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Team:TMU-Tokyo/Notebook experiment/2nd
From 2013.igem.org
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<h1>Experiment > 2nd week</h1> | <h1>Experiment > 2nd week</h1> | ||
<Hr> | <Hr> | ||
| + | <Br> | ||
| + | <b>September 8th</b><Br> | ||
| + | PCR following fragment. (2nd PCR)<Br> | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>Fragment IF2</Td><Td>Fragment FS2</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>CmNR-Not<Br>FLP-R</Td><Td>CmC2<Br>FLP-L</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>PtetR1<Br>FLP</Td><Td>FLP<Br>Sm</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/b/bb/TMU-Tokyo_0908.jpg"><Br> | ||
| + | 1: Fragment IF2, 2: Fragment FS2<Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | PCR following fragment. (3rd PCR)<Br> | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>Fragment 1</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>lacA-IRR/IRL<Br>cynR-SaApR</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>IF2<Br>FS2</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/2/23/TMU-Tokyo_09081.jpg"><Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | <b>September 9th</b><Br> | ||
| + | PCR following fragment. (3rd PCR)<Br> | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>Fragment 1-A</Td><Td>Fragment 1-B</Td><Td>Fragment 1-C</Td><Td>Fragment 1-D</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>lacA-IRR/IRL<Br>cynR-SaApR</Td><Td>lacA-IRR/IRL<Br>cynR-SaApR</Td><Td>lacA-IRR/IRL<Br>cynR-SaApR</Td><Td>CmC2<Br>CmNR-Not</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>IF2<Br>FS</Td><Td>IF<Br>FS2</Td><Td>IF2<Br>FS2</Td><Td>IF2<Br>FS2</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | <b>September 10th</b><Br> | ||
| + | PCR Result (Fragment 1-A, 1-B, 1-C, 1-D)<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/1/13/TMU-Tokyo_0910.jpg"><Br> | ||
| + | 1: Fragment 1-A, 2: Fragment 1-B,<Br> | ||
| + | 3: Fragment 1-C, 4: Fragment 1-D<Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | Plasmid Extract attP (BBa_K617004)<Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | Vector Remodeling with PCR. Insertion Recognition sites (NruⅠ).<Br> | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>C3</Td><Td>attP1･2</Td><Td>A3</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>pSB1C3-NSP2<Br>pSB1C3-NXE2</Td><Td>attP-NruⅠR<Br>attP-NruⅠL</Td><Td>pSB1C3-NSP2<Br>pSB1C3-NXE2</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>attP</Td><Td>attP</Td><Td>IRR</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/b/b8/TMU-Tokyo_09101.jpg"><Br> | ||
| + | 1: C3, 2: attP1･2, 3: A3<Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | DNA Existract following fragment from gel. | ||
| + | <Table><Tr><Td>C3, A3, 1-A, 1-B, 1-3</Td></Tr></Table> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | <b>September 11th</b><Br> | ||
| + | Digestion | ||
| + | <Table><Tr><Td>pSB1C3, pSB1A3</Td></Tr></Table> | ||
| + | Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/b/b1/TMU-Tokyo_0911.jpg"><Br> | ||
| + | 1: C3, 2:A3<Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | Electro Pration<Br> | ||
| + | <Table><Tr><Td>Fragment 1, 2, 3 to <i>Escherichia coli</i> MG1655 red</Td></Tr></Table> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | PCR following vector<Br> | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>pSB1A3</Td><Td>pSB1C3</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>pSB1C3-NSP2<Br>pSB1C3-NXE2</Td><Td>pSB1C3-NSP2<Br>pSB1C3-NXE2</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>attP<Br>IRR</Td><Td>attP<Br>IRR</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/0/01/TMU-Tokyo_09111.jpg"><Br> | ||
| + | 1-2: pSB1A3, 3-7: pSB1C3<Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | <b>September 12th</b><Br> | ||
| + | Digestion | ||
| + | <Table><Tr><Td>pSB1A3, pSB1C3</Td></Tr></Table> | ||
| + | Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/5/56/TMU-Tokyo_0912.jpg"><Br> | ||
| + | 1-2: pSB1A3, 3-6: pSB1C3<Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | Ligation following vector and fragment.<Br> | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Plasmid name</Td><Td>Fragment 1</Td><Td>Fragment 2</Td><Td>Fragment 3 - Ap</Td><Td>Fragment 3 - Cm</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Vector</Td><Td>pSB1C3</Td><Td>pSB1C3</Td><Td>pSB1C3</Td><Td>pSB1A3</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>Fragment 1-C</Td><Td>Fragment 2</Td><Td>FRT-Ap</Td><Td>lacI-FRT-Cm</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | Electro Poration | ||
| + | <Table><Tr><Td>Fragment 1-C, 2 and FRT-Ap to <i>Escherichia coli</i> MG1655 red</Td></Tr></Table> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | |||
| + | PCR following fragment. (1st PCR)<Br> | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>pBAD</Td><Td>fimE</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>pBAD-N2<Br>pBAD-C3</Td><Td>pBAD-fimE<Br>fimE-C-EcoRⅠ</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>pBAD-MycHis</Td><Td>fimE (BBa_K137007)</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | |||
| + | |||
| + | |||
| + | <b>September 13th</b><Br> | ||
| + | Electro Poration Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/2/20/TMU-Tokyo_0912_1.jpg"><Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/2/2b/TMU-Tokyo_0912_2.jpg"><Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/4/42/TMU-Tokyo_0912_3.jpg"><Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | Digestion following fragment. | ||
| + | <Table><Tr><Td>attP, Km-Blunt</Td></Tr></Table> | ||
| + | Result<Br> | ||
| + | <IMG Src="https://static.igem.org/mediawiki/2013/5/54/TMU-Tokyo_0912_00.jpg"><Br> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | PCR following fragment. | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>Km-Blunt</Td><Td>fimE</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>Km-BluntN<Br>Km-BluntC</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>pACYC177</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | |||
| + | |||
| + | |||
| + | <b>September 14th</b><Br> | ||
| + | PCR following fragment. | ||
| + | <div style="text-align:center;"> | ||
| + | <Table cellpadding="5"> | ||
| + | <Tr> | ||
| + | <Td>Fragment</Td><Td>Fragment 1</Td><Td>Fragment 2</Td><Td>Fragment 3 - Ap</Td><Td>Fragment 3 - Cm</Td><Td>pBAD-fimE</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Primer</Td><Td>lacA-IRR/IRL<Br>cynR-SaApR</Td><Td>araCHC<Br>araA-TcC</Td><Td>lacI-R(FRT)2<Br>mhpA-SaApR</Td><Td>R(SaApR)-lacIN<Br>mhpA-CmC6</Td><Td>pBAD-N2<Br>fimE-C-EcoRⅠ</Td> | ||
| + | </Tr> | ||
| + | <Tr> | ||
| + | <Td>Templete</Td><Td>IF2<Br>FS2</Td><Td>Fragment 2-C<Br>Fragment 2-D</Td><Td>pSG76A2</Td><Td>lacI<Br>FRT-Cm</Td><Td>pBAD<Br>fimE</Td> | ||
| + | </Tr> | ||
| + | </Table> | ||
| + | </div> | ||
| + | <Br> | ||
| + | <Br> | ||
| + | Electro Poration<Br> | ||
| + | <Table><Tr><Td>Fragment 2 (vector: pSB1C3) to <i>Escherichia coli</i> MG1655 red</Td></Tr></Table> | ||
| + | <Br> | ||
| + | <Br> | ||
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Latest revision as of 19:37, 27 September 2013
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 |
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