Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W7"

 
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<html>
 
<html>
 
<head>
 
<head>
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<title> Week 7: 8/8 - 8/14</title>
+
<style>
 +
p {text-align: center;}
 +
</style>
 
</head>
 
</head>
 
<body>
 
<body>
  
−
<div class="center">
+
<div align="center">
−
<h3> I left you some of the commonly used templates down there under 8/8. Just fill them in as needed. I'll only really have wifi for a few hours on two days between Saturday and Thursday. If you need anything, message me on Facebook and I'll get back to you as soon as I can. </h3>
+
<br>
−
<h4> With the warmest love and regards, </h4>
+
<hr>
−
<h4>~<font color="red">J</font><font color="orange">O</font><font color="yellow">S</font><font color="green">H</font><font color="blue">U</font><font color="indigo">A</font></h4>
+
<br>
 +
</div>
 +
 
  
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h1> Week 7: 8/8 - 8/14</h1>
+
<h2> 8/8 </h2>
 +
</div>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Sequencing </h4>
 
<ul>
 
<ul>
−
<hr>
+
<table>
 +
<tr>
 +
<th> T1 </th>
 +
<th> T2 </th>
 +
</tr>
 +
<tr>
 +
<td> 5.34 uL H2O </td>
 +
<td> 5.34 uL H2O</td>
 +
</tr>
 +
<tr>
 +
<td> 1 uL CR-1 Primer 1 </td>
 +
<td> 1 uL CR-1 Primer 2 </td>
 +
</tr>
 +
<td> 1.66 uL DNA (301.6 ng/uL) </td>
 +
<td>  1.66 uL DNA (301.6 ng/uL) </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<h5> PCR Table Template: </h5>
 
  
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> Diluted BioBrick MiniPreps for PCR </h4>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> Contents </th>
+
<th> BioBrick Name </th>
−
<th> ul </th>
+
<th> Concentration (ng/uL) </th>
 +
<th> Contents (DNA + water) </th>
 
</tr>
 
</tr>
−
<td> Phire </td>
+
<tr>
−
<td> # </td>
+
<td> K592009 </td>
 +
<td> 76.9 </td>
 +
<td> 1 uL + 6.69 uL water </td>
 +
</tr>
 +
<tr>
 +
<td> B0015 </td>
 +
<td> 51.7 </td>
 +
<td> 1 uL + 4.17 uL water </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> H<sub>2</sub>O</td>
+
<td> K1033910 </td>
−
<td> # </td>
+
<td> 156.5 </td>
 +
<td> 1 uL + 14.65 uL water </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 50 uM F Primer </td>
+
<td> R0082 </td>
 +
<td>  49.5 </td>
 +
<td> 1 uL + 3.95 uL water </td>
 +
</tr>
 +
<tr>
 +
<td> C0012 </td>
 +
<td> 150.8 </td>
 +
<td> 1 uL + 14.08 uL water </td>
 +
</tr>
 +
<tr>
 +
<td> J61100 </td>
 +
<td> 123.0 </td>
 +
<td> 1 uL + 11.3 uL water </td>
 +
</tr>
 +
<tr>
 +
<td> R0011 </td>
 +
<td> 56.7 </td>
 +
<td> 1 uL + 4.67 uL water </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> PCR of each BioBrick parts  (25 uL Q5 MasterMix rxns) </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th>  Contents </th>
 +
<th> uL </th>
 +
</tr>
 +
<tr>
 +
<td> 10 uM VF2 primer </td>
 +
<td> 1.25 uL </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM VR primer </td>
 +
<td> 1.25 uL  </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td>  0.5 uL </td>
 +
</tr>
 +
<tr>
 +
<td> Q5 MM </td>
 +
<td>  12.5 uL </td>
 +
</tr>
 +
<tr>
 +
<td> Water </td>
 +
<td>  19.5 uL </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
 
 +
<div align="center">
 +
<br>
 +
<hr>
 +
<br>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<div align="center">
 +
<h2> 8/9 </h2>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> Gel run: </h5>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li>Ladder</li>
 +
<li>R0011</li>
 +
<li>B001</li>
 +
<li>K1033</li>
 +
<li>R008</li>
 +
<li>J61100</li>
 +
<li>C0012</li>
 +
<li>K592</li>
 +
</ol>
 +
 
 +
<ul>
 +
<li>Didn't work</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> Nanodrop of Miniprepped Plamids: </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Tube # </th>
 +
<th> Concentration (ng/ul) </th>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> 277.3 </td>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 
<td> # </td>
 
<td> # </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 50 uM R Primer </td>
+
<td> 2 </td>
 
<td> # </td>
 
<td> # </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> #ug/ul Construct </td>
+
<td> 2 </td>
 
<td> # </td>
 
<td> # </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
−
<h4> PCR Settings Template: </h4>
+
<div class="column full_size">
 +
<br>
 +
</div>
  
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> PCR of Minprepped Plasmids </h5>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Q5 Buffer</td>
 +
<td> 5uL </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 16.25 uL </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 1.25 uL </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 1.25 uL </td>
 +
</tr>
 +
<tr>
 +
<td> Construct </td>
 +
<td> 0.5 uL </td>
 +
</tr>
 +
<tr>
 +
<td>  Q5 Polymerase </td>
 +
<td> 0.25 uL </td>
 +
</tr>
 +
<tr>
 +
<td>  Q5 Polymerase </td>
 +
<td> 0.25 uL </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> PCR Settings: </h4>
 
<ul>
 
<ul>
 
<table>
 
<table>
Line 62: Line 254:
 
<td> Initial Denaturation </td>
 
<td> Initial Denaturation </td>
 
<td> 98 </td>
 
<td> 98 </td>
−
<td> # </td>
+
<td> 30s </td>
 
</tr>
 
</tr>
 
<tr>  
 
<tr>  
 
<td> Denaturation </td>
 
<td> Denaturation </td>
 
<td> 98 </td>
 
<td> 98 </td>
−
<td> # </td>
+
<td> 20s </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> Annealing </td>
 
