NYMU Taipei/Lab Notes/2007 8 30
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== 2nd phase assembly== | == 2nd phase assembly== | ||
| - | * RBS + cinR | + | * '''Objective: RBS + cinR''' |
| - | * RBS + HSL | + | * '''Objective: RBS + HSL''' |
*Enzyme Digestion (RBS) | *Enzyme Digestion (RBS) | ||
**RBS: 11ul | **RBS: 11ul | ||
| - | **SpeI: 1ul | + | **SpeI: 1ul (10,000 U/ml) |
| - | **PstI: 1ul | + | **PstI: 1ul (20,000 U/ml) |
**10x buffer: 2, 2ul | **10x buffer: 2, 2ul | ||
**10x BSA: 2ul | **10x BSA: 2ul | ||
| Line 14: | Line 14: | ||
*Enzyme Digestion (cinR) | *Enzyme Digestion (cinR) | ||
**cinR: 11ul | **cinR: 11ul | ||
| - | **XbaI: 1ul | + | **XbaI: 1ul (20,000 U/ml) |
**PstI: 1ul | **PstI: 1ul | ||
**10x buffer: 3, 2ul | **10x buffer: 3, 2ul | ||
| Line 44: | Line 44: | ||
**HSL: 1ul | **HSL: 1ul | ||
**10x buffer: 1ul | **10x buffer: 1ul | ||
| - | **T4 ligase (Promega): 1ul | + | **T4 ligase (Promega): 1ul (3 U/ul) |
**H2O: 6ul | **H2O: 6ul | ||
| + | *Transformation | ||
| + | **Competent cell: 50ul | ||
| + | **DNA: 2ul | ||
---- | ---- | ||
| - | * | + | ==Who attend?== |
| + | * 立翔 | ||
Latest revision as of 07:27, 1 September 2007
2nd phase assembly
- Objective: RBS + cinR
- Objective: RBS + HSL
- Enzyme Digestion (RBS)
- RBS: 11ul
- SpeI: 1ul (10,000 U/ml)
- PstI: 1ul (20,000 U/ml)
- 10x buffer: 2, 2ul
- 10x BSA: 2ul
- H2O: 3ul
- Total: 20ul
- 37°C, 2h
- Heat inactivation: 65°C 20min
- Enzyme Digestion (cinR)
- cinR: 11ul
- XbaI: 1ul (20,000 U/ml)
- PstI: 1ul
- 10x buffer: 3, 2ul
- 10x BSA: 2ul
- H2O: 3ul
- Total: 20ul
- 37°C, 2h
- Heat inactivation: 65°C 20min
- Enzyme Digestion (HSL)
- HSL: 4ul
- XbaI: 1ul
- PstI: 1ul
- 10x buffer: 3, 2ul
- 10x BSA: 2ul
- H2O: 10ul
- Total: 20ul
- 37°C, 2h
- Heat inactivation: 65°C 20min
- Plasmid extraction: H_IGFBP3, H_IGFBP7, H_INS, M_IDE
- From 5ml LB medium to 50ul EB
- Ligation (RBS+cinR)
- RBS: 1ul
- cinR: 1ul
- 10x buffer: 1ul
- T4 ligase (Promega): 1ul
- H2O: 6ul
- Ligation (RBS+HSL)
- RBS: 1ul
- HSL: 1ul
- 10x buffer: 1ul
- T4 ligase (Promega): 1ul (3 U/ul)
- H2O: 6ul
- Transformation
- Competent cell: 50ul
- DNA: 2ul
Who attend?
- 立翔
