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

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R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76/0&#8451;       
R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76/0&#8451;       
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        KOD plus Neo
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KOD plus Neo
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Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart)  
Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart)  
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        Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
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Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
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asmRFP(on pHN1257)  
asmRFP(on pHN1257)  
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        5&micro;L DNA to DH5&alpha; Turbo
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5&micro;L to DH5&alpha; Turbo
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asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;
asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;
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        Kapa-Taq
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Kapa-Taq
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asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)  
asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)  
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        2.5&micro;L each to DH5&alpha; Turbo
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2.5&micro;L each to DH5&alpha; Turbo
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Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)1. 2mL LB with 100&micro;L 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally.
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Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
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1. 2mL LB with 100&micro;L 100mM IPTG  
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2. 2mL LB However, IPTG was too much to grow normally.
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Latest revision as of 15:25, 9 September 2015

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...

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/Plac-failed-experiment"