<td> Annealing </td>
−
<td> # </td>
+
<td> 56 </td>
−
<td> # </td>
+
<td> 15s </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> Extension </td>
 
<td> Extension </td>
 
<td> 72 </td>
 
<td> 72 </td>
−
<td> # </td>
+
<td> 30s </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> Final Extension </td>
 
<td> Final Extension </td>
 
<td> 72 </td>
 
<td> 72 </td>
−
<td> # </td>
+
<td> 60s </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
−
<h4> Nanodrop Template: </h4>
+
<div align="center">
 +
<br>
 +
<hr>
 +
<br>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<div align="center">
 +
<h2> 8/10 </h2>
 +
<br>
 +
<h4> 25 ul PCR of Miniprepped Plasmids for Project</h4>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Q5 Buffer </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> Q5 Polymerase </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 16.25 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 1.25 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 1.25 </td>
 +
</tr>
 +
<tr>
 +
<td> Construct </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Phase </th>
 +
<th> Temperature (°C) </th>
 +
<th> Time (sec)</th>
 +
</tr>
 +
<tr>
 +
<td> Initial Denaturation </td>
 +
<td> 98 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 20 </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 56</td>
 +
<td> 15 </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<div align="center">
 +
<h5> PCR was run on the following construct concentrations in ng/ul</h5>
 +
<ul>
 +
<li> 273</li>
 +
<li>176.5</li>
 +
<li>296.6</li>
 +
<li>311.5</li>
 +
<li>301.6</li>
 +
<li>301.6</li>
 +
<li> 259</li>
 +
<li> 366.6</li>
 +
</ul>
 +
<ul>
 +
<li> PCRs did not work → Changed annealing temperature to 66°C</li>
 +
<li> Re-ran the first 5 constructs on the above list </li>
 +
</ul>
 +
</div>
 +
 
 +
<br>
 +
 
 +
 
 +
<h4> 25 ul PCR of Miniprepped Plasmid for BioBrick Portion </h4>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> Content </th>
+
<th> Contents </th>
−
<th> Concentration (ng/ul) </th>
+
<th> ul </th>
−
<th> 260/280 </th>
+
</tr>
 +
<td> Q5 Master Mix </td>
 +
<td> 12.5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Construct </td>
+
<td> H<sub>2</sub>O</td>
−
<td> # </td>
+
<td> 9.5 </td>
−
<td> # </td>
+
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Vector </td>
+
<td> 10 uM F Primer </td>
−
<td> # </td>
+
<td> 1.25 </td>
−
<td> # </td>
+
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 1.25 </td>
 +
</tr>
 +
<tr>
 +
<td> Template </td>
 +
<td> 0.5 </td>
 +
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
  
−
<h4> Ligation Template </h4>
+
<ul>
 +
<li> This setup was used for all BioBrick Parts </li>
 +
<li> Ran with the "Sunil" Program with a 66°C annealing temperature</li>
 +
 
 +
<div align="center">
 +
<br>
 +
<hr>
 +
<br>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<div align="center">
 +
<h2> 8/11 </h2>
 +
</div>
 +
 
 +
<h4> Ran a gel of BioBrick Plasmid PCRs from last night </h4>
 +
<h6> Lanes:</h6>
 +
<ol>
 +
<li> BOOi5</li>
 +
<li> ROO82 </li>
 +
<li> ROO11 </li>
 +
<li> Ja1100</li>
 +
<li> Ladder </li>
 +
<li> K1033910 </li>
 +
<li> K592009 </li>
 +
<li> COO12</li>
 +
</ol>
 +
<ul>
 +
<li> Used 8ul of each product was used in each gel </li>
 +
</ul>
 +
</div>
 +
 
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> 25ul PCR Run </h4>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> Content </th>
+
<th> Contents </th>
 
<th> ul </th>
 
<th> ul </th>
 +
</tr>
 +
<td> Q5 Buffer </td>
 +
<td> 5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 10X T4 Buffer </td>
+
<td> Q5 Polymerase </td>
−
<td> # </td>
+
<td> 0.25 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Vector </td>
+
<td> H<sub>2</sub>O</td>
−
<td> # (# ng) </td>
+
<td> 15.75 </td>
 
</tr>
 
</tr>
−
<td> Construct </td>
+
<tr>
−
<td> # (# ng) </td>
+
<td> 10 uM F Primer </td>
 +
<td> 1.25 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> H<sub>2</sub>O</td>
+
<td> 10 uM R Primer </td>
−
<td> # </td>
+
<td> 1.25 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Ligase </td>
+
<td> 366.6ug/ul Construct </td>
−
<td> #</td>
+
<td> 1ul </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> PCR Settings - 35 Cycles </h5>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Phase </th>
 +
<th> Temperature (°C) </th>
 +
<th> Time (sec)</th>
 +
</tr>
 +
<tr>
 +
<td> Initial Denaturation </td>
 +
<td> 98 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 20 </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 65 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 
</div>
 
</div>
  
−
<!--  THE DIV ABOVE THIS ENCLOSES EVERYTHING FROM 8/8 UP UNTIL HERE. PLEASE REMOVE THAT DIV LATER ON ONCE YOU FILL IN THOSE TEMPLATES. -->
+
<div class="column full_size">
−
<!-- This line and the all caps line right above this are unseen on the wiki. It's just an HTML comment. I'll remove it when I get back -->
+
<h5> Dilution of 366.6 Plasmid made </h5>
 +
<p> (1ul)(366.6ng/ul) = X(1n/ul) </p>
 +
<br>
  
 +
<h4> Gel of Plasmid PCR</h4>
 +
<h6>Lanes </h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> Jonathan Plasmid </li>
 +
<li> Ryan Plasmid - 366.6 dilution </li>
 +
</ol>
  
