"
Team:TMU-Tokyo/Notebook experiment/2nd
From 2013.igem.org
Experiment > 2nd week
September 8th
PCR following fragment. (2nd PCR)
| Fragment | Fragment IF2 | Fragment FS2 |
| Primer | CmNR-Not FLP-R | CmC2 FLP-L |
| Templete | PtetR1 FLP | FLP Sm |
1: Fragment IF2, 2: Fragment FS2
PCR following fragment. (3rd PCR)
| Fragment | Fragment 1 |
| Primer | lacA-IRR/IRL cynR-SaApR |
| Templete | IF2 FS2 |
September 9th
PCR following fragment. (3rd PCR)
| Fragment | Fragment 1-A | Fragment 1-B | Fragment 1-C | Fragment 1-D |
| Primer | lacA-IRR/IRL cynR-SaApR | lacA-IRR/IRL cynR-SaApR | lacA-IRR/IRL cynR-SaApR | CmC2 CmNR-Not |
| Templete | IF2 FS | IF FS2 | IF2 FS2 | IF2 FS2 |
September 10th
PCR Result (Fragment 1-A, 1-B, 1-C, 1-D)
1: Fragment 1-A, 2: Fragment 1-B,
3: Fragment 1-C, 4: Fragment 1-D
Plasmid Extract attP (BBa_K617004)
Vector Remodeling with PCR. Insertion Recognition sites (NruⅠ).
| Fragment | C3 | attP1･2 | A3 |
| Primer | pSB1C3-NSP2 pSB1C3-NXE2 | attP-NruⅠR attP-NruⅠL | pSB1C3-NSP2 pSB1C3-NXE2 |
| Templete | attP | attP | IRR |
1: C3, 2: attP1･2, 3: A3
DNA Existract following fragment from gel.
| C3, A3, 1-A, 1-B, 1-3 |
September 11th
Digestion
| pSB1C3, pSB1A3 |
1: C3, 2:A3
Electro Pration
| Fragment 1, 2, 3 to Escherichia coli MG1655 red |
PCR following vector
| Fragment | pSB1A3 | pSB1C3 |
| Primer | pSB1C3-NSP2 pSB1C3-NXE2 | pSB1C3-NSP2 pSB1C3-NXE2 |
| Templete | attP IRR | attP IRR |
1-2: pSB1A3, 3-7: pSB1C3
September 12th
Digestion
| pSB1A3, pSB1C3 |
1-2: pSB1A3, 3-6: pSB1C3
Ligation following vector and fragment.
| Plasmid name | Fragment 1 | Fragment 2 | Fragment 3 - Ap | Fragment 3 - Cm |
| Vector | pSB1C3 | pSB1C3 | pSB1C3 | pSB1A3 |
| Fragment | Fragment 1-C | Fragment 2 | FRT-Ap | lacI-FRT-Cm |
Electro Poration
| Fragment 1-C, 2 and FRT-Ap to Escherichia coli MG1655 red |
PCR following fragment. (1st PCR)
| Fragment | pBAD | fimE |
| Primer | pBAD-N2 pBAD-C3 | pBAD-fimE fimE-C-EcoRⅠ |
| Templete | pBAD-MycHis | fimE (BBa_K137007) |
September 13th
Electro Poration Result
Digestion following fragment.
| attP, Km-Blunt |
PCR following fragment.
| Fragment | Km-Blunt | fimE |
| Primer | Km-BluntN Km-BluntC |
|
| Templete | pACYC177 |
September 14th
PCR following fragment.
| Fragment | Fragment 1 | Fragment 2 | Fragment 3 - Ap | Fragment 3 - Cm | pBAD-fimE |
| Primer | lacA-IRR/IRL cynR-SaApR | araCHC araA-TcC | lacI-R(FRT)2 mhpA-SaApR | R(SaApR)-lacIN mhpA-CmC6 | pBAD-N2 fimE-C-EcoRⅠ |
| Templete | IF2 FS2 | Fragment 2-C Fragment 2-D | pSG76A2 | lacI FRT-Cm | pBAD fimE |
Electro Poration
| Fragment 2 (vector: pSB1C3) to Escherichia coli MG1655 red |
Back to last week<< >>Go to next week
