From 2013.igem.org
Aug 17
Colony PCR
Nakamoto
| Sample | base pair
|
| 8/15 BBa_K112806(lysis2) -(1) | 828
|
| NC | --
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 1min | 30cycles
|
| Sample | base pair
|
| 8/10 BBa_Q0400 -(1) | 3995
|
| NC | --
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 30s | 30cycles
|
Electrophoresis
No name
| Lane | Sample
|
| 1 | 1kbp ladder
|
| 2 | K112806(lysis2) -1
|
| 3 | NC
|
| 4 | Q04400(tetR) -1
|
| 5 | NC
|
| 6 | 1kbp ladder
|
Liquid Culture
Nakamoto
| Sample | medium
|
| 8/15 BBa_K112806(lysis2) -(1) | Plusgrow medium(+CP)
|
| 8/15 BBa_Q04400(tetR) -(1) | Plusgrow medium(+Amp)
|
| 8/16 pBR322 -(1) | Plusgrow medium(+Amp)
|
Master Plate
Nakamoto
| Number | Use LB plate (+CP)
|
| 1 | BBa_K112806(lysis2) -(1)
|
| Number | Use LB plate (+Kana)
|
| 1 | BBa_Q04400(tetR) -(1)
|
Miniprep
Nakamoto and Murakami
| DNA | concentration[µg/mL] | 260/280 | 260/230
|
| K748002(lysis3) -(1) | 330 | 1.66 | 2.08
|
| B0015(DT) -(1) | 188 | 1.73 | 1.82
|
| R0061(Plux) -(1) | 76 | 1.64 | 1.30
|
| K117000(lysis1) -(1) | 286 | 1.71 | 2.01
|
| K117000(lysis1) -(2) | 248 | 1.69 | 1.78
|
| I732020(RBS-lacZα-DT) -(1) | 198 | 1.71 | 1.93
|
| I13504(RBS-GFP-DT) -(1) | 248 | 1.69 | 2.03
|
| I13504(RBS-GFP-DT) -(2) | 246 | 1.55 | 1.73
|
| I0462(RBS-luxR-DT) -(1) | 326 | 1.65 | 1.74
|
Restriction Enzyme Digestion
Murata, Murakami, Nakamoto, Ashida, Matsumoto and Yoshida
| | 8/11 J23100 -(2) | SpeI | PstI | Buffer | MilliQ | total
|
| 2 cuts | 4.0µL | 1.0µL | 1.0µL | 3.0µL | 21µL | 30µL
|
| 1 cut | 1.3µL | 0.2µL | -- | 1.0µL | 7.5µL | 10µL
|
| 1 cut | 1.3µL | -- | 0.2µL | 1.0µL | 7.5µL | 10µL
|
| NC | 1.3µL | -- | -- | 1.0µL | 7.7µL | 10µL
|
| | 8/15 B0034(RBS) -(2) | SpeI | PstI | Buffer | MilliQ | total
|
| 2 cuts | 6.0µL | 1.0µL | 1.0µL | 3.0µL | 19µL | 30µL
|
| 1 cut | 2.0µL | 0.2µL | -- | 1.0µL | 6.8µL | 10µL
|
| 1 cut | 2.0µL | -- | 0.2µL | 1.0µL | 6.8µL | 10µL
|
| NC | 2.0µL | -- | -- | 1.0µL | 7.0µL | 10µL
|
| | 8/17 K117000(lysis1) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total
|
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL
|
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL
|
| | 8/17 K748002(lysis3) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total
|
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL
|
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL
|
| | 8/17 I13504(RBS-GFP-DT) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total
|
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL
|
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL
|
| | 8/17 I0462(RBS-luxR-DT) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total
|
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL
|
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL
|
| | 8/17 I732020(RBS-lacZα-DT) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total
|
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL
|
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL
|
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL
|
| | 8/11 R6061 -(1) | SpeI | PstI | Buffer | MilliQ | total
|
| 2 cuts | 8.0µL | 1.0µL | 1.0µL | 3.0µL | 17µL | 30µL
|
| 1 cut | 3.0µL | 0.2µL | -- | 1.0µL | 5.8µL | 10µL
|
| 1 cut | 3.0µL | -- | 0.2µL | 1.0µL | 5.8µL | 10µL
|
| NC | 3.0µL | -- | -- | 1.0µL | 6.0µL | 10µL
|
- incubate at 37 °C, for 1 h
Electrophoresis
No name
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | J23100 -② | SpeI | PstI
|
| 2 | J23100 -② | SpeI | --
|
| 3 | J23100 -② | -- | PstI
|
| 4 | J23100 -② | -- | --
|
| 5 | B0034(RBS) -(2) | SpeI | PstI
|
| 6 | B0034(RBS) -(2) | SpeI | --
|
| 7 | B0034(RBS) -(2) | -- | PstI
|
| 8 | B0034(RBS) -(2) | -- | --
|
| 9 | 1kb ladder | -- | --
|
| 10 | K11700(lysis1) -(1) | XbaI | PstI
|
| 11 | K11700(lysis1) -(1) | XbaI | --
|
| 12 | K11700(lysis1) -(1) | -- | PstI
|
| 13 | K11700(lysis1) -(1) | -- | --
|
| 14 | K748002(lysis3) -(1) | XbaI | PstI
|
| 15 | K748002(lysis3) -(1) | XbaI | --
|
| 16 | K748002(lysis3) -(1) | -- | PstI
|
| 17 | K748002(lysis3) -(1) | -- | --
|
| Lane | Sample | Enzyme1 | Enzyme2
|
| 18 | I13504(RBS-GFP-DT) -(1) | XbaI | PstI
|
| 19 | I13504(RBS-GFP-DT) -(1) | XbaI | --
|
| 20 | I13504(RBS-GFP-DT) -(1) | -- | PstI
|
| 21 | I13504(RBS-GFP-DT) -(1) | -- | --
|
| 22 | I0462(RBS-luxR-DT) -(1) | XbaI | PstI
|
| 23 | I0462(RBS-luxR-DT) -(1) | XbaI | --
|
| 24 | I0462(RBS-luxR-DT) -(1) | -- | PstI
|
| 25 | I0462(RBS-luxR-DT) -(1) | -- | --
|
| 26 | 1kb ladder | -- | --
|
| 27 | I732020(RBS-lacZα-DT) -(1) | XbaI | PstI
|
| 28 | I732020(RBS-lacZα-DT) -(1) | XbaI | --
|
| 29 | I732020(RBS-lacZα-DT) -(1) | -- | PstI
|
| 30 | I732020(RBS-lacZα-DT) -(1) | -- | --
|
| 31 | R0061(Plux) -(1) | SpeI | PstI
|
| 32 | R0061(Plux) -(1) | SpeI | --
|
| 33 | R0061(Plux) -(1) | -- | PstI
|
| 34 | R0061(Plux) -(1) | -- | --
|
Miniprep
Murata
| DNA | concentration[µg/mL] | 260/280 | 260/230
|
| 8/17 PBR322(TaKaRa) -(1) | 47 | 1.68 | 1.55
|
| 8/17 K112806(lysis2) -(1) | 64 | 1.67 | 1.70
|
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