From 2007.igem.org

• Enzyme Digestion (2h)
  • Restriction enzymes: 1ul each
  • Incubate for 2h
  • Heat inactivation at 65°C, 20min
• Gel Separation (0.5h)
  • 100V, 30min
• Gel Extraction (0.5h)
  • Qiagene GelExtraction Kit
• Ligation (3hr)
  • T4 ligase: 1ul
  • Incubate at RT for 3h
• Plate Culture and Transformation(16h)
  • DNA: 2ul
  • Competent cell: 50ul
• Liquid Culture (16h)
  • 5ml LB
• Plasmid Extraction (1hr)
  • Qiagene Miniprep Kit
• Enzyme digestion to ligation formula
  • phptest