−
<h4> Multiple Digests Template: </h4>
+
<h5> Ran a PCR purification</h5>
 +
<ul>
 +
<li> Used the setup from 8/10 </li>
 +
<li> Also purified the BioBrick PCRs from 8/10 </li>
 +
</ul>
 +
 
 +
<h4> Nanodropped the products </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Concentration (ng/ul) </th>
 +
<th> 260/280 </th>
 +
</tr>
 +
<tr>
 +
<td> BOO15 </td>
 +
<td> 15.1 </td>
 +
<td> 1.72 </td>
 +
</tr>
 +
<tr>
 +
<td> ROO15.1 </td>
 +
<td> 15.1 </td>
 +
<td> 1.85 </td>
 +
</tr>
 +
<tr>
 +
<td> ROOd1 </td>
 +
<td> 11.8 </td>
 +
<td> 1.71 </td>
 +
</tr>
 +
<tr>
 +
<td> J61100 </td>
 +
<td> 11.3 </td>
 +
<td> 1.61 </td>
 +
</tr>
 +
<tr>
 +
<td> K59200 </td>
 +
<td> 20.3 </td>
 +
<td> 1.76 </td>
 +
</tr>
 +
<tr>
 +
<td> K103340 </td>
 +
<td>21.7 </td>
 +
<td> 1.82 </td>
 +
</tr>
 +
<tr>
 +
<td> COO12 </td>
 +
<td> 23.4 </td>
 +
<td> 1.9 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<h4> Gel of BioBrick PCR </h4>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> COO12</li>
 +
<li> ROO11 </li>
 +
<li> K1033910 </li>
 +
<li> J61100 </li>
 +
<li> K592009 </li>
 +
<li> ROO82 </li>
 +
<li> BOO15 </li>
 +
</ol>
 +
 
 +
<br>
 +
 
 +
<h4> Digest of Project Plasmids </h4>
 +
</div>
  
 
<div class="column half_size">
 
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h5> This </h5>
+
<h5> 277.3 ng/ul Template </h5>
−
</div>
+
 
<ul>
 
<ul>
 
<table>
 
<table>
Line 159: Line 627:
 
<tr>  
 
<tr>  
 
<td> H<sub>2</sub>O </td>
 
<td> H<sub>2</sub>O </td>
−
<td> #</td>
+
<td> 40.89</td>
−
<td> # </td>
+
<td> 41.39 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Cutsmart </td>
+
<td> 3.1 Buffer </td>
−
<td> # </td>
+
<td> 5 </td>
−
<td> # </td>
+
<td> 5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> EcoRI-HF </td>
+
<td> NotI </td>
−
<td> # </td>
+
<td> 0.5 </td>
−
<td> X </td>
+
−
</tr>
+
−
<tr>
+
−
<td> XbaI </td>
+
−
<td># </td>
+
 
<td> X </td>
 
<td> X </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> DNA </td>
 
<td> DNA </td>
−
<td> # </td>
+
<td> 3.61 </td>
−
<td> # </td>
+
<td> 3.61 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 
</div>
 
</div>
  
 
<div class="column half_size">
 
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h5> Is </h5>
+
<h5> 176.5 ng/ul </h5>
−
</div>
+
 
<ul>
 
<ul>
 
<table>
 
<table>
Line 199: Line 662:
 
<tr>  
 
<tr>  
 
<td> H<sub>2</sub>O </td>
 
<td> H<sub>2</sub>O </td>
−
<td> #</td>
+
<td> 38.83</td>
−
<td> # </td>
+
<td> 39.33 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Cutsmart </td>
+
<td> 3.1 </td>
−
<td> # </td>
+
<td> 5 </td>
−
<td> # </td>
+
<td> 5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> EcoRI-HF </td>
+
<td> NotI </td>
−
<td> # </td>
+
<td> 0.5 </td>
−
<td> X </td>
+
−
</tr>
+
−
<tr>
+
−
<td> XbaI </td>
+
−
<td># </td>
+
 
<td> X </td>
 
<td> X </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> DNA </td>
 
<td> DNA </td>
−
<td> # </td>
+
<td> 5.67 </td>
−
<td> # </td>
+
<td> 5.67 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 
</div>
 
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
−
<br>
+
<div align="center">
 +
<ul>
 +
<li> Band expected at 6500bp </li>
 +
</div>
 
</div>
 
</div>
  
 
<div class="column half_size">
 
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h5> A </h5>
+
<h5> 296.6 ng/ul </h5>
−
</div>
+
 
<ul>
 
<ul>
 
<table>
 
<table>
Line 243: Line 704:
 
<tr>  
 
<tr>  
 
<td> H<sub>2</sub>O </td>
 
<td> H<sub>2</sub>O </td>
−
<td> #</td>
+
<td> 41.13</td>
−
<td> # </td>
+
<td> 41.63 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Cutsmart </td>
+
<td> 3.1 </td>
−
<td> # </td>
+
<td> 5</td>
−
<td> # </td>
+
<td> 5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> EcoRI-HF </td>
+
<td> EcoRI-HF</td>
−
<td> # </td>
+
<td> 0.5</td>
−
<td> X </td>
+
−
</tr>
+
−
<tr>
+
−
<td> XbaI </td>
+
−
<td># </td>
+
 
<td> X </td>
 
<td> X </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> DNA </td>
 
<td> DNA </td>
−
<td> # </td>
+
<td> 3.37 </td>
−
<td> # </td>
+
<td> 3.37 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 
</div>
 
</div>
  
 
<div class="column half_size">
 
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h5> Thing </h5>
+
<h5> 311.5 ng/ul </h5>
−
</div>
+
 
<ul>
 
<ul>
 
<table>
 
<table>
Line 283: Line 739:
 
