Difference between revisions of "Team:Tec-Monterrey/Notebook"

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            <li><a href="#page-top"><span class="label">Header<br><br></span></a></li>
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              Notebook
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          </div>
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          <div class="header-subtitle">
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              Everyday activities by the team
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          </div>
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          </div>
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      </div>
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   </header>
 
   </header>
  
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   <section class="notebook-wrapper">
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<div class="articulo">
 +
   <section id="logbook" class="notebook-wrapper">
 +
    <div class="body-title">Logbook</div>
 
     <ul>
 
     <ul>
 
       <li>
 
       <li>
−
         <div class="collapsible-header">May</div>
+
         <div class="collapsible-header">July</div>
 
         <div class="collapsible-body">
 
         <div class="collapsible-body">
 
             <ul class="collapsible-list">
 
             <ul class="collapsible-list">
 
               <li>
 
               <li>
−
                 <div class="collapsible-header">May 3</div>
+
                 <div class="collapsible-header">Week 3: 9-15 </div>
 
                 <div class="collapsible-body">
 
                 <div class="collapsible-body">
−
                   This is text for May 3
+
                    
 +
<h2><strong>Pre-Interlab: Transformation Practice</strong><span style="font-weight: 400;"> (p. 25, 07/13/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p>&nbsp;</p>
 +
<p><strong>Antibiotic stock solutions</strong><span style="font-weight: 400;"> (Carlos)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Ampicillin 100 mg/ml</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Chloramphenicol 35 mg/ml</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Kanamycin 35 mg/ml</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>LB Medium (without agar) </strong><span style="font-weight: 400;">(Jes&uacute;s)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">25 g/L, 500 ml of medium</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>LB Medium (with agar) </strong><span style="font-weight: 400;">(Jes&uacute;s)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">25 g/L, 500 ml of medium</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">15 g/L of agar, 500 ml of medium</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Medium LB and silica spheres are put inside autoclave</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">121&deg;C for 15 minutes</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Materials left inside fume hood, with 15 min. of UV light</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Bain-marie prepared, 42&deg;C</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Pre-Interlab: Transformation Practice</strong><span style="font-weight: 400;"> (p. 25, 07/13/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><strong>Transformations </strong><span style="font-weight: 400;">(Samantha)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">4 Petris, 23K + antibiotic, 8P + antibiotic, 23K w/o antibiotic, competent w/o antibiotic</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Positive control: 8P, dish 3, kit 2018: BBa_I20270 (GFP)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">1: 23K, dish 3, kit 2018: BBa_I763007 (RFP)</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>DNA from plates to Eppendorfs</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">1: 50 &mu;L CC, 1 &mu;L 23K</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2: 50 &mu;L CC, 1 &mu;L 23K</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">3: 50 &mu;L CC, 1 &mu;L 8P</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">4: 50 &mu;L CC</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Plates </strong><span style="font-weight: 400;">(Victor)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">20 ml LB with agar for all plates</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">20 &mu;L of Chloramphenicol when needed</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Heat Shock </strong><span style="font-weight: 400;">(Samantha)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">1-4: 1 minute, 42&deg;C, then 5 minutes on ice + 200 &mu;L LB to each tube</span></li>
 +
</ul>
 +
<p><span style="font-weight: 400;">Note</span><span style="font-weight: 400;">: iGEM protocol says 450 ml of SOC, but instead we used 200 &mu;L of LB</span></p>
 +
<p>&nbsp;</p>
 +
<p><strong>Incubations </strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">37&deg;C and 220 rpm, during 1 hour (6:03-7:03 pm)</span></li>
 +
</ul>
 +
 
 
                 </div>  
 
                 </div>  
 
               </li>
 
               </li>
 
               <li>
 
               <li>
−
                 <div class="collapsible-header">May 6</div>
+
                 <div class="collapsible-header">Week 4: 16-22</div>
 
                 <div class="collapsible-body">
 
                 <div class="collapsible-body">
−
                  This is text for May 6
+
 
 +
<h2><strong>Interlab: Phase 1 and Preparations</strong><span style="font-weight: 400;"> (p. 26-27, 07/16/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><strong>SOC medium preparation </strong><span style="font-weight: 400;">(NCb BioLabs, Thermo Fisher)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2% Tryptone</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">0.5% yeast extract</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM NaCl</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2.5 mM KCl</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM MgCl</span><span style="font-weight: 400;">2</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM MgSO</span><span style="font-weight: 400;">4</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">20 mM Glucose</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>SOB medium preparation</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2% Tryptone</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">0.5% yeast extract</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM NaCl</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2.5 mM KCl</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM MgCl</span><span style="font-weight: 400;">2</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM MgSO</span><span style="font-weight: 400;">4</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Buffer CCMB80 1L preparation</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM KOAc ph 7.0</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">80 mM CaCl</span><span style="font-weight: 400;">2</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">20 mM MnCl</span><span style="font-weight: 400;">2</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">10 mM MgCl</span><span style="font-weight: 400;">2</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Growth</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">In 200 ml of SOC</span></li>
 +
</ul>
 +
<p><span style="font-weight: 400;">Notes</span><span style="font-weight: 400;">: iGEM protocol says 250 ml SOB, instead 200 ml were used</span></p>
 +
<p><span style="font-weight: 400;">There&rsquo;s less overnight, so initial absorbance will be less than 0.1</span></p>
 +
<p>&nbsp;</p>
 +
<p><strong>Optical density</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">3:00 pm, 0.3</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">3:52 pm, 0.465</span></li>
 +
</ul>
 +
<p><span style="font-weight: 400;">Note</span><span style="font-weight: 400;">: For buffer resuspension, 20 ml were used instead of 80 ml</span></p>
 +
<p>&nbsp;</p>
 +
<p><strong>RFP transformation after 16 hours</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">12 hours: 1-2 colonies, low expression</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">16 hours: 3-4 colonies, medium expression</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Second competents batch</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Previous results were successful, so iGEM (buffer CCMB80) protocol will be repeated.</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Absorbance before the first resuspension: 0.482</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Resuspension in 20 ml of buffer</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Absorbance after resuspension: 0.444</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Interlab: Plate 7 Transformations</strong><span style="font-weight: 400;"> (p. 28-29, 07/17/18-07/18/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<table>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<p><strong>Devices</strong></p>
 +
</td>
 +
<td>
 +
<p><strong>Wells</strong></p>
 +
</td>
 +
<td>
 +
<p><strong>Eppendorf</strong></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">Ctrl(-).BBa_R0040</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2D, 7-4D, 7-21D, 6-20D, 2-6F</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">(-)</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">Ctrl(+).BBa_I20270</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2B, 7-4B, 7-21B, 6-20B, 3-8F</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">(+)</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">D1.BBa_J364000</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2F, 7-4F, 7-21F, 6-20F</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">1</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">D2.BBa_J364001</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2H, 7-4H. 7-21H, 6-20H</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">2</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">D3.BBa_J364002</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2J, 7-4J, 7-21J, 6-20J</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">3</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">D4.BBa_J364003</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2L, 7-4L</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">4</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">D5.BBa_J364004</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2N, 7-4N</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">5</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><span style="font-weight: 400;">D6.BBa_J364005</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">7-2P, 7-4P</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">6</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p>&nbsp;</p>
 +
<p><strong>DNA Resuspension </strong><span style="font-weight: 400;">(Samantha)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">With respect to the wells on plate 7</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Competent cells</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">50 &mu;L to each Eppendorf duplicate</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Resuspended DNA</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2 &mu;L were added to each Eppendorf duplicate</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Colonies</strong></p>
 +
<p><br /><br /></p>
 +
<table>
 +
<tbody>
 +
<tr>
 +
<td>&nbsp;</td>
 +
<td>
 +
<p><span style="font-weight: 400;">1.1</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">1.2</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">1.3</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">2.1</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">2.2</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">2.3</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">3.1</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">3.2</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">3.3</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">4.1</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">4.2</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">4.3</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><strong>D3</strong></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">388</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">417</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">381</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">536</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">491</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">523</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">498</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">452</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">575</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">548</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">559</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">300</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><strong>D4</strong></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">71</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">167</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">213</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">223</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">200</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">197</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">255</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">242</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">165</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">116</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">269</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">281</span></p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><strong>D5</strong></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">15</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">52</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">61</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">65</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">50</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">218</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">55</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">78</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">50</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">56</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">79</span></p>
 +
</td>
 +
<td>
 +
<p><span style="font-weight: 400;">97</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 
                 </div>  
 
