Team:HokkaidoU Japan/Notebook/Promoter K1084001~010-B0034-lacZ-B0015

From 2013.igem.org

Maestro E.coli

Notebook

K1084001-010-B0034-lacZ-B0015

  • Start

  • PCR & PCR purification

    K1084007-B0034-mRFP1-B0015
    100b up, random-oripro-r, KOD plus Neo
  • Digestion

    Cut random promoter(PCR product) with EcoRI, SpeI(using 10xH buffer)
    Cut K1084007-B0034-mRFP1-B0015 on pSB1C3 with EcoRI, SpeI(using 10xH buffer)
  • Antarctic phosphatase

    pSB1C3
  • Ligation

    Ligate random promoter with pSB1C3
  • Transformation

    random promoter on pSB1C3
    1µL DNA to DH5α
  • Colony PCR

    random promoter on pSB1C3
  • Culture

    K1084007-B0034-mRFP1-B0015 on pSB1C3
  • Mini-prep

    K1084007-B0034-mRFP1-B0015 on pSB1C3
  • PCR

    K1084007:100b up, oripro-r, KOD plus Neo
    pSB1C3:Suf-I-F, XE-R, KOD plus Neo
  • EtOH precipitation

    K1084007, pSB1C3
  • Digestion

    Cut K1084007 with EcoRI, SpeI(using 10xH buffer)
    Cut pSB1C3 with EcoRI, SpeI(using 10xH buffer)
  • Antarctic phosphatase

    K1084007
  • Ligation

    Ligate K1084007 with pSB1C3
  • Transformation

    K1084007 on pSB1C3
    1µL DNA to JM109
  • Transformation

    random promoter on pSB1C3
    1µL DNA to JM109
  • Colony PCR

    random promoter on pSB1C3
  • Ligation

    Ligate random promoter with pSB1C3
  • Transformation

    random promoter on pSB1C3
  • Colony PCR

    random promoter on pSB1C3
  • Sequencing

    random promoter on pSB1C3
  • Digestion

    Cut I732020 with XbaI, PstI(using 10xM buffer)
    Cut R0040-B0034-mRFP1-B0015 on pSB1C3 with EcoRI, PstI(using 10xH buffer)
  • Antarctic phosphatase

    pSB1C3
  • Ligation

    Ligate random promoter with B0034-lacZα-B0015, pSB1C3
  • Transformation

    random promoter-B0034-lacZα-B0015 on pSB1C3
    5µL DNA to JM109
  • Colony PCR

    random promoter-B0034-lacZα-B0015 on pSB1C3
  • Culture

    random promoter-B0034-lacZα-B0015 on pSB1C3

    random promoter:K1084001~010
  • Sequencing

    K1084001~010-B0034-lacZα-B0015
    200b dn
  • β-galactosidase assay

    K1084001~010-B0034-lacZα-B0015 on pSB1C3
  • Complete!