From 2007.igem.org

• PCR (H_INS_A)
  • Template (H_INS): 1 ul
  • 10x PCR buffer: 5 ul
  • 2.5mM dNTP: 2 ul
  • H_INSA-F (10pM/ul): 1.5 ul
  • H_INSA-R (10pM/ul): 1.5 ul
  • Pfu(Promega): 0.5 ul
  • H2O: 38.5 ul
  • Total: 50ul
• PCR (H_INS_B)
  • Template (H_INS): 1 ul
  • 10x PCR buffer: 5 ul
  • 2.5mM dNTP: 2 ul
  • H_INSB-F (10pM/ul): 1.5 ul
  • H_INSB-R (10pM/ul): 1.5 ul
  • Pfu: 0.5 ul
  • H2O: 38.5 ul
  • Total: 50ul
• program:PETT2
  • 95°C:5-10min
  • 95°C:1min(*)
  • 55°C:1min(*)
  • 72°C:1min(*)
  • (*) 35 cycles
  • 72°C:10min
  • 4°C:∞
• H_INS:28-30ul/each well
• marker:5ul
• H_INS_A

• • from left to right
  • well 1:empty
  • well 2,3:INS_A 5
  • well 4,5:INS_A 4
  • well 6,7:INS_A 3
  • well 8,9:INS_A 2
  • well 10,11:INS_A 1
  • well 12:marker
• H_INS_B

• • from left to right
  • well 1:empty
  • well 2,3:INS_B 5
  • well 4,5:INS_B 4
  • well 6,7:INS_B 3
  • well 8,9:INS_B 2
  • well 10,11:INS_B 1
  • well 12:marker
• QlAquick Gel Extraction Kit
  • A1:0.08g
  • A3:0.15g
  • B1:0.12g
  • B2:0.12g
  • B3:0.16g
  • B4:0.11g
  • B5:0.17g
• RE Digestion
  • H_INS_A/B:11ul
  • XbaI:1ul
  • PstI:1ul
  • 10X buffer:2ul
  • 10X BSA:2ul
  • ddH2O:3ul
  • total:20ul
• QlAquick Gel Extraction Kit
• measuring the con.of H_INS_A/B with the spectrophotometer
  • INS_A1:13ug/ul
  • INS_B1:4ug/ul
  • INS_B2:0ug/ul
  • INS_B3:0ug/ul
  • INS_B4:0ug/ul
  • INS_B5:0ug/ul
• Ligation(H_INS_A)
  • H_INS_A1:4ul
  • 10X ligase:1 ul
  • 10X buffer:1 ul
  • ddH2O:4ul
  • total: 10ul
• Ligation(H_INS_B)
  • H_INS_B1:8ul
  • 10X ligase:1 ul
  • 10X buffer:1 ul
  • ddH2O:0ul
  • total: 10ul
• ligation: room temp.:15 mins
• transformation
  • ligated DNA:2ul
  • compenent cell:50ul
• apply the cells on the agar plate(Amp)
• culture plate failed due to lack of vector adding in the ligation process