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Team:TMU-Tokyo/Notebook experiment/4th
From 2013.igem.org
Experiment > 4th week
September 22th
Mini prep following plasmid.
| fimE-pBAD(in pSB1A3), pSB1C3-Km, pSB1A3-Km |
1: pSB1C3-Km, 2-4: pSB1A3-Km, 5-6: fimE-pBAD
Check PCR.
| Fragment | (control) | Fragment 3 - ApCm |
| Primer | 141-49 SaApL | 141-49 SaApL |
| Templete | Escherichia coli MG1655 red (Cell suspention) | (Cell suspention) |
1-9: Fragment 3 - ApCm
Check PCR.
| Fragment | (control) | Fragment 3 - ApCm | Fragment 3 - ApCm |
| Primer | 2 sets of the others | 141-1 141-20 | 141-1 141-74 |
| Templete | Escherichia coli MG1655 red (Cell suspention) | (Cell suspention) |
1-7: 141-1 and 141-20, 8: control(141-1 and 141-20),
9-15: 141-1 and 141-74, 16: control(141-1 and 141-74),
Digestion following plasmid.
| pSB1C3-Km-Blunt |
P1 Preparation following fragment.
| Fragment 1, 2 |
September 23th
PCR following fragment.
| Fragment | Fragment 3 - ApCm |
| Primer | mhpA-CmC6 SaApL |
| Templete | Fragment 3 - Cm Ap(SaApR-SaApL) |
1: Fragment 3 - Cm, 2: Fragment 3 - ApCm,
3: pSB1C3-Km-Blunt
Check PCR.
| Fragment | Fragment 3 - Ap (control) | Fragment 3 - ApCm |
| Primer | CmNR-Not SaApL | CmNR-Not SaApL |
| Templete | (Cell suspention) | (Cell suspention) |
1-31: Fragment 3 - ApCm
Electro Pration
| Fragment 3 - Cm to Escherichia coli MG1655 red. |
P1 Preparation
| Fragment 3 - ApCm |
September 24th
P1 transdunction
| Fragment 1, 2 |
| Fragment | Fragment 3 - Ap (control) | Fragment 3 - ApCm |
| Primer | CmNR-Not 141-1 | CmNR-Not 141-1 |
| Templete | (Cell suspention) | (Cell suspention) |
1-16: Fragment 3 - ApCm
P1 transduction
| Fragment 2 to Fragment 1 |
| Fragment 1 to Fragment 2 |
Get Fragment 1-2
Electro Poration
| Fragment 1, 2, 3 - Ap, 3 - Cm, 3 - ApCm (in pSBC3-Km) to Escherichia coli DH1 |
September 25th
Check PCR.
| Fragment | Fragment 3 - ApCm (control) | Fragment 3 - ApCm |
| Primer | 141-49 141-1 | CmNR-Not 141-1 |
| Templete | (Cell suspention) | (Cell suspention) |
1-7: 141-49 and 141-1, 8: control(141-49 and 141-8),
9-15: 141-1 and CmNR-Not, 16: control(141-1 and CmNR-Not)
September 26th
P1 transduction
| Fragment 3 to Fragment 1-2 |
Check PCR
| Fragment | Fragment 1-2-3 | Fragment 1-2 (control) |
| Primer | CmNR-Not 141-1 | 141-49 141-1 |
| Templete | (Cell suspention) | (Cell suspention) |
1-7: Fragment 1-2-3, 8: cotrol, 9-10: Fragment 1,
11-13: Fragment 2, 14-15: Fragment 3 - Ap, 16-17: Fragment 3 - Cm
18-19: Fragment 3 - AmCm
September 27th
Assay
Pythagorean devices
| LB 2ml + arabinose -10% 0.1ml | + with the inoculating loop 1655 ins①~③ No.1 + with the inoculating loop 1655 ins①~③ No.2 + with the inoculating loop 1655 ins①~③ No.3 + with 1655 |
| LB 2ml | + with the inoculating loop 1655 ins①~③ No.1 + with the inoculating loop 1655 ins①~③ No.2 + with the inoculating loop 1655 ins①~③ No.3 + with 1655 |
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