                 </div>  
 
               </li>
 
               </li>
Line 114: Line 585:
 
       </li> <!-- parent list item ends -->
 
       </li> <!-- parent list item ends -->
 
       <li>
 
       <li>
−
         <div class="collapsible-header">May</div>
+
         <div class="collapsible-header">August</div>
 
         <div class="collapsible-body">
 
         <div class="collapsible-body">
 
             <ul class="collapsible-list">
 
             <ul class="collapsible-list">
 
               <li>
 
               <li>
−
                 <div class="collapsible-header">May 3</div>
+
                 <div class="collapsible-header">Week 2: 6-12</div>
 
                 <div class="collapsible-body">
 
                 <div class="collapsible-body">
−
                  This is text for May 3
+
 
 +
<h2><strong>Overnights</strong><span style="font-weight: 400;"> (p. 29, 08/07/18-08/08/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Arnulfo)</span></p>
 +
<p><span style="font-weight: 400;">BL21 Cas1Cas2 WT 1 colony</span></p>
 +
<p><span style="font-weight: 400;">BL21 Cas1Cas2 WT 4 colonies</span></p>
 +
<p><span style="font-weight: 400;">DH5⍺ Cas1Cas2 tags 4 colonies</span></p>
 +
<p><span style="font-weight: 400;">DH5⍺ Cas1Cas2 tags 1 colony</span></p>
 +
<p><span style="font-weight: 400;">DH5⍺ Cas1Cas2 WT colony</span></p>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">Note</span><span style="font-weight: 400;">: First two overnights showed no growth, DNA plasmid </span><span style="font-weight: 400;"><br /></span><span style="font-weight: 400;">extraction protocol was conducted on the other 3 overnights.</span></p>
 +
<p>&nbsp;</p>
 +
<p><strong>Minipreps </strong><span style="font-weight: 400;">(Carlos)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Marked solutions protocol on kits was followed</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Low amounts of DNA were obtained from extraction</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Minipreps Digestion </strong><span style="font-weight: 400;">(p. 31, 08/09/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Arnulfo)</span></p>
 +
<p><span style="font-weight: 400;">NEB Protocol: For plasmid DNA, Invitrogen protocol was followed.</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Step 4: Nomenclature mistake while tagging samples.</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Step 6: N9 was added and accidentally thrown out along with the filter</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Competent Cell Test Kit </strong><span style="font-weight: 400;">(p. 32, 08/10/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Carlos, Victor, Norma)</span></p>
 +
<p><span style="font-weight: 400;">iGEM Transformation Interlab Test 2018. 2 &mu;L of DNA on each tube to reach the same concentration proposed on the protocol.</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Competent BL21 Cells (10:20 pm), 20 minutes of overnights and buffer CCMB80</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">Note</span><span style="font-weight: 400;">: OD was not measured at the beginning</span></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Transformations </strong><span style="font-weight: 400;">(p. 32, 08/10/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Resuspend DNA from kit with 10 &mu;L of H</span><span style="font-weight: 400;">2</span><span style="font-weight: 400;">O (turns red)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Place competent cells on ice (10 min)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Pipette 50 &mu;L of competent cells in a 1.5 ml tube + 1 &mu;L of resuspended DNA</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Pipette 1 &mu;L of control DNA in 2 ml tube (resuspending on ice)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Close 1.5 ml tubes, mixing by inversion and incubating 30 min</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Heat shock, 42&deg;C, 45 s</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Incubate on ice, 5 min</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Pipette 950 &mu;L of SOC medium in each transformation</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Incubate at 37&deg;C for 2 hours at 200-300 rpm</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Pipette 100 &mu;L of each transformation on a Petri dish</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Centrifugate at 6800 g for 3 min. Throw out 800 &mu;L of leftover</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Resuspend cells on 100 &mu;L leftovers and pipette each transformation on Petris</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Incubate transformations overnight at 37&deg;C</span></li>
 +
</ul>
 +
<p><span style="font-weight: 400;"> &nbsp;</span></p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Overnights LB + Str</strong><span style="font-weight: 400;"> (p. 33, 08/10/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Carlos)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">20 ml sterile LB + Str 15 &mu;L (100 &mu;g/&mu;L stock)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Cultivate on a handle, starting 3:00 pm</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>IDTE buffer solution</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Tris and EDTA calculations</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>LB Medium (with agar) </strong><span style="font-weight: 400;">(Sof&iacute;a)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">LB: 25 g/L on 500 ml medium</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Agar: 15 g/L on 500 ml medium</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Minipreps </strong><span style="font-weight: 400;">(p. 33-34, 08/11/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Lizeth, Ana, Andr&eacute;s)</span></p>
 +
<p><span style="font-weight: 400;">Invitrogen Academic Lab Kit 2018</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Using Cas1Cas2 WT and Cas1Cas2 Tag</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">DNA concentration using Nanodrop</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Digestion with Xba1, using NEB protocol</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">Bands between 4000 and 5000 bps were obtained, which match with plasmids.</span></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
 
