Team:TU Eindhoven/Notebook

From 2014.igem.org

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     <div id="july" class="container july">
     <div id="july" class="container july">
       <h1>July</h1>
       <h1>July</h1>
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    <h2>June 30th - July 6th</h2>
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<h1>
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Testing the SPAAC reaction in general</h1>
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<p>In this step, the SPAAC reaction will be tested with just the unnatural amino acid pAzF and DBCO-PEG (~10 kDa).&nbsp;</p>
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<p>Phosphate buffer (100 mL, 20mM trisodium phosphate, pH 7.4):</p>
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100 mL demi water</p>
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 tablet PBS <em>(Phosphate Buffer Saline)</em></p>
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<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>
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Calibration curve and examination of the absorption wavelength of DBCO (306 nm)</h4>
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<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>
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<table border="1" cellpadding="0" cellspacing="0">
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<tbody>
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<tr>
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<td style="width:308px;">
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<p>Photometric mode</p>
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</td>
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<td style="width:308px;">
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<p>Abs</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Response</p>
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</td>
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<td style="width:308px;">
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<p>Medium</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>UV/Vis bandwith</p>
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</td>
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<td style="width:308px;">
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<p>1.0 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Scan Speed</p>
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</td>
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<td style="width:308px;">
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<p>40 nm/min</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Start</p>
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</td>
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<td style="width:308px;">
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<p>340 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>End</p>
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</td>
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<td style="width:308px;">
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<p>190 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Data interval</p>
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</td>
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<td style="width:308px;">
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<p>0.5 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Vertical size</p>
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</td>
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<td style="width:308px;">
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<p>Auto</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Scan mode</p>
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</td>
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<td style="width:308px;">
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<p>Continuos</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Accamulation/cycle</p>
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</td>
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<td style="width:308px;">
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<p>Accamulation</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>No. Of cycles</p>
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</td>
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<td style="width:308px;">
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<p>2</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Temperatuur</p>
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</td>
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<td style="width:308px;">
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<p>20 ˚C</p>
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</td>
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</tr>
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</tbody>
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</table>
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<p><br />
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These measurements showed that 1.8 mL is sufficient to measure absorption.</p>
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<p>Next, a stock-solution of DBCO-PEG (10 kDa) is made (142.9 &micro;M):</p>
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4.5 mL phosphate buffer</p>
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 6.43 mg DBCO-PEG (10 kDa)</p>
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<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>
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<table border="1" cellpadding="0" cellspacing="0">
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<tbody>
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<tr>
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<td style="width:308px;">
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<p>Photometric mode</p>
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</td>
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<td style="width:308px;">
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<p>Abs</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Response</p>
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</td>
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<td style="width:308px;">
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<p>Medium</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>UV/Vis bandwith</p>
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</td>
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<td style="width:308px;">
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<p>1.0 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Scan Speed</p>
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</td>
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<td style="width:308px;">
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<p>40 nm/min</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Start</p>
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</td>
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<td style="width:308px;">
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<p>340 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>End</p>
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</td>
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<td style="width:308px;">
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<p>220 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Data interval</p>
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</td>
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<td style="width:308px;">
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<p>0.5 nm</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Vertical size</p>
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</td>
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<td style="width:308px;">
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<p>Auto</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Scan mode</p>
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</td>
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<td style="width:308px;">
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<p>Continuos</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Accamulation/cycle</p>
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</td>
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<td style="width:308px;">
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<p>Accamulation</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>No. Of cycles</p>
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</td>
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<td style="width:308px;">
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<p>2</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Temperatuur</p>
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</td>
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<td style="width:308px;">
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<p>37 ˚C</p>
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</td>
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</tr>
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<tr>
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<td style="width:308px;">
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<p>Stirrer</p>
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</td>
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<td style="width:308px;">
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<p>150 rpm</p>
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</td>
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</tr>
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</tbody>
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</table>
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<p>&nbsp;</p>
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<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>
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</p>
 
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  </br>
 
  <h2>July 7th - July 13th</h2>
  <h2>July 7th - July 13th</h2>
  <p>Deze week hebben we ook hele leuke dingen gedaan.</p>
  <p>Deze week hebben we ook hele leuke dingen gedaan.</p>

Revision as of 08:17, 16 July 2014

TU Eindhoven iGEM Team Notebook

Notebook

Hallo hallo

February

March

April

May

June

July

July 7th - July 13th

Deze week hebben we ook hele leuke dingen gedaan.

August

September

October

Created by iGEM Team TU Eindhoven 2014

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