From 2007.igem.org

• Plasmid extraction: RBS+cinR 1, RBS+HSL 1, RBS+HSL 2, RBS+HSL 3 from 5ml LB culture to 50ul EB
• The concentration was measured with spectrophotometer at 260nm
  • RBS+cinR 1: 8ug/ul
  • RBS+HSL 1: 3ug/ul
  • RBS+HSL 2: 8ug/ul
  • RBS+HSL 3: 6ug/ul
• Enzyme digest RBS+cinR 1
  • RBS+cinR 1: 14ul
  • SpeI: 1ul
  • PstI 1ul
  • 10x buffer 2: 2ul
  • 10x BSA: 2ul
  • 37°C water bath, 2h
  • Heat inactivation: 65°C, 20min
• Enzyme digest RBS+HSL 2
  • RBS+HSL 2: 14ul
  • XbaI: 1ul
  • PstI: 1ul
  • 10x buffer 3: 2ul
  • 10x BSA: 2ul
  • 37°C water bath, 2h
  • Heat inactivation: 65°C, 20min
• Enzyme digest RBS+HSL 3
  • RBS+HSL 3: 14ul
  • XbaI: 1ul
  • PstI: 1ul
  • 10x buffer 3: 2ul
  • 10x BSA: 2ul
  • 37°C water bath, 2h
  • Heat inactivation: 65°C, 20min
• Check RBS+cinR 1 with BsaBI digestion
  • RBS+cinR 1: 3ul
  • BsaBI: 1ul
  • 10x buffer 2: 2ul
  • H2O: 14ul
  • 60°C dry bath, 1h

• Well 1: 100pb ladder
• Well 2 3: "RBS+HSL 2" XbaI, PstI
• Well 4 5: "RBS+HSL 3" XbaI, PstI
• Well 6 7: "RBS+cinR 1" SpeI, PstI
• Well 8: "RBS+cinR 1" BsaBI
• Comment
  • There seems to be some incomplete digestion of RBS+HSL 2, possibly result from too many DNA.
  • BsaBI digestion test of RBS+cinR failed. Redo ligation RBS+cinR.
  • Todo: BsaBI digestion of cinR
• Ligation (RBS+cinR+RBS+HSL)
  • RBS+cinR: 1ul
  • RBS+HSL 3: 3ul
  • 10x buffer A: 1ul
  • 10x ligase (Yeastern, 2U/ul): 1ul
  • H2O: 4ul
  • RT 20min
• Ligation (RBS+cinR ver. 2)
  • RBS: 1ul
  • cinR: 1ul
  • 10x buffer A: 1ul
  • 10x ligase (Yeastern, 2U/ul): 1ul
  • H2O: 6ul
  • RT 20min