 
                 </div>  
 
                 </div>  
 
               </li>
 
               </li>
 
               <li>
 
               <li>
−
                 <div class="collapsible-header">May 6</div>
+
                 <div class="collapsible-header">Week 3: 13-19</div>
 
                 <div class="collapsible-body">
 
                 <div class="collapsible-body">
−
                  This is text for May 6
+
 
 +
<h2><strong>Plates with P1, P2 and Construct </strong><span style="font-weight: 400;">(p. 34, 08/13/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Ana, Mar&iacute;a, Alan, Valeria)</span></p>
 +
<p>&nbsp;</p>
 +
<p><strong>Miniprep of construct</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Using Invitrogen Academic Lab Kit 2018</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Nanodrop was left for tomorrow</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Transformations </strong><span style="font-weight: 400;">(p. 35, 08/14/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Samantha, Victor)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 - Cas1Cas2Flag, 2 &mu;L DNA</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 - Cas1Cas2WT, 2 &mu;L DNA</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">70 1 &mu;L CCBL21 (Alan)</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>SOC medium preparation</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Openwetware protocol of SOB medium + sterilized glucose for SOC</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Minipreps </strong><span style="font-weight: 400;">(p. 35-36, 08/16/18-08/17/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Sof&iacute;a, Arnulfo, Nora)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Cobalt (P1), Test 1 and Test 2 (P1-1 &amp; P1-2)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Lead(P2), Test 1 and Test 2 (P2-1 &amp; P2-2)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Construct, Test 1 and Test 2 (C1 &amp; C2)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Total: 6 minipreps </span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestion</strong><span style="font-weight: 400;"> (Ana, Jes&uacute;s)</span></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Electrophoresis </strong><span style="font-weight: 400;">(p. 37, 08/16/18-08/18/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Alan)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">EtBr was handled according to safety protocols since first trial had some mistakes</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Plasmid DNA Digestion</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Buffer 10x and BSA 100x</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Cas1 and Cas2 gel</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Miniprep of RT-his Flag WT</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">P1 in DH5⍺, Construct in DH5⍺, P1 in BL21</span></li>
 +
</ul>
 +
 
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <div class="collapsible-header">Week 4: 20-26</div>
 +
                <div class="collapsible-body">
 +
 
 +
 
 +
 
 +
<h2><strong>BL21 Flag and RT-his Transformation </strong><span style="font-weight: 400;">(p. 37, 08/20/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Bain-marie, 10 s</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Ice, 5 min, then incubation</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Plating of 100 &mu;L, 800 &mu;L removed from medium</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">Note</span><span style="font-weight: 400;">: Incubation was done 35 min. After icing without SOC</span></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Transformations </strong><span style="font-weight: 400;">(p. 38, 08/22/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Adri&aacute;n)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 - Flag(str) and RT-His (IDT)(AMP), WT</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21WT - RT-His (IDT)(AMP)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">NEB protocol will be used</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">Note</span><span style="font-weight: 400;">: Bain-marie is now set at 42&deg;C with &ldquo;control temperature&rdquo; button </span></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Minipreps Digestion </strong><span style="font-weight: 400;">(p. 38, 08/24/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Carlos)</span></p>
 +
<p><span style="font-weight: 400;">NEB Protocol for the following plates:</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 P1, DH5⍺ WT, DH5⍺ Flag, DH5⍺ P1, DH5⍺ Rt-His, DH5⍺ construct, BL21 WT</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>SOC medium preparation (400 ml)</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Transformations </strong><span style="font-weight: 400;">(p. 39, 08/25/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Roberto, Ana, Nora)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 - 50 &mu;L</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Flag (streptomycin) - 5 &mu;L</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">RT (ampicillin) - 5 &mu;L</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestions </strong><span style="font-weight: 400;">(Jes&uacute;s, Victor)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">NEB protocol, p. 38</span></li>
 +
</ul>
 +
 
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <div class="collapsible-header">Week 5: 27-31</div>
 +
                <div class="collapsible-body">
 +
 
 +
 
 +
<h2><strong>Overnights </strong><span style="font-weight: 400;">(p. 39, 08/27/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Jes&uacute;s, Victor)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">8:00 pm, set on LB: BL21 WT + RT-His 1, BL21 WT + RT-His 2, BL21 His + Flag</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">8:30 am, few growth on overnights</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">9:25 am, inoculation on SOC, 15 ml</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">11:10 am, no significant growth on overnights nor on SOC</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Streptomycin solution </strong><span style="font-weight: 400;">(p. 40, 08/29/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Esteban, Sof&iacute;a)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">50 mg/ml, online protocols</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Glycerol stocks</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">500 &mu;L BL21 and glycerol 50%</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Transformations</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">RT-His (IDT)</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>BL21 Minipreps </strong><span style="font-weight: 400;">(p. 40, 08/29/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Carlos, Nora)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">RT-His and BL21 - Cas1Cas2: Flag, both pellets were red</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Nanodrop</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Band = 4711</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Heatshok Transformation</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Different antibiotics will be used: 15 &mu;L and 20 &mu;L of strepto &amp; ampicillin</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">In order to analyze results and standardize antibiotic concentrations</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestion and gels for minipreps</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">NEB protocol, WT-RT</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Minipreps </strong><span style="font-weight: 400;">(p. 41, 08/30/18-08/31/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Ana, Arnulfo)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">DNA concentrations for BL21 RT-His, Bl212 Cas1Cas2 Flag, BL21 Cas1Cas2 WT and BL21 Cas1Cas2 WT RT-His</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestion</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">NEB protocol, WT-RT for 50 &mu;L</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Electrophoresis </strong><span style="font-weight: 400;">(Adri&aacute;n)</span></p>
 +
 
 +
                </div>
 +
              </li>
 +
            </ul> <!-- children ul list ends -->
 +
        </div> <!-- collapsible-body ends -->
 +
      </li> <!-- parent list item ends -->
 +
      <li>
 +
        <div class="collapsible-header">September</div>
 +
        <div class="collapsible-body">
 +
            <ul class="collapsible-list">
 +
              <li>
 +
                <div class="collapsible-header">Week 2: 3-9</div>
 +
                <div class="collapsible-body">
 +
 
