCR)
| Fragment | Fragment 1-A | Fragment 1-B | Fragment 2-C | Fragment 2-D |
| Primer | CmNR-Stu FLP-R | FLP-L CmC | araC2 RTcN-hbiF | pBAD-hbiF TcC3 |
| Templete | PtetR FLP | FLP Sm | araC-P hbiF | hbiF Tc |
1: Fragment 1-A, 2: Fragment 1-B,
3: Fragment 2-C, 4: Fragment 2-D
Check following gel extract product.
| PtetR1, FLP, Sm, araC-P, hbiF, Tc, lacI, FRT-Cm, FRT-Ap, Km-Blunt |
1: PtetR1, 2: FLP, 3: Sm, 4: araC-P, 5: hbiF,
6: Tc, 7: lacI, 8: FRT-Cm, 9: FRT-Ap, 10: Km-Blunt
September 6th
PCR following fragment. (2nd PCR)
| Fragment | Fragment 1-A | Fragment 1-B | Fragment 2-A | Fragment 2-B |
| Primer | CmNR-Stu FLP-R | FLP-L CmC | FLP-L CmC | FLP-L cynR-SaApL |
| Templete | PtetR FLP | FLP Sm | Sm FLP | Sm FLP |
1: Fragment 1-A, 2: Fragment 2-B
(1-B and 2-A was failed in PCR)
PCR following fragment. (2nd PCR)
| Fragment | Fragment IF | Fragment FS |
| Primer | lacA-IRR/IRL FLP-R | FLP-L cynR-SaApR |
| Templete | PtetR FLP | FLP Sm |
September 7th
PCR Result (Fragment IF, FS)
1: Fragment IF, 2: Fragment FS
PCR following fragment. (3rd PCR)
| Fragment | Fragment 1 | Fragment 2 |
| Primer | lacA-IRR/IRL cynR-SaApR | araCHC araA-TcC |
| Templete | Fragment IF Fragment FS | Fragment 2-B Fragment 2-C |
1: Fragment 1, 2: Fragment 2
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