Team:HokkaidoU Japan/Notebook/Pre experiment/mRFP-expression-construct

From 2014.igem.org

Notebook
Lab Documents

mRFP Expression Plasmid Construction

  • Start

  • Get genes

    pSB6A1

  • Transformation

    pSB6A1 1µL to JM109

  • Liquid Culture

    pSB6A1

  • Mini-prep

    pSB6A1

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 from our stock 100up_F Tm:70.4℃ & 200dn_R Tm:66.6℃
    KOD plus Neo

  • Digestion

    Cut R0040-B0034-E1010-B0015 with EcoRI, PstI (using 10×H buffer)
    Cut pSB6A1 with EcoRI, PstI (using 10×H buffer)

  • Gel Extraction

    R0040-B0034-E1010-B0015 pSB6A1

  • Ethanol precipetation

    R0040-B0034-E1010-B0015 pSB6A1

  • Dephosphorylation

    R0040-B0034-E1010-B0015

  • Ligation

    Ligate R0040-B0034-E1010-B0015 with pSB6A1

  • Transformation

    R0040-B0034-E1010-B0015(on pSB6A1) 5µL to DH5α Turbo

  • Liquid Culture

    R0040-B0034-E1010-B0015(on pSB6A1)

  • Mini-prep

    R0040-B0034-E1010-B0015(on pSB6A1)

  • Complete!

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