<tr>  
 
<tr>  
 
<td> H<sub>2</sub>O </td>
 
<td> H<sub>2</sub>O </td>
−
<td> #</td>
+
<td> 41.29</td>
−
<td> # </td>
+
<td> 41.29 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Cutsmart </td>
+
<td> 3.1</td>
−
<td> # </td>
+
<td> 5 </td>
−
<td> # </td>
+
<td> 5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> EcoRI-HF </td>
 
<td> EcoRI-HF </td>
−
<td> # </td>
+
<td> 0.5 </td>
−
<td> X </td>
+
−
</tr>
+
−
<tr>
+
−
<td> XbaI </td>
+
−
<td># </td>
+
 
<td> X </td>
 
<td> X </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> DNA </td>
 
<td> DNA </td>
−
<td> # </td>
+
<td> 3.21 </td>
−
<td> # </td>
+
<td> 3.21 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 
</div>
 
</div>
−
 
+
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<div align="center">
 
<ul>
 
<ul>
 +
<li> Band expected at 300bp and 6200 </li>
 +
<li> Result = 1 band at 5600 </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div align="center">
 +
<br>
 
<hr>
 
<hr>
 +
<br>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h2> 8/12 </h2>
 +
<h4> Gels of project plasmid </h4>
 +
<ul>
 +
<li> Loaded with 20ul of each product in wells </li>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<h5> Gel 1 </h5>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> 277.3 Control </li>
 +
<li> 277.3 Experimental </li>
 +
<li> Ladder </li>
 +
<li> 176.5 Experimental </li>
 +
<li> 176.5 Control </li>
 +
</ol>
 +
</div>
 +
 +
<div class="column half_size">
 +
<h5> Gel 1 </h5>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Empty </li>
 +
<li> 296.6 Control</li>
 +
<li> 296.6 Experimental </li>
 +
<li> Ladder </li>
 +
<li> 311.5 Experimental </li>
 +
<li> 311.5 Control </li>
 +
</ol>
 
</div>
 
</div>
  
Line 320: Line 815:
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h2> 8/8 </h2>
+
<br>
 +
<br>
 +
<h4> Digested and nanodropped BioBrick parts </h4>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<h4> Sequencing </h4>
+
<div class="column half_size">
 +
<div align="center">
 +
<h4> Nanodropped the products </h4>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> T1 </th>
+
<th> Content </th>
−
<th> T2 </th>
+
<th> Concentration (ng/ul) </th>
 +
<th> 260/280 </th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 5.34 uL H2O </td>
+
<td> BOO15 </td>
−
<td> 5.34 uL H2O</td>
+
<td> 11.9 </td>
 +
<td> 1.39 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 1 uL CR-1 Primer 1 </td>
+
<td> ROO11 </td>
−
<td> 1 uL CR-1 Primer 2 </td>
+
<td> 10.2 </td>
 +
<td> 1.38 </td>
 
</tr>
 
</tr>
−
<td> 1.66 uL DNA (301.6 ng/uL) </td>
+
<tr>
−
<td> 1.66 uL DNA (301.6 ng/uL) </td>
+
<td> ROO82 </td>
 +
<td> 18.4 </td>
 +
<td> 1.64 </td>
 +
</tr>
 +
<tr>
 +
<td> J61100 </td>
 +
<td> 14.2 </td>
 +
<td> 1.35 </td>
 +
</tr>
 +
<tr>
 +
<td> K59200 </td>
 +
<td> 9.5 </td>
 +
<td> 1.46 </td>
 +
</tr>
 +
<tr>
 +
<td> K103340 </td>
 +
<td>13.4 </td>
 +
<td> 1.47 </td>
 +
</tr>
 +
<tr>
 +
<td> COO12 </td>
 +
<td> 13.6 </td>
 +
<td> 1.47 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
−
<h4> Diluted BioBrick MiniPreps for PCR </h4>
+
<div class="column half_size">
 +
<div align="center">
 +
<h4> Gel of PEP352GAP + Jonathan Plasmids </h4>
 +
</div>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> 547 </li>
 +
<li> 498.9 </li>
 +
<li> Ladder </li>
 +
<li> Balazsi </li>
 +
<li> Transformed Balazsi </li>
 +
</ol>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
 
 +
<div align="center">
 +
<h4> Digests of BioBrick Parts - Round 1 </h4>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> ROO11 </h5>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> BioBrick Name </th>
+
<th> Content </th>
−
<th> Concentration (ng/uL) </th>
+
<th> ul </th>
−
<th> Contents (DNA + water) </th>
+
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> K592009 </td>
+
<td> Cutsmart </td>
−
<td> 76.9 </td>
+
<td> 5.5 </td>
−
<td> 1 uL + 6.69 uL water </td>
+
 
</tr>
 
</tr>
−
<tr>  
+
<tr>
−
<td> B0015 </td>
+
<td> SpeI </td>
−
<td> 51.7 </td>
+
<td> 0.5 </td>
−
<td> 1 uL + 4.17 uL water </td>
+
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> K1033910 </td>
+
<td> DNA </td>
−
<td> 156.5 </td>
+
<td> 49 </td>
−
<td> 1 uL + 14.65 uL water </td>
+
 
</tr>
 
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> J61100 </h5>
 +
<ul>
 +
<table>
 
<tr>
 
<tr>
−
<td> R0082 </td>
+
<th> Content </th>
−
<td> 49.5 </td>
+
<th> ul </th>
−
<td> 1 uL + 3.95 uL water </td>
+
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> C0012 </td>
+
<td> Cutsmart </td>
−
<td> 150.8 </td>
+
<td> 3.98 </td>
−
<td> 1 uL + 14.08 uL water </td>
+
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> J61100 </td>
+
<td> XbaI </td>
−
<td> 123.0 </td>
+
<td> 0.5 </td>
−
<td> 1 uL + 11.3 uL water </td>
+
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> R0011 </td>
+
<td> DNA </td>
−
<td> 56.7 </td>
+
<td> 35.2 </td>
−
<td> 1 uL + 4.67 uL water </td>
+
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
 +
<div class="column full_size">
 +
<br>
 +
</div>
  
