From 2013.igem.org
Sep 17
Liquid Culture
Tatsui and Hirano
| Sample | medium
|
| Spi-DT | LB medium(+CP)
|
| Pcon-Spi-DT,Pcon-pT181-anti-spi-DT | LB medium(+Amp)
|
PCR
Hirano
| Pcon-tet ap-DT(VF) | BigDye | 5x buffer | primer(VF) | MilliQ | total
|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96°C | 96°C | 50°C | 60°C | --
|
| 2m | 10s | 5s | 4m | 30cycles
|
| Pcon-tet ap-DT(VR) | BigDye | 5x buffer | primer(VR) | MilliQ | total
|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96°C | 96°C | 50°C | 60°C | --
|
| 2m | 10s | 5s | 4m | 30cycles
|
Ligation
No Name
| state | Vector | Inserter | Ligation High ver.2
|
| 8/28 | name | xx µg/mL | name | xx µg/mL | xx µg/mL
|
| NC | name | xx µg/mL | name | xx µg/mL | xx µg/mL
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | pSB1C3 | E+S
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| pSB1C3 | 10.0 | 1.84 | 0.40
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | pSB4K5 | X+P
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| pSB4K5 | 7.6 | 1.81 | 0.32
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | Pcon-RBS-tetR-DT | X+P
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| Pcon-RBS-tetR-DT | 8.0 | 2.40 | 1.14
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | pSB1C3 | E+P
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| pSB1C3 | 9.1 | 2.17 | 0.35
|
PCR
No name
| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA_fwd primer | SasA_rev primer | MilliQ | total
|
| | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| °C | °C | °C | °C | --
|
| （◎Д◎） | | | |
|
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