Team:HokkaidoU Japan/Notebook/Pre experiment/Plac-failed-experiment

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Notebook
Lab Documents

Wrong Plac failed Experiment

  • Start

  • Get anti-sense vector

    pHN1257 Great thanks to N. Nakashima

  • Transformation

    R0010-B0034-E1010-B0015(on pSB6A1) from destribution kit 1µL to JM109

  • Liquid Culture

    R0010-B0034-E1010-B0015(on pSB6A1)

  • Mini-prep

    R0010-B0034-E1010-B0015(on pSB6A1)

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76/0℃
    KOD plus Neo

  • Ehanol precipetation

    asmRFP

  • Digestion

    Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart)
    Cut asmRFP with NcoI, XhoI (using 10×Cut Smart)

  • Gel Extraction

    pHN1257 & asmRFP

  • Ethanol precipetation

    pHN1257 & asmRFP

  • Ligation

    Ligate asmRFP with pHN1257

  • Transformation

    asmRFP(on pHN1257)
    5µL to DH5α Turbo

  • Colony PCR

    asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃
    Kapa-Taq

  • Liquid Culture

    asmRFP(on pHN1257)

  • Mini-prep

    asmRFP(on pHN1257)

  • DoubleTransformation

    asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
    2.5µL each to DH5α Turbo

  • Asssay(unsuccess)

    Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
    1. 2mL LB with 100µL 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally.

  • Assay(unsuccess)

    Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) E. coli was early growth 1. 2mL LB with 20µL 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination.

  • Failure...

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