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

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  <ul class="steps">
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    <li class="step start">
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      <div class="step-tile"><h2>Start</h2></div>
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    </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Annealing two primers to synthesize oripro(K1084007)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sun Feb 24</time> </li> </ul> <div class="step-recipe"> Anneal ut-tg-oripro(K1084007)-f and ut-tg-oripro(K1084007)-r </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Annealing two primers to synthesize oripro(K1084007)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sun Feb 24</time> </li> </ul> <div class="step-recipe"> Anneal ut-tg-oripro(K1084007)-f and ut-tg-oripro(K1084007)-r </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Culture</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Feb 25</time> </li> </ul> <div class="step-recipe"> pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Culture</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Feb 25</time> </li> </ul> <div class="step-recipe"> pSB1C3 </div> </div> </div> </li>
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<li class="step"> <div class="step-tile"> <h2 class="step-title">Culture for &beta;-galactosidase assay</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 19</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-lacZ&alpha;-B0015 on pSB1AK3<br> R0040-B0034-lacZ&alpha;-B0015 on pSB1A2<br> NC:K1084007 on pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Culture for &beta;-galactosidase assay</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 19</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-lacZ&alpha;-B0015 on pSB1AK3<br> R0040-B0034-lacZ&alpha;-B0015 on pSB1A2<br> NC:K1084007 on pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">&beta;-galactosidase assay</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 20</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-lacZ&alpha;-B0015 on pSB1AK3<br> R0040-B0034-lacZ&alpha;-B0015 on pSB1A2<br> NC:K1084007 on pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">&beta;-galactosidase assay</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 20</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-lacZ&alpha;-B0015 on pSB1AK3<br> R0040-B0034-lacZ&alpha;-B0015 on pSB1A2<br> NC:K1084007 on pSB1C3 </div> </div> </div> </li>
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      <div class="step-tile"><h2>Complete!</h2></div>
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Revision as of 06:10, 27 September 2013

Maestro E.coli

Notebook

  • Start

  • 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
  • Complete!