 +
 
 +
<h2><strong>Ligation and Transformation </strong><span style="font-weight: 400;">(p. 42, 09/04/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Andr&eacute;s, Samantha, Jes&uacute;s, Sof&iacute;a)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;"> CAM, 2 plates</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">PO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;"> CAM, 2 plates</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Construct CAM, 2 plates</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BFP CAM, 1 plate</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Stop CAM, 1 plate</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">RBS AMP, 1 plate</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>EDTA 0.5M and Tris-HCl 1M solutions were prepared</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>LB and SOC preparations </strong><span style="font-weight: 400;">(p. 42, 09/05/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Andr&eacute;s, Esteban, Sof&iacute;a)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">LB + agar (300 ml)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">SOC medium (300 ml)</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Ligation and Transformation</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Competent cells and autoclaving </strong><span style="font-weight: 400;">(p. 42, 09/08/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Alan, Samantha, Sof&iacute;a)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Small, medium and large tips were autoclaved</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Ligations from 09/05/18 were plated and incubated</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestion Minipreps Data </strong><span style="font-weight: 400;">(Carlos)</span></p>
 +
 
 +
 
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <div class="collapsible-header">Week 3: 10-16</div>
 +
                <div class="collapsible-body">
 +
     
 +
 
 +
<h2><strong>Minipreps </strong><span style="font-weight: 400;">(p. 38, 09/10/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">DH5⍺ (NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">, PO</span><span style="font-weight: 400;">4</span><span style="font-weight: 400;">, Construct) and BL21 &nbsp;(NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">, PO</span><span style="font-weight: 400;">4</span><span style="font-weight: 400;">, Construct) &nbsp;with the kit</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">15 min on ice instead of 30 min</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">60 s on HS instead of 10 s</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2 min on ice instead of 5 min</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>SOC medium preparation (400 ml)</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Minipreps for Construct, NO</strong><strong>3</strong><strong>, PO</strong><strong>4</strong> <span style="font-weight: 400;">(p. 44, 09/13/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Construct: CD DH5⍺ 17.1, CB BL21 7.2</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">: ND1 14.6, ND2 16.5, NB1 7.3, NB2 12.9</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">PO</span><span style="font-weight: 400;">4</span><span style="font-weight: 400;">: PD1 11.8, PD2 14.7, PB1 9.7, PB2 14.8</span></li>
 +
</ul>
 +
 
 +
 
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <div class="collapsible-header">Week 4: 17-23</div>
 +
                <div class="collapsible-body">
 +
 
 +
 
 +
<h2><strong>TAE TX 50x (200 ml) </strong><span style="font-weight: 400;">(p. 44, 09/17/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Tris-base, acetic acid and EDTA</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">50x was not prepared, stock was taken to do TAE 1x</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">2 min on ice instead of 5 min</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>SOC medium preparation (400 ml)</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Digestion </strong><span style="font-weight: 400;">(p. 44, 09/18/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Carlos)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">For CD, CB, PD1, PB1, PB2, ND1, ND2, NB1, and NB2</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">For WT, RT-His and Flag</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Overnights </strong><span style="font-weight: 400;">(p. 45-46, 09/19/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Overnights with duplicates on plates: DH5⍺-PO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">, DH5⍺-NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">, BL21-PO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">, BL21-NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">, &nbsp;DH5⍺-Construct, DH5⍺-Construct</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>PCR Reaction</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Stock 100 &mu;M</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Minipreps and PCR Repetition </strong><span style="font-weight: 400;">(Nora, Victor)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">DNA dilution for each target and concentration</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>New Transformations Protocols </strong><span style="font-weight: 400;">(p. 46, 09/20/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Cells on ice, 10 min</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">50 &mu;L cells + 1-5 &mu;L DNA</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Resuspend, put on ice 15 min, heat shock 60 s, 2 min on ice</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">950 &mu;L SOC</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Incubate at 37&deg;C, 60 min, 250 rpm</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Preheat plates and add antibiotic</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Minipreps</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">RT minipreps showed a very dense yellowish fluid, it should be centrifuged more time</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestions</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">RT, Flag 69.6, Flag 56.2 &nbsp;&nbsp;</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Cas1Cas2 Plates </strong><span style="font-weight: 400;">(p. 47, 09/22/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">For Flag, WT (DH5⍺)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Summary of previous results</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>PCR Gel </strong><span style="font-weight: 400;">(Ana)</span></p>
 +
<p>&nbsp;</p>
 +
<p><strong>Minipreps</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">DH5⍺ Cas1Cas2 Flag</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">DH5⍺ Flag</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">DH5⍺ WT</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 RT-His</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 RT-His</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Overnight minipreps </strong><span style="font-weight: 400;">(p. 47-48, 09/23/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><strong>Minipreps (PO and NO</strong><strong>3</strong><strong>)</strong></p>
 +
<p><strong>Cas and RT </strong><span style="font-weight: 400;">(Arnulfo, Adri&aacute;n, Sof&iacute;a)</span></p>
 +
<p><strong>PCR Dilutions</strong></p>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestions and Backbone</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;"> 50 &mu;L</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">PO</span><span style="font-weight: 400;">4</span><span style="font-weight: 400;"> 25 &mu;L</span></li>
 +
</ul>
 +
 
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <div class="collapsible-header">Week 5: 24-30</div>
 +
                <div class="collapsible-body">
 +
 
 +
 
 +
<h2><strong>Digestion </strong><span style="font-weight: 400;">(p. 48, 09/24/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Nora, Ana)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">For RT-His</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Enzyme Master Mix (50 &mu;L)</strong></p>
 +
<p><strong>Digest Plasmid Backbone</strong></p>
 +
<p><strong>Ligation</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Electrophoresis gel </strong><span style="font-weight: 400;">(p. 49-50, 09/25/18-09/26/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">A high amount of interference was observed in Nanodrop, &lambda; &lt; 230 nm, for minipreps from 23/09 and from 25/09</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Gel extraction</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">1.2 mL for 400 mg on both procedures</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Gel digestion</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Electrophoresis gel </strong><span style="font-weight: 400;">(p. 50-51, 09/28/18-09/29/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">A high amount of interference was observed in Nanodrop, &lambda; &lt; 230 nm, for minipreps from 23/09 and from 25/09</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Gel extraction</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">1.2 mL for 400 mg on both procedures</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Gel digestion</strong></p>
 +
<p><strong>SOC medium preparation </strong><span style="font-weight: 400;">(Esteban)</span></p>
 +
<p><strong>TAE 50x buffer preparation 250 ml </strong><span style="font-weight: 400;">(Esteban)</span></p>
 +
<p><strong>Digestion and overnights</strong></p>
 +
 
