From 2007.igem.org

Lab schedule

• mouse insulin gene cloning (8/6 to 8/7)
  • primer design for insulin II of Mus musculus, MN_008387.3 transcript
    • sequence for standard assembly (marked in green) and PCR reaction (marked in red)
    • primer for A chain
      • translation (90-110): GIVDQCCTSICSLYQLENYCN
      • transcript (341-403): GGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC, 63 bp
      • left: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGGGCATTGTAGATCAGTGCTG
      • right: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGTTGCAGTAGTTCTCCAGCT
      • Potential dimer formation! Do NOT use this pair of primers! We need a new design.
    • primer for B chain
      • translation (25-54): FVKQHLCGSHLVEALYLVCGERGFFYTPMS
      • transcript (146-235):TTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCC, 90 bp
      • left: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGTTTGTCAAGCAGCACCTTTGT
      • right: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGGACATGGGTGTGTAGAAGA
• purchase of BCRC human insulin clone (ordered not paid)
• cutting sites of iGEM 2007 parts
  • ompR binding ompC promoter
    • ompR binding site (C1, C2, C3) extraction
    • assembly to insulin
  • tar-EnvZ fusion construct: cut EnvZ part for our fusion construct
• antibiotics LB plate preparation
  • ampicillin
  • kanamycin