−
<h4> PCR of each BioBrick parts  (25 uL Q5 MasterMix rxns) </h4>
+
<div class="column half_size">
 +
<div align="center">
 +
<h5> K1033910 </h5>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> Contents </th>
+
<th> Content </th>
−
<th> uL </th>
+
<th> ul </th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 10 uM VF2 primer </td>
+
<td> Cutsmart </td>
−
<td> 1.25 uL </td>
+
<td> 3.98 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 10 uM VR primer </td>
+
<td> SpeI </td>
−
<td> 1.25 uL  </td>
+
<td> 0.5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> DNA </td>
 
<td> DNA </td>
−
<td> 0.5 uL </td>
+
<td> 37.3 </td>
 
</tr>
 
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> BOO15 </h5>
 +
<ul>
 +
<table>
 
<tr>
 
<tr>
−
<td> Q5 MM </td>
+
<th> Content </th>
−
<td> 12.5 uL </td>
+
<th> ul </th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Water </td>
+
<td> Cutsmart </td>
−
<td> 19.5 uL </td>
+
<td> 4.75 </td>
 +
</tr>
 +
<tr>
 +
<td> XbaI </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 42.0 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
−
 
+
</div>
 +
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<br>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> ROO82 </h5>
 
<ul>
 
<ul>
−
<hr>
+
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 3.1 </td>
 +
</tr>
 +
<tr>
 +
<td> SpeI </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 27.2 </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 +
</div>
 
</div>
 
</div>
  
−
<div class="column full_size">
+
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h2> 8/9 </h2>
+
<h5> 561100 </h5>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 3.98 </td>
 +
</tr>
 +
<tr>
 +
<td> XbaI </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 35.2</td>
 +
</tr>
 +
</table>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<h5> Gel ran </h5>
+
<div class="column full_size">
−
<h4>Lanes:</h4>
+
<br>
−
<ol>
+
</div>
−
<li>Ladder</li>
+
−
<li>R0011</li>
+
−
<li>B001</li>
+
−
<li>K1033</li>
+
−
<li>R008</li>
+
−
<li>J61100</li>
+
−
<li>C0012</li>
+
−
<li>K592</li>
+
−
</ol>
+
  
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> K592009</h5>
 
<ul>
 
<ul>
−
<li>Didn't work</li>
+
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5.9 </td>
 +
</tr>
 +
<tr>
 +
<td> SpeI </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 52.6</td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
−
<h4> Nanodrop of Miniprepped Plamids: </h4>
+
<div class="column half_size">
 +
<div align="center">
 +
<h5> 561100 </h5>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> Tube # </th>
+
<th> Content </th>
−
<th> Concentration (ng/ul) </th>
+
<th> ul </th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 1 </td>
+
<td> Cutsmart </td>
−
<td> 277.3 </td>
+
<td> 3.98 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 2 </td>
+
<td> XbaI </td>
−
<td> # </td>
+
<td> 0.5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 2 </td>
+
<td> DNA </td>
−
<td> # </td>
+
<td> 35.2</td>
 
</tr>
 
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<br>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> COO12 </h5>
 +
<ul>
 +
<table>
 
<tr>
 
<tr>
−
<td> 2 </td>
+
<th> Content </th>
−
<td> # </td>
+
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 4.3 </td>
 +
</tr>
 +
<tr>
 +
<td> SpeI </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 38.5</td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
−
 
+
<div class="column half_size">
−
<h5> PCR of Minprepped Plasmids </h5>
+
<div align="center">
 +
<h5> BOO15</h5>
 
<ul>
 
<ul>
 
<table>
 
<table>
 
<tr>
 
<tr>
−
<th> Contents </th>
+
<th> Content </th>
 
<th> ul </th>
 
<th> ul </th>
 
</tr>
 
</tr>
−
<td> Q5 Buffer</td>
+
<tr>
−
<td> 5uL </td>
+
<td> Cutsmart </td>
 +
<td> 4.75 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> H<sub>2</sub>O</td>
+
<td> XbaI </td>
−
<td> 16.25 uL </td>
+
<td> 0.5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 10 uM F Primer </td>
+
<td> DNA </td>
−
<td> 1.25 uL </td>
+
<td> 42.0</td>
 
</tr>
 
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<br>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> PCR of IDT Construct </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<ul>
 +
<table>
 
<tr>
 
<tr>
−
<td> 10 uM R Primer </td>
+
<th> Contents </th>
−
<td> 1.25 uL </td>
+
<th> ul </th>
 +
</tr>
 +
<td> Phire </td>
 +
<td> 25 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Construct </td>
+
<td> H<sub>2</sub>O</td>
−
<td> 0.5 uL </td>
+
<td> 22 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Q5 Polymerase </td>
+
<td> 50 uM F Primer </td>
−
<td> 0.25 uL </td>
+
<td> 1 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Q5 Polymerase </td>
+
<td> 50 uM R Primer </td>
−
<td> 0.25 uL </td>
+
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA Template </td>
 +
<td> 1 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
−
<h4> PCR Settings: </h4>
+
<div class="column half_size">
 +
<div align="center">
 
<ul>
 
<ul>
 
<table>
 
<table>
Line 527: Line 1,222:
 
<td> Initial Denaturation </td>
 
<td> Initial Denaturation </td>
 
<td> 98 </td>
 
<td> 98 </td>
−
<td> 30s </td>
+
<td> 30</td>
 
</tr>
 
</tr>
 
<tr>  
 
<tr>  
 
<td> Denaturation </td>
 
<td> Denaturation </td>
 
<td> 98 </td>
 
<td> 98 </td>
−
<td> 20s </td>
+
<td> 20 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> Annealing </td>
 
