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
  • Transformation

    ligation products 5.0µL to DH5α
  • Transformation

    ligation products 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 B2K3)
  • 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!