From 2013.igem.org
Aug 18
Yoshida and Kojima
| Lane | DNA | Enzyme
|
| 1 | 100kb ladder | --
|
| 2 | 8/11 J23100 -② | --
|
| 3 | 8/11 J23100 -② | --
|
| 4 | -- | --
|
| 5 | 8/17 K117000(lysis1) -1 | --
|
| 6 | 8/17 K117000(lysis1) -1 | --
|
| 7 | -- | --
|
| 8 | 8/17 K748002(lysis3) -1 | --
|
| 9 | 8/17 K748002(lysis3) -1 | --
|
| 10 | -- | --
|
| 11 | 8/17 I13504(RBS-GFP-DT) -1 | --
|
| 12 | 8/17 I13504(RBS-GFP-DT) -1 | --
|
| 13 | 1kb ladder | --
|
| 14 | 8/17 I0462(RBS-luxR-DT) -1 | --
|
| 15 | 8/17 I0462(RBS-luxR-DT) -1 | --
|
| 16 | -- | --
|
| 17 | 8/17 I732020(RBS-lacZα-DT) -1 | --
|
| 18 | 8/17 I732020(RBS-lacZα-DT) -1 | --
|
fig after
fig after
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| 8/14 J231000 | 1.7 | 1.61 | 0.04
|
| 8/17 K748002(lysis3) -1 | 5.4 | 2.39 | 0.45
|
| 8/17 I13504(RBS-GFP-DT) -1 | 3.4 | 3.33 | 0.19
|
| 8/17 I0462(RBS-luxR-DT) -1 | 4.4 | 2.07 | 0.37
|
| 8/17 I732020(RBS-lacZα-DT) -1 | 2.9 | 2.17 | 0.29
|
Ligation
Ashida
| state | Vector | Inserter | Ligation High ver.2
|
| experiment | J23100 | 9.0 | RBS-lacZα-DT | 8.6 |
|
| experiment | J23100 | 9.0 | RBS-GFP-DT | 7.3 |
|
| experiment | RBS | 1 | lysis3 | 4.6 |
|
| experiment | Plux | 1 | GFP | 7.3 |
|
| experiment | J23100 | 9.0 | -- | -- |
|
| experiment | RBS | 1.0 | -- | -- |
|
| experiment | Plux | 1.0 | -- | -- |
|
incubate 16°C 1hour
Restriction Enzyme Digestion
Murata and Okazaki
| | 8/17K112806(lysis2)-1 | XbaI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 3.0µL | 30 µL
|
| 1 cuts | 1.0&mic6/8ro;L | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10 µL
|
| 1 cuts | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10 µL
|
Electrophoresis
Okazaki and Honda
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 100bp ladder | -- | --
|
| 2 | lysis1 | Xba1 | Pst1
|
| 3 | lysis1 | Xba1 | --
|
| 4 | lysis1 | -- | Pst1
|
| 5 | lysis1 | -- | --
|
| 6 | lysis2 | Xba1 | Pst1
|
| 7 | lysis2 | Xba1 | --
|
| 8 | lysis2 | -- | Pst1
|
| 9 | lysis2 | -- | --
|
| 10 | 100bp ladder | -- | --
|
Colony PCR
No name
| Sample | base pair
|
| 8/8 LBKana | --
|
| 8/16 Pbad araC | --
|
| NC | --
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 1min |
|
Liquid Culture
No name
| Sample | medium
|
| 7/8 LB Kana | Plusgrow(+Kana)
|
| 8/16 Pbad araC | Plusgrow(+Kana)
|
Medium Plate
No name
| volume | 200ml
|
| Bacto Trypton | 2g
|
| Bacto yeasy extract | 1g
|
| NaCl | 1g
|
| Agar Powder | 2g
|
Transformation
No name
| Name | Sample | Competent Cells | Total | Plate
|
| 8/18 J23100+RBS-LacZα-DT | 2µL | 20µL | 22µL | Amp
|
| 8/18 J23100+RBS-GFP-DT | 2µL | 20µL | 22µL | Amp
|
| 8/18 J23100+RBS-luxR-DT | 2µL | 20µL | 22µL | Amp
|
| RBS+lysis3 | 2µL | 20µL | 22µL | Amp
|
| Plux+GFP | 2µL | 20µL | 22µL | CP
|
| J23100 | 2µL | 20µL | 22µL | Amp
|
| RBS | 2µL | 20µL | 22µL | Amp
|
| Plux | 2µL | 20µL | 22µL | CP
|
Electrophoresis
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