NYMU Taipei/Lab Notes/2007 8 30

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< NYMU Taipei/Lab Notes(Difference between revisions)
 
Line 44: Line 44:
**HSL: 1ul
**HSL: 1ul
**10x buffer: 1ul
**10x buffer: 1ul
-
**T4 ligase (Promega): 1ul (3 U/ml)
+
**T4 ligase (Promega): 1ul (3 U/ul)
**H2O: 6ul
**H2O: 6ul
*Transformation
*Transformation

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

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