"
August/20 August 2009
From 2009.igem.org
1.digestion and ligation
digestion with restriction enzyme
K204007 , (K204003)
| Vector | Insert | ||
|---|---|---|---|
| RBS([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 1-2M]) | 10 | cinR([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F]) | 12 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 6 | dH2O | 4 |
| total | 20uL | total | 20uL |
K204008
| Vector | Insert | ||
|---|---|---|---|
| RBS([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 1-2M]) | 10 | LuxI([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 2-8O]) | 12 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 6 | dH2O | 4 |
| total | 20uL | total | 20uL |
↓
37°C, 2hr
↓
gel electrophoresis
UP : 1-2M , 1-14F , 1-2M , 2-8O DOWN : No.3-A,B,C,D , No.4-A,B,C,D , No.6-A,B,C,D , Vector
gel cut
↓
purification by [QIAquick Nucleotide Removal Kit]
↓
ligation
ligation DNA 44 10* buffer 5 ligation 1 total 50uL ↓ 16°C overnight
2. rapid check
③(RBS+cinR),K204003
③-C,D : OK
④(RBS+LasR),K204004
④-A,B,C : OK
⑥(RBS+cinI),K204006
⑥-A,B,C : OK
