NYMU Taipei/Lab Notes/2007 8 27
From 2007.igem.org
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**Final volume: 10ul | **Final volume: 10ul | ||
**RT 3h | **RT 3h | ||
| - | *Enzyme Digestion | + | *Enzyme Digestion (pOmpC) |
| - | **pOmpC: 14ul EYFP: 3ul | + | **pOmpC: 14ul |
| - | ** | + | **SpeI: 1ul |
| + | **PstI: 1ul | ||
| + | **10x buffer: 2, 2ul | ||
| + | **10x BSA: 2ul | ||
| + | **Total: 20ul | ||
| + | **37°C, 2h | ||
| + | **Heat inactivation: 65°C 20min | ||
| + | *Enzyme Digestion (EYFP) | ||
| + | **EYFP: 3ul | ||
| + | **XbaI: 1ul | ||
| + | **PstI: 1ul | ||
| + | **10x buffer: 3, 2ul | ||
| + | **10x BSA: 2ul | ||
| + | **H2O: 11ul | ||
| + | **Total: 20ul | ||
| + | **37°C, 2h | ||
**Heat inactivation: 65°C 20min | **Heat inactivation: 65°C 20min | ||
---- | ---- | ||
Revision as of 05:10, 30 August 2007
- plasmid concentration after liquid culture extraction
| part | concentration (ug/ul) |
|---|---|
| pOmpC | 6 |
| EYFP | 30 |
| ECFP | 23 |
| CinR | 15 |
| HSL | 49 |
| pCinHSL | 7 |
| pCinHSLRBS | 12 |
| RBS | 14 |
| D-term | 27 |
first assembly
- Ligation
- Insert:vector=3:1
- Vector: 300ng/ul(2ul pOmpC after enzyme digestion)
- Insert: 100ng/ul(2ul EYFP after enzyme digestion)
- Ligase: 1ul
- Final volume: 10ul
- RT 3h
- Enzyme Digestion (pOmpC)
- pOmpC: 14ul
- SpeI: 1ul
- PstI: 1ul
- 10x buffer: 2, 2ul
- 10x BSA: 2ul
- Total: 20ul
- 37°C, 2h
- Heat inactivation: 65°C 20min
- Enzyme Digestion (EYFP)
- EYFP: 3ul
- XbaI: 1ul
- PstI: 1ul
- 10x buffer: 3, 2ul
- 10x BSA: 2ul
- H2O: 11ul
- Total: 20ul
- 37°C, 2h
- Heat inactivation: 65°C 20min
Others
- Culture tar-EnvZ, pLacIQ in 5 ml LB liquid medium (Kan:20ug/ml)