<td> Annealing </td>
−
<td> 56 </td>
+
<td> 5 </td>
−
<td> 15s </td>
+
<td> 30</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> Extension </td>
 
<td> Extension </td>
 
<td> 72 </td>
 
<td> 72 </td>
−
<td> 30s </td>
+
<td> 30 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> Final Extension </td>
 
<td> Final Extension </td>
 
<td> 72 </td>
 
<td> 72 </td>
−
<td> 60s </td>
+
<td> 60 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
−
 
+
</div>
−
 
+
</div>
−
 
+
−
 
+
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<h4> Ran a gel on those products </h4>
 +
<h6> Lanes:</h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> Blank </li>
 +
<li> JK Construct Plasmid </li>
 +
<li> PCR </li>
 +
</ol>
 +
 +
<h4> Nanodrop of PCR </h4>
 
<ul>
 
<ul>
−
<hr>
+
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Concentration (ng/ul) </th>
 +
<th> 260/280 </th>
 +
</tr>
 +
<tr>
 +
<td> Construct </td>
 +
<td> 79.7 </td>
 +
<td> 1.77 </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 +
</div>
 +
 +
<div align="center">
 +
<br>
 +
<hr>
 +
<br>
 
</div>
 
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h2> 8/10 </h2>
+
<h2> 8/13 </h2>
 
</div>
 
</div>
 +
 +
<h4> Miniprep and gel run of Dean vector </h4>
 +
<ul>
 +
<li> Two preps used Epoch kit </li>
 +
<li> Two preps used Qiagen </li>
 +
</ul>
 
</div>
 
</div>
  
−
<div class="column full_size">
+
<div class="column half_size">
 +
<h5> Ran the plasmids on a gel</h5>
 +
<h6> Lanes:</h6>
 +
<ol>
 +
<li> 1st Epoch </li>
 +
<li> 2nd Epoch </li>
 +
<li> Ladder </li>
 +
<li> 1st Qiagen </li>
 +
<li> 2nd Qiagen </li>
 +
</ol>
 +
<ul>
 +
<li> The gel wells 4 and 5 were contaminated by a little bit of RNA </li>
 +
</ul>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<h4> Nanodrop of Miniprepped plasmids </h4>
 
<ul>
 
<ul>
−
<hr>
+
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Concentration (ng/ul) </th>
 +
<th> 260/280 </th>
 +
</tr>
 +
<tr>
 +
<td> 1st Epoch</td>
 +
<td> 133.4 </td>
 +
<td> 1.89 </td>
 +
</tr>
 +
<tr>
 +
<td> 2nd Epoch </td>
 +
<td> 142.6 </td>
 +
<td> 1.92 </td>
 +
</tr>
 +
<tr>
 +
<td> 1st Qiagen </td>
 +
<td> 188.5 </td>
 +
<td> 2.02 </td>
 +
</tr>
 +
<tr>
 +
<td> 2nd Qiagen </td>
 +
<td> 205.6 </td>
 +
<td> 1.97 </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 
</div>
 
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
−
<div align="center">
+
<div align="center">  
−
<h2> 8/11 </h2>
+
<h4> 50ul Digest of Plasmid and Insert </h4>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<div class="column full_size">
+
<div class="column half_size">
 +
<div align="center">
 +
<h5> Epoch 1 </h5>
 
<ul>
 
<ul>
−
<hr>
+
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O </td>
 +
<td> 36.5</td>
 +
<td> 37.5 </td>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> PvuII-HF </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> XhoI </td>
 +
<td> 0.5</td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 7.5 </td>
 +
<td> 7.5 </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 +
</div>
 
</div>
 
</div>
  
−
<div class="column full_size">
+
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h2> 8/12 </h2>
+
<h5> Epoch 2 </h5>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O </td>
 +
<td> 37</td>
 +
<td> 38</td>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> PvuII-HF </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> XhoI </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 7.01 </td>
 +
<td> 7.01 </td>
 +
</tr>
 +
</table>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<h2> Today was a depressing day <h2>
+
<br>
 +
<br>
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<div align="center">
 +
<h5> Insert </h5>
 
<ul>
 
<ul>
−
<hr>
+
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O </td>
 +
<td> 31.5</td>
 +
<td> 32.5</td>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> PvuII-HF </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> XhoI </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 12.55 </td>
 +
<td> 12.55 </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
 +
 +
<div align="center">
 +
<br>
 +
<hr>
 +
<br>
 
</div>
 
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h2> 8/13 </h2>
+
<h2> 8/14 </h2>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<div class="column full_size">
+
<div class="column half_size">
 +
<div align="center">
 +
<h4> Purified 8/13 digests with Qiagen PCR purificiation kit and ran a gel </h4>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Epoch 1 Control </li>
 +
<li> Epoch 1 Experimental </li>
 +
<li> Ladder </li>
 +
<li> Epoch 2 Control</li>
 +
<li> Epoch 2 Experimental</li>
 +
<li> Insert Experimental </li>
 +
<li> Insert Control </li>
 +
</ol>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> Nanodropped digest purifications </h4>
 
<ul>
 
<ul>
−
<hr>
+
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Concentration (ng/ul) </th>
 +
<th> 260/280 </th>
 +
</tr>
 +
<tr>
 +
<td> Epoch 1 </td>
 +
<td> 9.8 </td>
 +
<td> 2.81 </td>
 +
</tr>
 +
<tr>
 +
<td> Epoch 2 </td>
 +
<td> 9.4 </td>
 +
<td> 2.28 </td>
 +
</tr>
 +
<tr>
 +
<td> Insert </td>
 +
<td> 15.8 </td>
 +
<td> 1.98 </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>
 +
</div>
 
