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

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Revision as of 11:24, 14 October 2014

Wrong Plac failed Experiment

  • Start

  • Get anti-sense vector

    pHN1257 Great thanks to N. Nakashima

  • Transformation

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

  • Liquid Culture

    pSB6A1

  • Mini-prep

    pSB6A1

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 as RBS XhoI, as mRFP NcoI

  • Ehanol precipetation

    anti-sense(mRFP)

  • Digestion

    Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart) Cut anti-sense(mRFP) with NcoI, XhoI (using 10×Cut Smart)

  • Gel Extraction & Ethanol precipetation

    pHN1257 anti-sense(mRFP)

  • Ligation

    Ligate anti-sense(mFP) with pHN1257

  • Transformation

    anti-sense(mRFP) on pHN1257 5µL DNA to DH5α Turbo

  • Colony PCR

    anti-sense(mRFP) on pHN1257

  • Liquid Culture

    anti-sense(mRFP) on pHN1257

  • Mini-prep

    anti-sense(mRFP) on pHN1257

  • Transformation

    anti-sense(mRFP) on pHN1257 & pSB6A1 2.5µL each DNA to DH5α Turbo

  • Asssay(unsuccess)

    Culture anti-sense(mRFP) on pHN1257 & pSB6A1 1. 2mL LB with 100µL 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally.

  • Assay(unsuccess)

    Culture anti-sense(mRFP) on pHN1257 & 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"