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Team:TMU-Tokyo/Notebook experiment/3rd
From 2013.igem.org
Experiment > 3rd week
September 15th
PCR result
1: Fragment 1, 2: Fragment 2, 3: Fragment 3 - Ap,
4: Fragment 3- Cm, 5: pBAD-fimE
Ligation fragment and vector.
| Plasmid name | Fragment 1-A+B | Fragment 2 | Fragment 3 - Ap |
| Fragment | Fragment 1-A Fragment 1-B | Fragment 2 | Fragment 3 - Ap |
| Vector | pSB1C3 | pSB1C3 | pSB1C3 |
Transformation following plasmid to Escherichia coli MG1655 red.
| Fragment 1, 2, 3 - Ap |
September 16th
Transformation result.
Mini prep following plasmid.
| Fragment 1, 2, 3 - Ap, 3 - Cm, Km-Blunt |
1: Fragment 1, 2: Fragment 3 - Ap,
3-5: Fragment 3 - Cm, 6-8: Km-Blunt
PCR following fragment.
| Fragment | Fragment 1 | Fragment 2 | Fragment 3 - Ap | Fragment 3 - Cm |
| Primer | lacA-IRR/IRL cynR-SaApR | araCHC araA-TcC | lacI-R(FRT)2 mhpA-SaApR | p(SaApR)-lacIN mhpA-CmC6 |
| Templete | IF2 FS2 | Fragment 2-C Fragment 2-D | pSG76A2 | lacI FRT-Cm2 |
September 17th
PCR result.
1-2: Fragment 1, 3-4: Fragment 2,
5-6: Fragment 3 - Cm, 8-10: Fragment 3 - Ap
Electro Poration
| Fragment 1, 2, 3 - AP to Escherichia coli MG1655 red. |
PCR following fragment.
| Fragment | Fragment 1 | Fragment 2 | Fragment 3 - Ap | Fragment 3 - Cm |
| Primer | lacA-IRR/IRL cynR-SaApR | araCHC araA-TcC | lacI-R(FRT)2 mhpA-SaApR | p(SaApR)-lacIN mhpA-CmC6 |
| Templete | IF2 FS2 | Fragment 2-C Fragment 2-D | pSG76A2 | lacI FRT-Cm2 |
September 18th
PCR result.
1-3: Fragment 1, 4-6: Fragment 2,
7-9: Fragment 3 - Ap, 1-3: Fragment 3 - Cm
Mini prep following plasmid
| pSB1C3-Km, pSB1A3 |
PCR following fragment.
| Fragment | fimE | Fragment 1 | Fragment 2 | Fragment 3 - Cm |
| Primer | pBAD-N9 fimE-C-EcoRⅠ | lacA-IRR/IRL cynR-SaApR | araCHC araA-TcC | R(SaApR)-lacIN mhpA-CmC6 |
| Templete | pBAD fimE | IF2 FS2 | Fragment 2-C Fragment 2-D | lacI FRT-Cm |
September 19th
PCR result.
1: fimE, 2-3: Fragment 1, 4-5: Fragment 2,
6-7: Fragment 3 - Cm
PCR following fragment. (2nd PCR)
| Fragment | fimE(1N2) | fimE(2N2) | fimE(1N9) | fimE(2N9) |
| Primer | pBAD-N2 fimE-1 | pBAD-N2 fimE-2 | pBAD-N9 fimE-1 | pBAD-N9 fimE-2 |
| Templete | pBAD fimE | pBAD fimE | pBAD fimE | pBAD fimE |
1-3: 1N2, 4-6: 2N2, 7-9: 1N9, 10-12: 2N9
Electro Poration
| Fragment 1, 2, 3 - Ap to Escherichia coli MG1655 red. |
Mini prep following plasmid.
| pSB1A3, pSB1C3 |
1: pSB1A3, 2: pSB1C3
Digestion
| fimE-pBAD, pSB1C3-Km, pSB1A3 |
1: fimE, 2: pSB1C2-Km, 3: pSB1A3
September 20th
Ligation following vector and fragment.
| fimE-pBAD + pSB1A3 |
Check PCR.
| Fragment | (control) | Fragment 1 | Fragment 2 | Fragment 3 - Ap |
| Primer | 3 sets of the others | 141-64-1 FLP-R | 117-4 pBAD-C3 | 141-1 SaApR |
| Templete | Escherichia coli MG1655 red (Cell suspention) | (Cell suspention) | (Cell suspention) | (Cell suspention) |
1-5: Fragment 1, 6: control(fragment 1),
7-10: Fragment 2, 11: control(fragment 2),
12-15: Fragment 3, 16: control(fragment 3)
September 21th
Mini prep following plasmid.
| Km-Blunt in pSB1C3 |
1-3: Km-Blunt in pSB1C3
PCR following fragment.
| Fragment | Fragment 1 |
| Primer | lacA-IRR/IRL cynR-SaApR |
| Templete | IF2 FS2 |
1-2: Fragment 1
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