Team:HokkaidoU Japan/Notebook/Promoter WeakPromoter-I732020

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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!

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