Team:HokkaidoU Japan/Notebook/Promoter RandomPromtoer-B0034-LacZ-B0015

From 2013.igem.org

Maestro E.coli

Notebook

RandomPromtoer-B0034-LacZ-B0015

  • Start

  • Culture

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

    K1084007-B0034-lacZα-B0015 on pSB1C3
  • PCR & PCR purification

    random promoter
    random-oripro-f, 200b dn, KOD plus Neo
  • Digestion & Gel extraction

    Cut random promoter-B0034-lacZα-B0015 with XbaI, PstI (using 10×M buffer)
    Cut pSB1C3 with XbaI, PstI (using 10×M buffer)
  • EtOH precipitation

    random promoter-B0034-lacZα-B0015
    pSB1C3
  • Ligation

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

    random promoter-B0034-lacZα-B0015 on pSB1C3
    1µL DNA to DH5α
  • Culture

    Cultivation time for transformation product was elongated from 18 hrs to 24 hrs.
    About 10 blue colonies occurred after the cultivation.
  • Transformation

    random promoter-B0034-lacZα-B0015 on pSB1C3
    5µL DNA to DH5α
  • EtOH precipitation

    random promoter-B0034-lacZα-B0015 on pSB1C3(ligation product)
  • Transformation

    random promoter-B0034-lacZα-B0015 on pSB1C3
    1µL DNA to DH5α
  • Ligation

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

    Each samples were transformed and cultivated at 37°C or 30°C

    Ligation 30 min, cultivate at 37°C
    Ligation 30 min, cultivate at 30°C
    Ligation overnight, cultivate at 37°C
    Ligation overnight, cultivate at 30°C
  • Correction

    random promoter-B0034-lacZα-B0015 on pSB1C3
    In 5/20, 5 µL of DNA solution was used for transformation instead of 1 µL of DNA solution for transformation.
  • Culture

    random promoter-B0034-lacZα-B0015 on pSB1C3
  • Colony PCR

    random promoter-B0034-lacZα-B0015
    EX-F, PS-R
  • Culture

    Expression comparison
    random promoter-B0034-lacZα-B0015 on pSB1C3
  • Mini-prep

    random promoter-B0034-lacZα-B0015 on pSB1C3
  • PCR for sequencing

    random promoter-B0034-lacZα-B0015
    100b up, 200b dn, KOD Plus Neo
  • EtOH for sequencing

    random promoter-B0034-lacZα-B0015
  • Sequencing

    random promoter-B0034-lacZα-B0015
  • Complete!