 +
 
 +
                </div>
 +
              </li>
 +
            </ul> <!-- children ul list ends -->
 +
        </div> <!-- collapsible-body ends -->
 +
      </li> <!-- parent list item ends -->
 +
      <li>
 +
        <div class="collapsible-header">October</div>
 +
        <div class="collapsible-body">
 +
            <ul class="collapsible-list">
 +
              <li>
 +
                <div class="collapsible-header">Week 1: 1-7</div>
 +
                <div class="collapsible-body">
 +
 
 +
 
 +
<h2><strong>Minipreps </strong><span style="font-weight: 400;">(p. 51-52, 10/01/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><strong>Transformations</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">With construct and NO</span><span style="font-weight: 400;">3</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Digestion</strong></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Digestion </strong><span style="font-weight: 400;">(p. 53, 10/04/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">For RT-His, WT, and Flag</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Minipreps </strong><span style="font-weight: 400;">(p. 54, 10/06/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Esteban, Adri&aacute;n)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">With NO</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;"> (IPT)</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Stock 100 &mu;L</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Ligations IDT + 4B</strong></p>
 +
 
 +
 
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <div class="collapsible-header">Week 2: 8-14</div>
 +
                <div class="collapsible-body">
 +
 
 +
 
 +
<h2><strong>Inoculation Cas1Cas2 WT </strong><span style="font-weight: 400;">(p. 54, 10/10/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><span style="font-weight: 400;">(Samantha)</span></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">Agitation 37&deg;C, 11:10 am</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Transformations </strong><span style="font-weight: 400;">(Sof&iacute;a, Samantha)</span></p>
 +
<p><strong>Digestion and ligation</strong><span style="font-weight: 400;"> (Samantha, Andr&eacute;s)</span></p>
 +
<p>&nbsp;</p>
 +
<hr />
 +
<p>&nbsp;</p>
 +
<h2><strong>Western Blot </strong><span style="font-weight: 400;">(p. 55, 10/13/18)</span></h2>
 +
<p>&nbsp;</p>
 +
<p><strong>Overnights and IPTG inductions</strong></p>
 +
<ul>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 Cas1Cas2 Tags</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 Cas1Cas2 WT</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 RT-His</span></li>
 +
<li style="font-weight: 400;"><span style="font-weight: 400;">BL21 virgin</span></li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
 
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <div class="collapsible-header">Week 3: 15-17</div>
 +
                <div class="collapsible-body">
 +
<h1>WikiFreeze!</h1>
 
                 </div>  
 
                 </div>  
 
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     </ul> <!-- whole ul list ends -->
−
  </section>
 
−
 
 
−
  <section class="seccion-responsiva">
 
−
    <div class="body-title">Safety</div>
 
 
   </section>
 
   </section>
  
−
   <section class="seccion-responsiva">
+
   <section id="protocols" class="seccion-responsiva">
 
     <div class="body-title">Protocols</div>
 
     <div class="body-title">Protocols</div>
−
     <div class="pdf-container">
+
    <b>Note: </b> Even though we know that these protocols work, you should always compare them with other reliable sources before you conduct the experiment.
 +
     <div class="pdf-container"> <!-- Inicia un pdf -->
 
       <div class="pdf-heading">
 
       <div class="pdf-heading">
 +
        <div class="body-title">Reactant Preparation and Pipetting Techniques</div>
 
       </div>
 
       </div>
 
       <div class="pdf-collapse">
 
       <div class="pdf-collapse">
 
         <div class="pdf-body">
 
         <div class="pdf-body">
−
           <object data="https://static.igem.org/mediawiki/2018/2/2b/T--Tec-Chihuahua--Prot1.pdf" type="application/pdf" width="100%" height="60vh" align="middle" internalinstanceid="9"></object>
+
           <object data="https://static.igem.org/mediawiki/2018/2/2c/T--Tec-Monterrey--SAFETY_Reactants.pdf" type="application/pdf" width="100%" height="60vh" align="middle" internalinstanceid="9"></object>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
     </div>
+
     </div> <!-- termina un pdf --->
 +
    <div class="pdf-container"> <!-- Inicia un pdf -->
 +
      <div class="pdf-heading">
 +
        <div class="body-title">Competent Cells</div>
 +
      </div>
 +
      <div class="pdf-collapse">
 +
        <div class="pdf-body">
 +
          <object data="https://static.igem.org/mediawiki/2018/b/b6/T--Tec-Monterrey--SAFETY_Competent.pdf" type="application/pdf" width="100%" height="60vh" align="middle" internalinstanceid="9"></object>
 +
        </div>
 +
      </div>
 +
    </div> <!-- termina un pdf --->
 +
    <div class="pdf-container"> <!-- Inicia un pdf -->
 +
      <div class="pdf-heading">
 +
        <div class="body-title">Bacterial Transformation</div>
 +
      </div>
 +
      <div class="pdf-collapse">
 +
        <div class="pdf-body">
 +
          <object data="https://static.igem.org/mediawiki/2018/e/eb/T--Tec-Monterrey--SAFETY_Transform.pdf" type="application/pdf" width="100%" height="60vh" align="middle" internalinstanceid="9"></object>
 +
        </div>
 +
      </div>
 +
    <div class="pdf-container"> <!-- Inicia un pdf -->
 +
      <div class="pdf-heading">
 +
        <div class="body-title">Restriction analysis</div>
 +
      </div>
 +
      <div class="pdf-collapse">
 +
        <div class="pdf-body">
 +
          <object data="https://static.igem.org/mediawiki/2018/f/f1/T--Tec-Monterrey--SAFETY_Restriction_analysis.pdf" type="application/pdf" width="100%" height="60vh" align="middle" internalinstanceid="9"></object>
 +
        </div>
 +
      </div>
 +
    </div> <!-- termina un pdf --->
 +
    <div class="pdf-container"> <!-- Inicia un pdf -->
 +
      <div class="pdf-heading">
 +
        <div class="body-title">Miniprep</div>
 +
      </div>
 +
      <div class="pdf-collapse">
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        <div class="pdf-body">
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          <object data="https://static.igem.org/mediawiki/2018/6/6d/T--Tec-Monterrey--SAFETY_Miniprepp.pdf" type="application/pdf" width="100%" height="60vh" align="middle" internalinstanceid="9"></object>
 +
        </div>
 +
      </div>
 +
    </div> <!-- termina un pdf --->
 +
    <div class="pdf-container"> <!-- Inicia un pdf -->
 +
      <div class="pdf-heading">
 +
        <div class="body-title">DNA Gel Electrophoresis</div>
 +
      </div>
 +
      <div class="pdf-collapse">
 +
        <div class="pdf-body">
 +
          <object data="https://static.igem.org/mediawiki/2018/6/62/T--Tec-Monterrey--SAFETY_Electro.pdf" type="application/pdf" width="100%" height="60vh" align="middle" internalinstanceid="9"></object>
 +
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 +
      </div>
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    </div> <!-- termina un pdf --->
 +
    <div class="pdf-container"> <!-- Inicia un pdf -->
 +
      <div class="pdf-heading">
 +
        <div class="body-title">Gel Extraction and Ligation</div>
 +
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 +
      <div class="pdf-collapse">
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 +
    <div class="pdf-container"> <!-- Inicia un pdf -->
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      <div class="pdf-heading">
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        <div class="body-title">SDS Page Electrophoresis Protocol</div>
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 +
      <div class="pdf-collapse">
 +
        <div class="pdf-body">
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Latest revision as of 02:27, 18 October 2018

