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

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<h2>oripro(K1084007)-B0034-lacZ-B0015</h2>
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  <ul class="steps">
    <li class="step start">
      <div class="step-tile"><h2>Start</h2></div>
    </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Synthesize oripro</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">EtOH precipitation </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"> K1084007 </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"> I732020 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Feb 26</time> </li> </ul> <div class="step-recipe"> pSB1C3, I732020 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">PCR & Gel extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Feb 26, Wed Feb 27</time> </li> </ul> <div class="step-recipe"> I732020<br> 100b up, 200b dn, KOD plus NEO </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion & Gel extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Feb 27</time> </li> </ul> <div class="step-recipe"> Cut pSB1C3 with EcoRI, SpeI (using 10xH buffer) </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Feb 27</time> </li> </ul> <div class="step-recipe"> pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Ligation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Feb 28</time> </li> </ul> <div class="step-recipe"> Ligate pSB1C3 with K1084007 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Feb 28</time> </li> </ul> <div class="step-recipe"> K1084007 on pSB1C3<br> 5&micro;L DNA to DH5&alpha; </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Colony PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 1</time> </li> </ul> <div class="step-recipe"> K1084007 on pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> K1084007 on pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">PCR for Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2, Mon Mar 4, Tue Mar 5</time> </li> </ul> <div class="step-recipe"> I1084007<br> 100b up, 200b dn </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH for Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2, Mon Mar 4, Tue Mar 12</time> </li> </ul> <div class="step-recipe"> I1084007 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2, Mon Mar 4, Tue Mar 12</time> </li> </ul> <div class="step-recipe"> K1084007 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion & Gel extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5, Fri Mar 8</time> </li> </ul> <div class="step-recipe"> Cut I732020 with SpeI, XbaI (using 10xM buffer)<br> Cut R0040 with SpeI, PstI (using 10xH buffer)<br> Cut I732020(PCR product) with XbaI, PstI (using 10xM buffer) </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5, Fri Mar 8</time> </li> </ul> <div class="step-recipe"> I732020, R0040, I732020(PCR product) </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Ligation(over night)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5, Fri Mar 8</time> </li> </ul> <div class="step-recipe"> Ligate K1084007 with I732020<br> Ligate R0040 with I732020(PCR product) </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 6, Sat Mar 9</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-LacZ&alpha;-B0015 on pSB1AK3<br> 5&micro;L DNA to DH5&alpha;<br><br> R0040-B0034-LacZ&alpha;-B0015 on pSB1A2<br> 5&micro;L DNA to DH5&alpha; </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Colony PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Mar 7, Sun Mar 10</time> </li> </ul> <div class="step-recipe"> </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 8, Mon Mar 11</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-LacZ&alpha;-B0015 on pSB1AK3<br> R0040-B0034-LacZ&alpha;-B0015 on pSB1A2 </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 Mar 11</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-LacZ&alpha;-B0015 on pSB1AK3<br> R0040-B0034-LacZ&alpha;-B0015 on pSB1A2<br> (Add X-Gal to confirm &beta;-galactosidase activity)<br><br> X-Gal 50 mg<br> dimethylformamid 1 mL <br> (50 mg/mL)<br> <br> Diluted the X-gal stock 1 to 1 with DW<br> Pipetted 50 uL of this dilution onto the plate<br> Evenly distributed the solution onto the plate using a spreader<br> Dried for 30 min at 37C<br> Streaked these colonies as "single colony isolation" on each LBA plate </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">PCR for Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 12</time> </li> </ul> <div class="step-recipe"> K1084007, K1084007-B0034-lacZ&alpha;-B0015<br> 100b up, 200b dn </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH for Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 12</time> </li> </ul> <div class="step-recipe"> K1084007, K1084007-B0034-lacZ&alpha;-B0015 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 12</time> </li> </ul> <div class="step-recipe"> K1084007, K1084007-B0034-lacZ&alpha;-B0015 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Culture for assay</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 12</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 13 - Thu Mar 14</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-lacZ&alpha;-B0015 on pSB1AK3<br> R0040-B0034-lacZ&alpha;-B0015 on pSB1A2 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion & Gel extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 16</time> </li> </ul> <div class="step-recipe"> Cut I732020 with EcoRI, XbaI (using 10xM buffer)<br> Cut K184007 with EcoRI, SpeI (using 10xH buffer) </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 16</time> </li> </ul> <div class="step-recipe"> I732020, K1084007 </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>Sun Mar 17</time> </li> </ul> <div class="step-recipe"> R0040-B0034-mcherry-B0015 on pSB1A2<br> K1084007 on pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Mar 18</time> </li> </ul> <div class="step-recipe"> R0040-B0034-mcherry-B0015 on pSB1A2<br> K1084007 on pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion & Gel extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Mar 18</time> </li> </ul> <div class="step-recipe"> Cut K1084007- B0034-lacZ&alpha;-B0015 with XbaI, PstI (using 10xM buffer)<br> Cut K1084007 with XbaI, PstI (using 10xM buffer)<br> Cut R0040 with XbaI, PstI (using 10xM buffer) </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Mar 18</time> </li> </ul> <div class="step-recipe"> K1084007-B0034-lacZ&alpha;-B0015<br> K1084007<br> R0040-B0034-mcherry-B0015 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Ligation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Mar 18</time> </li> </ul> <div class="step-recipe"> Ligate K1084007-B0034-lacZ&alpha;-B0015 with pSB1A2<br> Ligate K1084007-B0034-lacZ&alpha;-B0015 with pSB1C3 </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</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 pSB1A2<br> K1084007-B0034-lacZ&alpha;-B0015 on pSB1C3<br> 5&micro;L DNA to DH5&alpha; </div> </div> </div> </li>
<li class="step"> <div class="step-tile"> <h2 class="step-title">Culture for 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 complete">
      <div class="step-tile"><h2>Complete!</h2></div>
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