Team:HokkaidoU Japan/Notebook/asB0034/asE1010 Target gene vector Exchange to 6A1

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Notebook
Lab Documents

E1010 Target gene vector exchange to pSB6A1

  • Start

  • PCR

    R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R Tm:72.6℃ (insert)
    KOD plus Neo

  • Digestion

    Cut vector with EcoRI, PstI (using 10×H buffer)
    Cut insert with EcoRI, PstI (using 10×M buffer)

  • Gel extraction

    digested vector & insert

  • Dephosphorylation

    Antarctic phosphorylate vector (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min

  • Ligation

    Ligate vector with insert

  • Transformation

    ligation products
    each of 5.0µL to DH5α

  • PCR

    R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R Tm:72.6℃ (insert) KOD plus Neo

  • Digestion

    Cut vector with EcoRI, PstI (using 10×H buffer)
    Cut insert with EcoRI, PstI (using 10×M buffer)

  • Gel extraction

    digested insert

  • Ethanol Precipitation

    digested insert

  • Digestion

    Cut vector with EcoRI, PstI (using 10×H buffer)

  • Dephosphorylation

    Antactic phosphorylate vector (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min

  • Ligation

    Ligate vector with insert

  • Transformation

    ligation products
    5.0µL DNA to DH5α

  • Colony PCR

    R0040-B0032-E1010-B0015(on pSB4C5) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    R0040-B0034-E1010-B0015(on pSB4C5) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    R0040-B0032-E1010-B0015(on pSB2K3) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    R0040-B0034-E1010-B0015(on pSB2K3) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    Kapa-Taq

  • Liquid Culture

    R0040-B0032-E1010-B0015(on pSB4C5)
    R0040-B0034-E1010-B0015(on pSB4C5)
    R0040-B0032-E1010-B0015(on pSB2K3)
    R0040-B0034-E1010-B0015(on pSB2K3)

  • Mini-Prep

    R0040-B0032-E1010-B0015(on pSB4C5)]
    R0040-B0034-E1010-B0015(on pSB4C5)
    R0040-B0032-E1010-B0015(on pSB2K3)
    R0040-B0034-E1010-B0015(on pSB2K3)

  • Complete!

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