Team:HokkaidoU Japan/Notebook/Promoter oripro(K1084007)-B0034-lacZ-B0015

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Maestro E.coli

Notebook

  • Annealing two primers to synthesize oripro(K1084007)

    Anneal ut-tg-oripro(K1084007)-f and ut-tg-oripro(K1084007)-r

  • Culture

    pSB1C3

  • EtOH precipitation

    K1084007

  • Culture

    I732020

  • Mini-prep

    pSB1C3, I732020

  • PCR & Gel extraction

    I732020
    100b up, 200b dn, KOD plus NEO

  • Digestion & Gel extraction

    Cut pSB1C3 with EcoRI, SpeI (using 10xH buffer)

  • EtOH precipitation

    pSB1C3

  • Ligation

    Ligate pSB1C3 with K1084007

  • Transformation

    K1084007 on pSB1C3
    5µL DNA to DH5α

  • Colony PCR

    K1084007 on pSB1C3

  • Mini-prep

    K1084007 on pSB1C3

  • PCR for Sequencing

    I1084007
    100b up, 200b dn

  • EtOH for Sequencing

    I1084007

  • Sequencing

    K1084007

  • Digestion & Gel extraction

    Cut I732020 with SpeI, XbaI (using 10xM buffer)
    Cut R0040 with SpeI, PstI (using 10xH buffer)
    Cut I732020(PCR product) with XbaI, PstI (using 10xM buffer)

  • EtOH precipitation

    I732020, R0040, I732020(PCR product)

  • Ligation(over night)

    Ligate K1084007 with I732020
    Ligate R0040 with I732020(PCR product)

  • Transformation

    K1084007-B0034-LacZα-B0015 on pSB1AK3
    5µL DNA to DH5%alpha;

    R0040-B0034-LacZα-B0015 on pSB1A2
    5µL DNA to DH5%alpha;

  • Colony PCR

    ‚È‚ñ‚©‘‚±‚€‚æ

  • Mini-prep

    K1084007-B0034-LacZα-B0015 on pSB1AK3
    R0040-B0034-LacZα-B0015 on pSB1A2

  • Culture

    K1084007-B0034-LacZα-B0015 on pSB1AK3
    R0040-B0034-LacZα-B0015 on pSB1A2
    (Add X-Gal to confirm β-galactosidase activity)

    X-Gal 50 mg
    dimethylformamid 1 mL
    (50 mg/mL)

    Diluted the X-gal stock 1 to 1 with DW
    Pipetted 50 uL of this dilution onto the plate
    Evenly distributed the solution onto the plate using a spreader
    Dried for 30 min at 37C
    Streaked these colonies as "single colony isolation" on each LBA plate

  • PCR for Sequencing

    K1084007, K1084007-B0034-lacZα-B0015
    100b up, 200b dn

  • EtOH for Sequencing

    K1084007, K1084007-B0034-lacZα-B0015

  • Sequencing

    K1084007, K1084007-B0034-lacZα-B0015

  • Culture for β-galactosidase assay

    K1084007-B0034-lacZα-B0015 on pSB1AK3
    R0040-B0034-lacZα-B0015 on pSB1A2
    NC:K1084007 on pSB1C3

  • β-galactosidase assay

    K1084007-B0034-lacZα-B0015 on pSB1AK3
    R0040-B0034-lacZα-B0015 on pSB1A2

  • Digestion & Gel extraction

    Cut I732020 with EcoRI, XbaI (using 10xM buffer)
    Cut K184007 with EcoRI, SpeI (using 10xH buffer)

  • EtOH precipitation

    I732020, K1084007

  • Culture

    R0040-B0034-mcherry-B0015 on pSB1A2
    K1084007 on pSB1C3

  • Mini-prep

    R0040-B0034-mcherry-B0015 on pSB1A2
    K1084007 on pSB1C3

  • Digestion & Gel extraction

    Cut K1084007- B0034-lacZα-B0015 with XbaI, PstI (using 10xM buffer)
    Cut K1084007 with XbaI, PstI (using 10xM buffer)
    Cut R0040 with XbaI, PstI (using 10xM buffer)

  • EtOH precipitation

    K1084007-B0034-lacZα-B0015
    K1084007
    R0040-B0034-mcherry-B0015

  • Ligation

    Ligate K1084007-B0034-lacZα-B0015 with pSB1A2
    Ligate K1084007-B0034-lacZα-B0015 with pSB1C3

  • Transformation

    K1084007-B0034-lacZα-B0015 on pSB1A2
    K1084007-B0034-lacZα-B0015 on pSB1C3
    5µL DNA to DH5α

  • Culture for β-galactosidase assay

    K1084007-B0034-lacZα-B0015 on pSB1AK3
    R0040-B0034-lacZα-B0015 on pSB1A2
    NC:K1084007 on pSB1C3

  • β-galactosidase assay

    K1084007-B0034-lacZα-B0015 on pSB1AK3
    R0040-B0034-lacZα-B0015 on pSB1A2
    NC:K1084007 on pSB1C3

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