Team:HokkaidoU Japan/Notebook/asB0034/asB0034,32-construction

From 2014.igem.org

Notebook
Lab Documents

Anti-Sense B0034 (B0032) Construction

  • Start

  • Primer Annealing

    asB0034_F Tm: 68.7℃ & asB0034_R Tm: 68.7℃
    asB0032_F Tm: 71.6℃ & asB0032_R Tm: 70.8℃

  • Digestion

    Cut vector with NcoI, XhoI (using 10×cut smart buffer)

  • Gel extraction

    digested pSB1A3

  • Ethanol Precipitation

    digested pSB1A3

  • Ligation

    Ligate pSB1A3 with R0010-stem-asB0034-stem-B0015
    Ligate pSB1A3 with R0010-stem-asB0032-stem-B0015

  • Transformation

    ligation products
    5.0µL to DH5α

  • Colony PCR

    R0010-stem-asB0034-stem-B0015(on pSB1A3) 100up_F Tm:62.8℃ & asB0034_R Tm:68.7℃
    R0010-stem-asB0032-stem-B0015(on pSB1A3) 100up_F Tm:74.5℃ & asB0032_R Tm:70.8℃
    Kapa-Taq

  • Liquid Culture

    R0010-stem-asB0034-stem-B0015(on pSB1A3)
    R0010-stem-asB0032-stem-B0015(on pSB1A3)

  • Mini-Prep

    R0010-stem-asB0034-stem-B0015(on pSB1A3)
    R0010-stem-asB0032-stem-B0015(on pSB1A3)

  • Complete!

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