Team:HokkaidoU Japan/Notebook/Promoter WeakPromoter-I732020

From 2013.igem.org

Maestro E.coli

Notebook

Weak Promoter-I732020

  • Start

  • Transformation

    J23112, J23103, J23117, J23106, J23111 from distribution kit
    each of 1µL DNA to DH5α
  • Culture

    J23112, J23103, J23117, J23106, J23111
    I732020 from our stock
  • Mini-prep

    J23112, J23103, J23117, J23106, J23111, I732020
  • PCR

    I732020
    100up, 200dn, KOD Plus Neo
  • Digestion

    Cut (J23112, J23103, J23117, J23106, J23111) with SpeI, PstI (using 10×H buffer)
    Cut I732020 with XbaI, PstI (using 10×M buffer)
  • Gel extraction

    J23112, J23103, J23117, J23106, J23111
  • Antarctic phosphatase

    J23112, J23103, J23117, J23106, J23111
  • Ligation

    Ligate (J23112, J23103, J23117, J23106, J23111) with I732020
  • Transformation

    (J23112, J23103, J23117, J23106, J23111)-I732020
    each of 1µL DNA to DH5α
  • Colony PCR & Culture

    (J23112, J23103, J23117, J23106, J23111)-I732020
  • Mini-prep

    (J23112, J23103, J23117, J23106, J23111)-I732020
  • PCR

    (J23112, J23103, J23117, J23106, J23111)-I732020
    EX-f, PS-r, KOD FX NEO
  • PCR purification

    (J23112, J23103, J23117, J23106, J23111)-I732020
  • Digestion

    Cut K1084007 on pSB1C3 with EcoRI, PstI (using 10×H buffer)
    Cut (J23112, J23103, J23117, J23106, J23111)-I732020 with EcoRI, PstI (using 10×H buffer)
  • Antarctic phophatase

    pSB1C3
  • Ligation

    Ligate pSB1C3 with (J23112, J23103, J23117, J23106, J23111)-I732020
  • Transformation

    (J23112, J23103, J23117, J23106, J23111)-I732020
  • Complete!