</div>
 
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<div align="center">
 +
<h4> Troubleshooting </h4>
 +
<ul>
 +
<li> To fix digests, sequential diests attempted. Digested with only XhoI and heat deactivated. Then digested with PvuII. Purified it and nanodropped it </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h2> 8/14 </h2>
+
<h5> Epoch 1 </h5>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O </td>
 +
<td> 37</td>
 +
<td> 37.5 </td>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> XhoI </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 7.5</td>
 +
<td> 7.5 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> Epoch 2 </h5>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O </td>
 +
<td> 37.5</td>
 +
<td> 38 </td>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> XhoI </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 7.01</td>
 +
<td> 7.01</td>
 +
</tr>
 +
</table>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<div align="center">
 
<ul>
 
<ul>
−
<hr>
+
<li> Placed in incubator </li>
 +
<ul>
 +
<li>XhoII heat inactivated at 65°C for 20 minutes</li>
 
</ul>
 
</ul>
 +
</ul>
 +
</div>
 +
 +
<div align="center">
 +
<br>
 +
<hr>
 +
<br>
 
</div>
 
</div>
  
 
</body>
 
</body>
 
</html>
 
</html>

Latest revision as of 07:52, 19 October 2016



8/8

Sequencing

    T1 T2
    5.34 uL H2O 5.34 uL H2O
    1 uL CR-1 Primer 1 1 uL CR-1 Primer 2
    1.66 uL DNA (301.6 ng/uL) 1.66 uL DNA (301.6 ng/uL)

Diluted BioBrick MiniPreps for PCR

    BioBrick Name Concentration (ng/uL) Contents (DNA + water)
    K592009 76.9 1 uL + 6.69 uL water
    B0015 51.7 1 uL + 4.17 uL water
    K1033910 156.5 1 uL + 14.65 uL water
    R0082 49.5 1 uL + 3.95 uL water
    C0012 150.8 1 uL + 14.08 uL water
    J61100 123.0 1 uL + 11.3 uL water
    R0011 56.7 1 uL + 4.67 uL water

PCR of each BioBrick parts (25 uL Q5 MasterMix rxns)

    Contents uL
    10 uM VF2 primer 1.25 uL
    10 uM VR primer 1.25 uL
    DNA 0.5 uL
    Q5 MM 12.5 uL
    Water 19.5 uL


8/9

Gel run:
Lanes:
  1. Ladder
  2. R0011
  3. B001
  4. K1033
  5. R008
  6. J61100
  7. C0012
  8. K592
  • Didn't work

Nanodrop of Miniprepped Plamids:

    Tube # Concentration (ng/ul)
    1 277.3
    2 #
    2 #
    2 #

PCR of Minprepped Plasmids
    Contents ul
    Q5 Buffer 5uL
    H2O 16.25 uL
    10 uM F Primer 1.25 uL
    10 uM R Primer 1.25 uL
    Construct 0.5 uL
    Q5 Polymerase 0.25 uL
    Q5 Polymerase 0.25 uL

PCR Settings:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30s
    Denaturation 98 20s
    Annealing 56 15s
    Extension 72 30s
    Final Extension 72 60s


8/10


25 ul PCR of Miniprepped Plasmids for Project

    Contents ul
    Q5 Buffer 5
    Q5 Polymerase 5
    H2O 16.25
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Construct 0.5
    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30
    Denaturation 98 20
    Annealing 56 15
    Extension 72 30
    Final Extension 72 60
PCR was run on the following construct concentrations in ng/ul
  • 273
  • 176.5
  • 296.6
  • 311.5
  • 301.6
  • 301.6
  • 259
  • 366.6
  • PCRs did not work → Changed annealing temperature to 66°C
  • Re-ran the first 5 constructs on the above list

25 ul PCR of Miniprepped Plasmid for BioBrick Portion

    Contents ul
    Q5 Master Mix 12.5
    H2O 9.5
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Template 0.5
  • This setup was used for all BioBrick Parts
  • Ran with the "Sunil" Program with a 66°C annealing temperature


    • 8/11

    Ran a gel of BioBrick Plasmid PCRs from last night

    Lanes:
    1. BOOi5
    2. ROO82
    3. ROO11
    4. Ja1100
    5. Ladder
    6. K1033910
    7. K592009
    8. COO12
    • Used 8ul of each product was used in each gel

    25ul PCR Run

      Contents ul
      Q5 Buffer 5
      Q5 Polymerase 0.25
      H2O 15.75
      10 uM F Primer 1.25
      10 uM R Primer 1.25
      366.6ug/ul Construct 1ul
    PCR Settings - 35 Cycles
      Phase Temperature (°C) Time (sec)
      Initial Denaturation 98 30
      Denaturation 98 20
      Annealing 65 30
      Extension 72 30
      Final Extension 72 60
    Dilution of 366.6 Plasmid made

    (1ul)(366.6ng/ul) = X(1n/ul)


    Gel of Plasmid PCR

    Lanes
    1. Ladder
    2. Jonathan Plasmid
    3. Ryan Plasmid - 366.6 dilution
    Ran a PCR purification
    • Used the setup from 8/10
    • Also purified the BioBrick PCRs from 8/10

    Nanodropped the products

      Content Concentration (ng/ul) 260/280
      BOO15 15.1 1.72
      ROO15.1 15.1 1.85
      ROOd1 11.8 1.71
      J61100 11.3 1.61
      K59200 20.3 1.76
      K103340 21.7 1.82
      COO12 23.4 1.9

    Gel of BioBrick PCR

    Lanes:
    1. COO12
    2. ROO11
    3. K1033910
    4. J61100
    5. K592009
    6. ROO82
    7. BOO15

