NYMU Taipei/Lab Notes/2007 8 30
From 2007.igem.org
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
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