Team:TU Eindhoven/Notebook

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<h2>Notebook</h2>
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          <li class="category active"><a href="#notebook">NOTEBOOK</a></li>
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<li class="category active"><a href="#january">JANUARY</a></li>
           <li class="category"><a href="#february">FEBRUARY</a></li>
           <li class="category"><a href="#february">FEBRUARY</a></li>
           <li class="category"><a href="#march">MARCH</a></li>
           <li class="category"><a href="#march">MARCH</a></li>
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<div class="main">
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    <div id="january" class="container january">
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      <h1 id="header1">January</h1>
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<p id="para">-Background research<br>
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</p>
     </div>
     </div>
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    <div id="notebook" class="container">
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<br><br>
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      <div class="header">
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        <h1>Notebook</h1>
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<p>Hallo hallo</p>
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      </div>
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    </div>
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     <div id="february" class="container february">
     <div id="february" class="container february">
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       <h1>February</h1>
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       <h1 id="header1">February</h1>
 +
<p id="para">-Background research<br>
 +
</p>
     </div>
     </div>
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 +
<br><br>
 +
     <div id="march" class="container march">
     <div id="march" class="container march">
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       <h1>March</h1>
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       <h1 id="header1">March</h1>
 +
<p id="para">
 +
-Selection of topic<br>
 +
-Further research<br>
 +
-Selection of projec<br>
 +
</p>
     </div>
     </div>
 +
 +
<br><br>
 +
     <div id="april" class="container april">
     <div id="april" class="container april">
-
       <h1>April</h1>
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       <h1 id="header1">April</h1>
 +
<p id="para">-Further research <br>
 +
</p>
     </div>
     </div>
 +
 +
<br><br>
 +
     <div id="may" class="container may">
     <div id="may" class="container may">
-
       <h1>May</h1>
+
       <h1 id="header1">May</h1>
 +
<p id="para"><b><u>General Activities</b></u><br>
 +
Regular team meetings<br>
 +
Looking for sponsors<br>
 +
Month planning<br>
 +
</p>
     </div>
     </div>
 +
 +
<br><br>
 +
     <div id="june" class="container june">
     <div id="june" class="container june">
-
       <h1>June</h1>
+
       <h1 id="header1">June</h1>
 +
<p id="para"><b><u>Wetlab</u></b><br>
 +
- Arrival of the biobricks<br>
 +
- The designs for the plasmids are approved and ordered Primers<br>
 +
- Preparation of culture media, agar plates, antibiotics and glycerol<br>
 +
- Amplification of PET-29a(+), pEVOL-pAzF, BBa_K811005(Penn) & CPX<br>
 +
 
 +
</p>
     </div>
     </div>
-
    <div id="july" class="container july">
 
-
      <h1>July</h1>
 
-
    <h2>June 30th - July 6th</h2>
 
-
<h1>
 
-
Testing the SPAAC reaction in general</h1>
 
-
<p>In this step, the SPAAC reaction will be tested with just the unnatural amino acid pAzF and DBCO-PEG (~10 kDa).&nbsp;</p>
 
-
<p>Phosphate buffer (100 mL, 20mM trisodium phosphate, pH 7.4):</p>
 
-
<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100 mL demi water</p>
 
-
<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 tablet PBS <em>(Phosphate Buffer Saline)</em></p>
 
-
<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3,74 g KCl (potassium chloride)</p>
 
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 500 &micro;L Tween 20</p>
 
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<h4>
 
-
Calibration curve and examination of the absorption wavelength of DBCO (306 nm)</h4>
 
-
<p>First, the amount of required substance has to be determined. &nbsp;This was done by filling the cuvette with consecutively 1.0, 1.8, 2.0 and 3.0 mL acetone. The settings of the measurement were as follows:</p>
 
-
<table border="1" cellpadding="0" cellspacing="0">
 
-
<tbody>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Photometric mode</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Abs</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Response</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Medium</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>UV/Vis bandwith</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>1.0 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Scan Speed</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>40 nm/min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Start</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>340 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>End</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>190 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Data interval</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>0.5 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Vertical size</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Auto</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Scan mode</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Continuos</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Accamulation/cycle</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Accamulation</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>No. Of cycles</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>2</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Temperatuur</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>20 ˚C</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p><br />
 
-
These measurements showed that 1.8 mL is sufficient to measure absorption.</p>
 
-
<p>Next, a stock-solution of DBCO-PEG (10 kDa) is made (142.9 &micro;M):</p>
 
-
<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.5 mL phosphate buffer</p>
 
-
<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 6.43 mg DBCO-PEG (10 kDa)</p>
 
-
<p>The first measurement has been performed to determine the cut-off value of the buffer. &nbsp;Hereafter, the concentration series of DBCO-PEG (10 kDa) are measured. The concentrations were subsequently 142.9, 100, 70, 49, 34.3, 24, 16.8 and 11.8 &mu;M. The measurements were performed with the following settings.</p>
 
-
<table border="1" cellpadding="0" cellspacing="0">
 
-
<tbody>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Photometric mode</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Abs</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Response</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Medium</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>UV/Vis bandwith</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>1.0 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Scan Speed</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>40 nm/min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Start</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>340 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>End</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>220 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Data interval</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>0.5 nm</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Vertical size</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Auto</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Scan mode</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Continuos</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Accamulation/cycle</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>Accamulation</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>No. Of cycles</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>2</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Temperatuur</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>37 ˚C</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="width:308px;">
 
-
<p>Stirrer</p>
 
-
</td>
 
-
<td style="width:308px;">
 
-
<p>150 rpm</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>With the obtained absorption spectra, a calibration curve can be made by fitting a line through the values for 306 nm. The slope of this line is the molar extinction coefficient and can be combined with the Beer-Lambert Law to determine the concentration of DBCO in the reaction mixture.</p>
 
