Team:HokkaidoU Japan/Notebook/RBS SD2,4,6


Maestro E.coli


SD2, 4, 6

  • Start

  • PCR

    LacZα-B0015 100up 200dn
  • PCR

    R0040-SD8-LacZα-B0015 / SD8 100up SD(2/4/6) mutation revers
  • PCR purification

    R0040-SD(6/4/2) / SD(6/4/2)
  • Digestion

    Cut LacZα-B-0015 EcoRI and XbaI (using 10×M buffer) , PCR products EcoRI and SpeI (using 10×H buffer)
  • Ethanol precipitation

    R0040-SD(6/4/2) / SD(6/4/2)
  • Gel extraction

  • Ethanol precipitation

  • Ligation

    Ligate R0040-SD(6/4/2) / SD(6/4/2) with LacZα-B0015
    Used X-cut-vector for inserts with R0040, and S-cut-vector for insrts without R0040
  • Transformation

    R0040-SD(6/4/2)LacZα-B0015 / SD(6/4/2)
    each of 4µL DNA to DH5α
  • Sequence

    SD(6/4/2) on pSB1C3
  • Complete!