Logbook
  • July
    • Week 3: 9-15

      Pre-Interlab: Transformation Practice (p. 25, 07/13/18)

       

       

      Antibiotic stock solutions (Carlos)

      • Ampicillin 100 mg/ml
      • Chloramphenicol 35 mg/ml
      • Kanamycin 35 mg/ml

       

      LB Medium (without agar) (Jesús)

      • 25 g/L, 500 ml of medium

       

      LB Medium (with agar) (Jesús)

      • 25 g/L, 500 ml of medium
      • 15 g/L of agar, 500 ml of medium

       

      Medium LB and silica spheres are put inside autoclave

      • 121°C for 15 minutes
      • Materials left inside fume hood, with 15 min. of UV light
      • Bain-marie prepared, 42°C

       

       

       

      Pre-Interlab: Transformation Practice (p. 25, 07/13/18)

       

      Transformations (Samantha)

      • 4 Petris, 23K + antibiotic, 8P + antibiotic, 23K w/o antibiotic, competent w/o antibiotic
      • Positive control: 8P, dish 3, kit 2018: BBa_I20270 (GFP)
      • 1: 23K, dish 3, kit 2018: BBa_I763007 (RFP)

       

      DNA from plates to Eppendorfs

      • 1: 50 μL CC, 1 μL 23K
      • 2: 50 μL CC, 1 μL 23K
      • 3: 50 μL CC, 1 μL 8P
      • 4: 50 μL CC

       

      Plates (Victor)

      • 20 ml LB with agar for all plates
      • 20 μL of Chloramphenicol when needed

       

      Heat Shock (Samantha)

      • 1-4: 1 minute, 42°C, then 5 minutes on ice + 200 μL LB to each tube

      Note: iGEM protocol says 450 ml of SOC, but instead we used 200 μL of LB

       

      Incubations

      • 37°C and 220 rpm, during 1 hour (6:03-7:03 pm)
    • Week 4: 16-22

      Interlab: Phase 1 and Preparations (p. 26-27, 07/16/18)

       

      SOC medium preparation (NCb BioLabs, Thermo Fisher)

      • 2% Tryptone
      • 0.5% yeast extract
      • 10 mM NaCl
      • 2.5 mM KCl
      • 10 mM MgCl2
      • 10 mM MgSO4
      • 20 mM Glucose

       

      SOB medium preparation

      • 2% Tryptone
      • 0.5% yeast extract
      • 10 mM NaCl
      • 2.5 mM KCl
      • 10 mM MgCl2
      • 10 mM MgSO4

       

      Buffer CCMB80 1L preparation

      • 10 mM KOAc ph 7.0
      • 80 mM CaCl2
      • 20 mM MnCl2
      • 10 mM MgCl2

       

      Growth

      • In 200 ml of SOC

      Notes: iGEM protocol says 250 ml SOB, instead 200 ml were used

      There’s less overnight, so initial absorbance will be less than 0.1

       

      Optical density

      • 3:00 pm, 0.3
      • 3:52 pm, 0.465

      Note: For buffer resuspension, 20 ml were used instead of 80 ml

       

      RFP transformation after 16 hours

      • 12 hours: 1-2 colonies, low expression
      • 16 hours: 3-4 colonies, medium expression

       

      Second competents batch

      • Previous results were successful, so iGEM (buffer CCMB80) protocol will be repeated.
      • Absorbance before the first resuspension: 0.482
      • Resuspension in 20 ml of buffer
      • Absorbance after resuspension: 0.444

       

       

      Interlab: Plate 7 Transformations (p. 28-29, 07/17/18-07/18/18)

       

      Devices

      Wells

      Eppendorf

      Ctrl(-).BBa_R0040

      7-2D, 7-4D, 7-21D, 6-20D, 2-6F

      (-)

      Ctrl(+).BBa_I20270

      7-2B, 7-4B, 7-21B, 6-20B, 3-8F

      (+)

      D1.BBa_J364000

      7-2F, 7-4F, 7-21F, 6-20F

      1

      D2.BBa_J364001

      7-2H, 7-4H. 7-21H, 6-20H

      2

      D3.BBa_J364002

      7-2J, 7-4J, 7-21J, 6-20J

      3

      D4.BBa_J364003

      7-2L, 7-4L

      4

      D5.BBa_J364004

      7-2N, 7-4N

      5

      D6.BBa_J364005

      7-2P, 7-4P

      6

       

      DNA Resuspension (Samantha)

      • With respect to the wells on plate 7

       

      Competent cells

      • 50 μL to each Eppendorf duplicate

       

      Resuspended DNA

      • 2 μL were added to each Eppendorf duplicate

       

      Colonies



       

      1.1

      1.2

      1.3

      2.1

      2.2

      2.3

      3.1

      3.2

      3.3

      4.1

      4.2

      4.3

      D3

      388

      417

      381

      536

      491

      523

      498

      452

      575

      548

      559

      300

      D4

      71

      167

      213

      223

      200

      197

      255

      242

      165

      116

      269

      281

      D5

      15

      52

      61

      65

      50

      218

      55

      78

      50

      56

      79

      97
  • August
    • Week 2: 6-12

      Overnights (p. 29, 08/07/18-08/08/18)

       

      (Arnulfo)

      BL21 Cas1Cas2 WT 1 colony

      BL21 Cas1Cas2 WT 4 colonies

      DH5⍺ Cas1Cas2 tags 4 colonies

      DH5⍺ Cas1Cas2 tags 1 colony

      DH5⍺ Cas1Cas2 WT colony

       

      Note: First two overnights showed no growth, DNA plasmid
      extraction protocol was conducted on the other 3 overnights.