 +
<br><br>
 +
    <div id="july" class="container july">
 +
      <h1 id="header1">July</h1>
 +
<p id="para"><b><u>Wetlab</b></u><br>
 +
<b>Week 27: 30 June – 6 July</b><br>
 +
Plasmid amplification  and culturing BBa_K811005(Penn) + CPX<br>
 +
Vector and insert Digestions<br>
 +
BBa_K811005(Penn) + CPX insert ligate pET29a <br>
 +
Colony PCR <br>
 +
<br>
 +
<b>Week 28: 7 July – 13 July</b><br>
 +
Colony PCR <br>
 +
Site directed mutagenesis of CPX <br>
 +
<br>
 +
<b>Week 29: 14 July – 20 July</b><br>
 +
Site directed mutagenesis of CPX <br>
 +
Sequencing of CPX and BBa_K811005(Penn)<br>
 +
<br>
 +
<b>Week 30: 21 July – 27 July</b><br>
 +
Sequencing results came in and were as expected<br>
 +
Protein expression <br>
 +
FACS – DBCO-PEG4-5/6-TAMRA <br>
 +
<br>
 +
<b>Week 31: 28 July – August 3</b><br>
 +
Protein expression<br>
 +
FACS – DBCO-PEG10kDa <br>
 +
</p>
 +
    </div>
 +
<br><br>
 +
    <div id="august" class="container august">
 +
      <h1 id="header1">August</h1>
 +
<p id="para"><b><u>Wetlab</b></u><br>
 +
<b>Week 32: 4 August – 10 August</b><br>
 +
Protein expression <br>
 +
FACS – Antibody titration <br>
 +
Protein expression curve <br>
 +
<br>
 +
<b>Week 33: 11 August – 17 August</b><br>
 +
FACS - DBCO PEG-5kDa fluorescent <br>
 +
<br>
 +
<b>Week 34: 18 August – 24 August</b><br>
 +
Start Bio-bricking<br>
 +
Start silencing mutagenesis <br>
 +
<br>
 +
<b>Week 35: 25 August – 31 August</b><br>
</p>
</p>
-
  </br>
 
-
  <h2>July 7th - July 13th</h2>
 
-
  <p>Deze week hebben we ook hele leuke dingen gedaan.</p>
 
-
    </div>
 
-
<div id="august" class="container august">
 
-
      <h1>August</h1>
 
     </div>
     </div>
 +
   
 +
<br><br>
 +
     <div id="september" class="container september">
     <div id="september" class="container september">
-
       <h1>September</h1>
+
       <h1 id="header1">September</h1>
 +
<p id="para"><b><u>Wetlab</b></u><br>
 +
<b>Week 36: 1 September – 7 September</b><br>
 +
<br>
 +
<b>Week 37: 8 September – 14 September</b><br>
 +
<br>
 +
<b>Week 38: 15 September – 21 September</b><br>
 +
<br>
 +
<b>Week 39: 22 September – 28 September</b><br>
 +
</p>
     </div>
     </div>
 +
 +
<br><br>
 +
     <div id="october" class="container october">
     <div id="october" class="container october">
-
       <h1>October</h1>
+
       <h1 id="header1">October</h1>
 +
<p id="para"><b><u>Wetlab</b></u><br>
 +
<b>Week 40: 29 September – 5 October</b><br>
 +
<br>
 +
<b>Week 41: 6 October – 12 October</b><br>
 +
<br>
 +
<b>Week 42: 13 October – 19 October</b><br>
 +
</p>
     </div>
     </div>
 +
   
 +
<br><br>
 +
 +
 
 +
 +
 +
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 +
 +
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Latest revision as of 12:58, 25 August 2014

TU Eindhoven iGEM Team

Notebook

January

-Background research



February

-Background research



March

-Selection of topic
-Further research
-Selection of projec



April

-Further research



May

General Activities
Regular team meetings
Looking for sponsors
Month planning



June

Wetlab
- Arrival of the biobricks
- The designs for the plasmids are approved and ordered Primers
- Preparation of culture media, agar plates, antibiotics and glycerol
- Amplification of PET-29a(+), pEVOL-pAzF, BBa_K811005(Penn) & CPX



July

Wetlab
Week 27: 30 June – 6 July
Plasmid amplification and culturing BBa_K811005(Penn) + CPX
Vector and insert Digestions
BBa_K811005(Penn) + CPX insert ligate pET29a
Colony PCR

Week 28: 7 July – 13 July
Colony PCR
Site directed mutagenesis of CPX

Week 29: 14 July – 20 July
Site directed mutagenesis of CPX
Sequencing of CPX and BBa_K811005(Penn)

Week 30: 21 July – 27 July
Sequencing results came in and were as expected
Protein expression
FACS – DBCO-PEG4-5/6-TAMRA

Week 31: 28 July – August 3
Protein expression
FACS – DBCO-PEG10kDa



August

Wetlab
Week 32: 4 August – 10 August
Protein expression
FACS – Antibody titration
Protein expression curve

Week 33: 11 August – 17 August
FACS - DBCO PEG-5kDa fluorescent

Week 34: 18 August – 24 August
Start Bio-bricking
Start silencing mutagenesis

Week 35: 25 August – 31 August



September

Wetlab
Week 36: 1 September – 7 September

Week 37: 8 September – 14 September

Week 38: 15 September – 21 September

Week 39: 22 September – 28 September



October

Wetlab
Week 40: 29 September – 5 October

Week 41: 6 October – 12 October

Week 42: 13 October – 19 October



Created by iGEM Team TU Eindhoven 2014

Retrieved from "http://2014.igem.org/Team:TU_Eindhoven/Notebook"