Team:HokkaidoU Japan/Notebook/Promoter RandomPromtoer-B0034-mRFP1-B0015

From 2013.igem.org

Maestro E.coli

Notebook

RandomPromtoer-B0034-mRFP1-B0015

  • Start

  • PCR

    B0034-mRFP1-B0015
    100up, 200dn, KOD Plus Neo
  • Get K1084007 from stock

  • PCR purification

    B0034-mRFP1-B0015
  • Digestion

    Cut K1084007 with SpeI, PstI (using 10×H buffer)
    Cut B0034-mRFP1-B0015 with XbaI, PstI (using 10×M buffer)
  • Ligation

    Ligate K1084007 with B0034-mRFP1-B0015
  • Transformation

    K1084007-B0034-mRFP1-B0015
    1µL DNA to DH5α
  • Colony PCR

    K1084007-B0034-mRFP1-B0015
  • PCR

    K1084007(colony)
    ranori-f, 200dn, KOD FX Neo
    We got random promter.

    B0034-LacZα-B0015(colony)
    100up, 200dn, KOD FX Neo
  • Digestion

    Cut random promoter with PstI, SpeI (using 10×H buffer)
    Cut B0034-LacZα-B0015 with XbaI, PstI (using 10×M buffer)
  • Ligation

    Ligate random promoter with B0034-LacZα-B0015
  • Transformation

    1µL DNA to JM109
    to X-Gal plate
  • Culture

    random promoter-B0034-LacZα-B0015
    to X-Gal 50mg/mL, X-Gal 5mg/mL and LBC
    We got some blue colonies with different levels of blue.
  • Culture

    random promoter-B0034-LacZα-B0015 ×10 samples
    random promoter-B0034-mRFP1-B0015 ×9 samples
    K1084007-B0034-mRFP1-B0015
  • Mini-prep

    random promoter-B0034-LacZα-B0015 ×10 samples
    random promoter-B0034-mRFP1-B0015 ×9 samples
    K1084007-B0034-mRFP1-B0015
  • Culture

    random promoter-B0034-mRFP1-B0015
    K1084007-B0034-mRFP1-B0015
    random promoter-B0034-mRFP1-B0015 ×20 samples
  • PCR

    random promoter-B0034-mRFP1-B0015 ×2 samples
    random promoter-B0034-LacZα-B0015 ×2 samples
    100up, 200dn, KOD FX Neo
  • PCR

    J23112, J23103, J23117, J23106, J23111, J23100
    100up, 200dn, KOD FX Neo
  • Digestion

    Cut (J23112, J23103, J23117, J23106, J23111, J23100) with EcoRI, PstI (using 10×H buffer)
    Cut B0034-mRFP1-B0015 with EcoRI, PstI (using 10×H buffer)
  • Antarctic phophatase

    B0034-mRFP1-B0015
  • Ligation

    Ligate (J23112, J23103, J23117, J23106, J23111, J23100) with B0034-mRFP1-B0015
  • Transformation

    (J23112, J23103, J23117, J23106, J23111, J23100)-B0034-mRFP1-B0015
    1µL DNA to JM109
    pre culture
  • Culture

    (J23112, J23103, J23117, J23106, J23111, J23100) with B0034-mRFP1-B0015
    We picked up some colonies to a X-Gal LBC plate. We confirmed that colonies were white.
  • Transformation

    R0040-B0034-mRFP1-B0015
  • Culture

    (J23112, J23103, J23117, J23106, J23111, J23100, R0040, R0010)-B0034-mRFP1-B0015
  • Transformation

    random promoter-B0034-mRFP1-B0015
    1µL DNA to JM109 to X-Gal plate
  • PCR & PCR purification

    J23112, J23103, J23117(colony)
    100up, 200dn, KOD FX Neo
  • Digestion

    Cut J23112, J23103, J23117 with EcoRI, PstI (using 10×H buffer)
    Cut random promoter-B0034-LacZα-B0015 on pSB1C3 with EcoRI, PstI (using 10×M buffer)
  • Antarctic phosphatase

    pSB1C3
  • Ligation

    Ligate (J23112, J23103, J23117) with pSB1C3
  • Transformation

    1µL DNA to JM109 to 10 X-Gal plates
  • Culture

    We cultured some colonies from Jul 19 and 23 plates to X-Gal plate.
  • Assay

    Assay for RFP expression
  • Culture

    random promoter-B0034-mRFP1-B0015
  • Mini-prep

    random promoter-B0034-mRFP1-B0015(cultured Aug 8)
  • Complete!