       

      Minipreps (Carlos)

      • Marked solutions protocol on kits was followed
      • Low amounts of DNA were obtained from extraction

       

       

      Minipreps Digestion (p. 31, 08/09/18)

       

      (Arnulfo)

      NEB Protocol: For plasmid DNA, Invitrogen protocol was followed.

      • Step 4: Nomenclature mistake while tagging samples.
      • Step 6: N9 was added and accidentally thrown out along with the filter

       

       

      Competent Cell Test Kit (p. 32, 08/10/18)

       

      (Carlos, Victor, Norma)

      iGEM Transformation Interlab Test 2018. 2 μL of DNA on each tube to reach the same concentration proposed on the protocol.

      • Competent BL21 Cells (10:20 pm), 20 minutes of overnights and buffer CCMB80

       

      Note: OD was not measured at the beginning

       

       

      Transformations (p. 32, 08/10/18)

       

      • Resuspend DNA from kit with 10 μL of H2O (turns red)
      • Place competent cells on ice (10 min)
      • Pipette 50 μL of competent cells in a 1.5 ml tube + 1 μL of resuspended DNA
      • Pipette 1 μL of control DNA in 2 ml tube (resuspending on ice)
      • Close 1.5 ml tubes, mixing by inversion and incubating 30 min
      • Heat shock, 42°C, 45 s
      • Incubate on ice, 5 min
      • Pipette 950 μL of SOC medium in each transformation
      • Incubate at 37°C for 2 hours at 200-300 rpm
      • Pipette 100 μL of each transformation on a Petri dish
      • Centrifugate at 6800 g for 3 min. Throw out 800 μL of leftover
      • Resuspend cells on 100 μL leftovers and pipette each transformation on Petris
      • Incubate transformations overnight at 37°C

       

       

      Overnights LB + Str (p. 33, 08/10/18)

       

      (Carlos)

      • 20 ml sterile LB + Str 15 μL (100 μg/μL stock)
      • Cultivate on a handle, starting 3:00 pm

       

      IDTE buffer solution

      • Tris and EDTA calculations

       

      LB Medium (with agar) (Sofía)

      • LB: 25 g/L on 500 ml medium
      • Agar: 15 g/L on 500 ml medium

       

       

      Minipreps (p. 33-34, 08/11/18)

       

      (Lizeth, Ana, Andrés)

      Invitrogen Academic Lab Kit 2018

      • Using Cas1Cas2 WT and Cas1Cas2 Tag
      • DNA concentration using Nanodrop
      • Digestion with Xba1, using NEB protocol

       

      Bands between 4000 and 5000 bps were obtained, which match with plasmids.

       

       

    • Week 3: 13-19

      Plates with P1, P2 and Construct (p. 34, 08/13/18)

       

      (Ana, María, Alan, Valeria)

       

      Miniprep of construct

      • Using Invitrogen Academic Lab Kit 2018
      • Nanodrop was left for tomorrow

       

       

      Transformations (p. 35, 08/14/18)

       

      (Samantha, Victor)

      • BL21 - Cas1Cas2Flag, 2 μL DNA
      • BL21 - Cas1Cas2WT, 2 μL DNA
      • 70 1 μL CCBL21 (Alan)

       

      SOC medium preparation

      • Openwetware protocol of SOB medium + sterilized glucose for SOC

       

       

      Minipreps (p. 35-36, 08/16/18-08/17/18)

       

      (Sofía, Arnulfo, Nora)

      • Cobalt (P1), Test 1 and Test 2 (P1-1 & P1-2)
      • Lead(P2), Test 1 and Test 2 (P2-1 & P2-2)
      • Construct, Test 1 and Test 2 (C1 & C2)
      • Total: 6 minipreps

       

      Digestion (Ana, Jesús)

       

       

      Electrophoresis (p. 37, 08/16/18-08/18/18)

       

      (Alan)

      • EtBr was handled according to safety protocols since first trial had some mistakes

       

      Plasmid DNA Digestion

      • Buffer 10x and BSA 100x
      • Cas1 and Cas2 gel

       

      Miniprep of RT-his Flag WT

      • P1 in DH5⍺, Construct in DH5⍺, P1 in BL21
    • Week 4: 20-26

      BL21 Flag and RT-his Transformation (p. 37, 08/20/18)

       

      • Bain-marie, 10 s
      • Ice, 5 min, then incubation
      • Plating of 100 μL, 800 μL removed from medium

       

      Note: Incubation was done 35 min. After icing without SOC

       

       

      Transformations (p. 38, 08/22/18)

       

      (Adrián)

      • BL21 - Flag(str) and RT-His (IDT)(AMP), WT
      • BL21WT - RT-His (IDT)(AMP)
      • NEB protocol will be used

       

      Note: Bain-marie is now set at 42°C with “control temperature” button

       

       

      Minipreps Digestion (p. 38, 08/24/18)

       

      (Carlos)

      NEB Protocol for the following plates:

      • BL21 P1, DH5⍺ WT, DH5⍺ Flag, DH5⍺ P1, DH5⍺ Rt-His, DH5⍺ construct, BL21 WT

       

      SOC medium preparation (400 ml)

       

       

      Transformations (p. 39, 08/25/18)

       

      (Roberto, Ana, Nora)

      • BL21 - 50 μL
      • Flag (streptomycin) - 5 μL
      • RT (ampicillin) - 5 μL

       

      Digestions (Jesús, Victor)

      • NEB protocol, p. 38
    • Week 5: 27-31

      Overnights (p. 39, 08/27/18)

       

      (Jesús, Victor)

      • 8:00 pm, set on LB: BL21 WT + RT-His 1, BL21 WT + RT-His 2, BL21 His + Flag
      • 8:30 am, few growth on overnights
      • 9:25 am, inoculation on SOC, 15 ml
      • 11:10 am, no significant growth on overnights nor on SOC

       

       

      Streptomycin solution (p. 40, 08/29/18)

       

      (Esteban, Sofía)

      • 50 mg/ml, online protocols

       

      Glycerol stocks

      • 500 μL BL21 and glycerol 50%

       

      Transformations

      • RT-His (IDT)

       

       

      BL21 Minipreps (p. 40, 08/29/18)

       

      (Carlos, Nora)

      • RT-His and BL21 - Cas1Cas2: Flag, both pellets were red

       

      Nanodrop

      • Band = 4711

       

      Heatshok Transformation

      • Different antibiotics will be used: 15 μL and 20 μL of strepto & ampicillin
      • In order to analyze results and standardize antibiotic concentrations

       

      Digestion and gels for minipreps

      • NEB protocol, WT-RT

       

       

      Minipreps (p. 41, 08/30/18-08/31/18)