    Digest of Project Plasmids

    277.3 ng/ul Template
      Content Experimental ul Control ul
      H2O 40.89 41.39
      3.1 Buffer 5 5
      NotI 0.5 X
      DNA 3.61 3.61
    176.5 ng/ul
      Content Experimental ul Control ul
      H2O 38.83 39.33
      3.1 5 5
      NotI 0.5 X
      DNA 5.67 5.67
    • Band expected at 6500bp
    296.6 ng/ul
      Content Experimental ul Control ul
      H2O 41.13 41.63
      3.1 5 5
      EcoRI-HF 0.5 X
      DNA 3.37 3.37
    311.5 ng/ul
      Content Experimental ul Control ul
      H2O 41.29 41.29
      3.1 5 5
      EcoRI-HF 0.5 X
      DNA 3.21 3.21
    • Band expected at 300bp and 6200
    • Result = 1 band at 5600


    8/12

    Gels of project plasmid

    • Loaded with 20ul of each product in wells
    Gel 1
    Lanes:
    1. 277.3 Control
    2. 277.3 Experimental
    3. Ladder
    4. 176.5 Experimental
    5. 176.5 Control
    Gel 1
    Lanes:
    1. Empty
    2. 296.6 Control
    3. 296.6 Experimental
    4. Ladder
    5. 311.5 Experimental
    6. 311.5 Control


    Digested and nanodropped BioBrick parts

    Nanodropped the products

      Content Concentration (ng/ul) 260/280
      BOO15 11.9 1.39
      ROO11 10.2 1.38
      ROO82 18.4 1.64
      J61100 14.2 1.35
      K59200 9.5 1.46
      K103340 13.4 1.47
      COO12 13.6 1.47

    Gel of PEP352GAP + Jonathan Plasmids

    Lanes:
    1. 547
    2. 498.9
    3. Ladder
    4. Balazsi
    5. Transformed Balazsi

    Digests of BioBrick Parts - Round 1

    ROO11
      Content ul
      Cutsmart 5.5
      SpeI 0.5
      DNA 49
    J61100
      Content ul
      Cutsmart 3.98
      XbaI 0.5
      DNA 35.2

    K1033910
      Content ul
      Cutsmart 3.98
      SpeI 0.5
      DNA 37.3
    BOO15
      Content ul
      Cutsmart 4.75
      XbaI 0.5
      DNA 42.0

    ROO82
      Content ul
      Cutsmart 3.1
      SpeI 0.5
      DNA 27.2
    561100
      Content ul
      Cutsmart 3.98
      XbaI 0.5
      DNA 35.2

    K592009
      Content ul
      Cutsmart 5.9
      SpeI 0.5
      DNA 52.6
    561100
      Content ul
      Cutsmart 3.98
      XbaI 0.5
      DNA 35.2

    COO12
      Content ul
      Cutsmart 4.3
      SpeI 0.5
      DNA 38.5
    BOO15
      Content ul
      Cutsmart 4.75
      XbaI 0.5
      DNA 42.0

    PCR of IDT Construct

      Contents ul
      Phire 25
      H2O 22
      50 uM F Primer 1
      50 uM R Primer 1
      DNA Template 1
      Phase Temperature (°C) Time (sec)
      Initial Denaturation 98 30
      Denaturation 98 20
      Annealing 5 30
      Extension 72 30
      Final Extension 72 60

    Ran a gel on those products

    Lanes:
    1. Ladder
    2. Blank
    3. JK Construct Plasmid
    4. PCR

    Nanodrop of PCR

      Content Concentration (ng/ul) 260/280
      Construct 79.7 1.77


    8/13

    Miniprep and gel run of Dean vector

    • Two preps used Epoch kit
    • Two preps used Qiagen
    Ran the plasmids on a gel
    Lanes:
    1. 1st Epoch
    2. 2nd Epoch
    3. Ladder
    4. 1st Qiagen
    5. 2nd Qiagen
    • The gel wells 4 and 5 were contaminated by a little bit of RNA

    Nanodrop of Miniprepped plasmids

      Content Concentration (ng/ul) 260/280
      1st Epoch 133.4 1.89
      2nd Epoch 142.6 1.92
      1st Qiagen 188.5 2.02
      2nd Qiagen 205.6 1.97

    50ul Digest of Plasmid and Insert

    Epoch 1
      Content Experimental ul Control ul
      H2O 36.5 37.5
      Cutsmart 5 5
      PvuII-HF 0.5 X
      XhoI 0.5 X
      DNA 7.5 7.5
    Epoch 2
      Content Experimental ul Control ul
      H2O 37 38
      Cutsmart 5 5
      PvuII-HF 0.5 X
      XhoI 0.5 X
      DNA 7.01 7.01


    Insert
      Content Experimental ul Control ul
      H2O 31.5 32.5
      Cutsmart 5 5
      PvuII-HF 0.5 X
      XhoI 0.5 X
      DNA 12.55 12.55


    8/14

    Purified 8/13 digests with Qiagen PCR purificiation kit and ran a gel

    Lanes:
    1. Epoch 1 Control
    2. Epoch 1 Experimental
    3. Ladder
    4. Epoch 2 Control
    5. Epoch 2 Experimental
    6. Insert Experimental
    7. Insert Control

    Nanodropped digest purifications

      Content Concentration (ng/ul) 260/280
      Epoch 1 9.8 2.81
      Epoch 2 9.4 2.28
      Insert 15.8 1.98

    Troubleshooting

    • To fix digests, sequential diests attempted. Digested with only XhoI and heat deactivated. Then digested with PvuII. Purified it and nanodropped it
    Epoch 1
      Content Experimental ul Control ul
      H2O 37 37.5
      Cutsmart 5 5
      XhoI 0.5 X
      DNA 7.5 7.5
    Epoch 2
      Content Experimental ul Control ul
      H2O 37.5 38
      Cutsmart 5 5
      XhoI 0.5 X
      DNA 7.01 7.01
    • Placed in incubator
      • XhoII heat inactivated at 65°C for 20 minutes