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

From 2013.igem.org

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<li class="step"> <div class="step-tile"> <h2 class="step-title">PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Jun 11</time> </li> </ul> <div class="step-recipe"> B0034-mRFP1-B0015<br> 100up, 200dn, KOD Plus Neo </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Jun 11</time> </li> </ul> <div class="step-recipe"> B0034-mRFP1-B0015<br> 100up, 200dn, KOD Plus Neo </div> </div> </div> </li>
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<li class="step"> <div class="step-tile"> <h2 class="step-title">Get K1084007 from our stock</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Jun 11</time> </li> </ul> <div class="step-recipe"> </div> </div> </div> </li>
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<li class="step"> <div class="step-tile"> <h2 class="step-title">Get K1084007 from stock</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Jun 11</time> </li> </ul> <div class="step-recipe"> </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">PCR purification</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Jun 12</time> </li> </ul> <div class="step-recipe"> B0034-mRFP1-B0015 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">PCR purification</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Jun 12</time> </li> </ul> <div class="step-recipe"> B0034-mRFP1-B0015 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Jun 12</time> </li> </ul> <div class="step-recipe"> Cut K1084007 with SpeI, PstI (using 10&times;H buffer)<br> Cut B0034-mRFP1-B0015 with XbaI, PstI (using 10&times;M buffer) </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Jun 12</time> </li> </ul> <div class="step-recipe"> Cut K1084007 with SpeI, PstI (using 10&times;H buffer)<br> Cut B0034-mRFP1-B0015 with XbaI, PstI (using 10&times;M buffer) </div> </div> </div> </li>

Revision as of 06:56, 27 September 2013

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!

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