       

      (Ana, Arnulfo)

      • DNA concentrations for BL21 RT-His, Bl212 Cas1Cas2 Flag, BL21 Cas1Cas2 WT and BL21 Cas1Cas2 WT RT-His

       

      Digestion

      • NEB protocol, WT-RT for 50 μL

       

      Electrophoresis (Adrián)

  • September
    • Week 2: 3-9

      Ligation and Transformation (p. 42, 09/04/18)

       

      (Andrés, Samantha, Jesús, Sofía)

      • NO3 CAM, 2 plates
      • PO3 CAM, 2 plates
      • Construct CAM, 2 plates
      • BFP CAM, 1 plate
      • Stop CAM, 1 plate
      • RBS AMP, 1 plate

       

      EDTA 0.5M and Tris-HCl 1M solutions were prepared

       

       

      LB and SOC preparations (p. 42, 09/05/18)

       

      (Andrés, Esteban, Sofía)

      • LB + agar (300 ml)
      • SOC medium (300 ml)

       

      Ligation and Transformation

       

       

      Competent cells and autoclaving (p. 42, 09/08/18)

       

      (Alan, Samantha, Sofía)

      • Small, medium and large tips were autoclaved
      • Ligations from 09/05/18 were plated and incubated

       

      Digestion Minipreps Data (Carlos)

    • Week 3: 10-16

      Minipreps (p. 38, 09/10/18)

       

      DH5⍺ (NO3, PO4, Construct) and BL21  (NO3, PO4, Construct)  with the kit

      • 15 min on ice instead of 30 min
      • 60 s on HS instead of 10 s
      • 2 min on ice instead of 5 min

       

      SOC medium preparation (400 ml)

       

       

      Minipreps for Construct, NO3, PO4 (p. 44, 09/13/18)

       

      • Construct: CD DH5⍺ 17.1, CB BL21 7.2
      • NO3: ND1 14.6, ND2 16.5, NB1 7.3, NB2 12.9
      • PO4: PD1 11.8, PD2 14.7, PB1 9.7, PB2 14.8
    • Week 4: 17-23

      TAE TX 50x (200 ml) (p. 44, 09/17/18)

       

      • Tris-base, acetic acid and EDTA
      • 50x was not prepared, stock was taken to do TAE 1x
      • 2 min on ice instead of 5 min

       

      SOC medium preparation (400 ml)

       

       

      Digestion (p. 44, 09/18/18)

       

      (Carlos)

      • For CD, CB, PD1, PB1, PB2, ND1, ND2, NB1, and NB2
      • For WT, RT-His and Flag

       

       

      Overnights (p. 45-46, 09/19/18)

       

      • Overnights with duplicates on plates: DH5⍺-PO3, DH5⍺-NO3, BL21-PO3, BL21-NO3,  DH5⍺-Construct, DH5⍺-Construct

       

      PCR Reaction

      • Stock 100 μM

       

      Minipreps and PCR Repetition (Nora, Victor)

      • DNA dilution for each target and concentration

       

       

      New Transformations Protocols (p. 46, 09/20/18)

       

      • Cells on ice, 10 min
      • 50 μL cells + 1-5 μL DNA
      • Resuspend, put on ice 15 min, heat shock 60 s, 2 min on ice
      • 950 μL SOC
      • Incubate at 37°C, 60 min, 250 rpm
      • Preheat plates and add antibiotic

       

      Minipreps

      • RT minipreps showed a very dense yellowish fluid, it should be centrifuged more time

       

      Digestions

      • RT, Flag 69.6, Flag 56.2   

       

       

      Cas1Cas2 Plates (p. 47, 09/22/18)

       

      • For Flag, WT (DH5⍺)
      • Summary of previous results

       

      PCR Gel (Ana)

       

      Minipreps

      • DH5⍺ Cas1Cas2 Flag
      • DH5⍺ Flag
      • DH5⍺ WT
      • BL21 RT-His
      • BL21 RT-His

       

       

      Overnight minipreps (p. 47-48, 09/23/18)

       

      Minipreps (PO and NO3)

      Cas and RT (Arnulfo, Adrián, Sofía)

      PCR Dilutions

       

      Digestions and Backbone

      • NO3 50 μL
      • PO4 25 μL
    • Week 5: 24-30

      Digestion (p. 48, 09/24/18)

       

      (Nora, Ana)

      • For RT-His

       

      Enzyme Master Mix (50 μL)

      Digest Plasmid Backbone

      Ligation

       

       

      Electrophoresis gel (p. 49-50, 09/25/18-09/26/18)

       

      • A high amount of interference was observed in Nanodrop, λ < 230 nm, for minipreps from 23/09 and from 25/09

       

      Gel extraction

      • 1.2 mL for 400 mg on both procedures

       

      Gel digestion

       

       

      Electrophoresis gel (p. 50-51, 09/28/18-09/29/18)

       

      • A high amount of interference was observed in Nanodrop, λ < 230 nm, for minipreps from 23/09 and from 25/09

       

      Gel extraction

      • 1.2 mL for 400 mg on both procedures

       

      Gel digestion

      SOC medium preparation (Esteban)

      TAE 50x buffer preparation 250 ml (Esteban)

      Digestion and overnights

  • October
    • Week 1: 1-7

      Minipreps (p. 51-52, 10/01/18)

       

      Transformations

      • With construct and NO3

       

      Digestion

       

       

      Digestion (p. 53, 10/04/18)

       

      • For RT-His, WT, and Flag

       

       

      Minipreps (p. 54, 10/06/18)

       

      (Esteban, Adrián)

      • With NO3 (IPT)
      • Stock 100 μL

       

      Ligations IDT + 4B

    • Week 2: 8-14

      Inoculation Cas1Cas2 WT (p. 54, 10/10/18)

       

      (Samantha)

      • Agitation 37°C, 11:10 am

       

      Transformations (Sofía, Samantha)

      Digestion and ligation (Samantha, Andrés)

       

       

      Western Blot (p. 55, 10/13/18)

       

      Overnights and IPTG inductions

      • BL21 Cas1Cas2 Tags
      • BL21 Cas1Cas2 WT
      • BL21 RT-His
      • BL21 virgin

       

    • Week 3: 15-17

      WikiFreeze!

Protocols
Note: Even though we know that these protocols work, you should always compare them with other reliable sources before you conduct the experiment.
Reactant Preparation and Pipetting Techniques
Competent Cells
Bacterial Transformation
Restriction analysis
Miniprep
DNA Gel Electrophoresis
Gel Extraction and Ligation
SDS Page Electrophoresis Protocol

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