Team:Marburg/Notebook:June

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{{:Team:Marburg/Template:ContentTitleNav}}
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Notebook
+
Notebook: June <html><a href="https://2013.igem.org/Team:Marburg/Notebook:July"><img src="https://static.igem.org/mediawiki/2013/7/71/Mr-igem-next-arrow.png" style="float:right;margin-left:5px !important;" alt="Next"></a>&nbsp;<a href="https://2013.igem.org/Team:Marburg/Notebook:Mai"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html>
-
{{:Team:Marburg/Template:ContentStart}}
+
{{:Team:Marburg/Template:ContentStartNav}}<html>
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="03-06-2013">03.06.2013</a>
 +
</h2>
 +
 
 +
<!-- Digest -->
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector &rarr; Digestion of combined 2 BioBricks.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>10 µl DNA template</li>
 +
<li>0,5 µl enzyme 1</li>
 +
<li>0,5 µl enzyme 2</li>
 +
<li>2 µl Cut Smart buffer</li>
 +
<li>7 µl bidest. H2O</li>
 +
</ul>
 +
<p>Samples:</p>
 +
<p>Digestion of pSB1A3 iBB1+5 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 +
<p>Digestion of pSB1A3 iBB6+3 with <i>EcoR</i>I-HF and <i>Spe</i>I-HF.</p>
 +
<p>The samples were incubated for 1 h at 37 °C.</p>
 +
<p>They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Ligation -->
 +
<fieldset class="experiment ligation">
 +
    <legend><a name="lig">Ligation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector &rarr; Ligation of two combined 2 BioBricks in pSB1C3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="lig">
 +
<li>45 ng vector DNA (pSB1C3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>140 ng iBB6+3 (<i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>150 ng iBB1+5 (<i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>2 µl 10x T4 DNA ligase buffer</li>
 +
<li>1 µl T4 DNA ligase</li>
 +
<li>ad 20 µl bidest. H2O</li>
 +
</ul>
 +
<p>The samples were incubated for 1 h at RT.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Transformation-->
 +
<fieldset class="experiment transformation">
 +
    <legend>Transformation</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector &rarr; Transformation of combined 4 BioBricks in pSB1C3 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Complete ligation samples were transformed into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB<sub>CM</sub>, overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Ligation -->
 +
<fieldset class="experiment ligation">
 +
    <legend><a name="lig">Ligation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of BioBricks &rarr; Ligation of single BioBricks into pSB1C3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="lig">
 +
<li>45 ng vector DNA (pSB1C3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>X ng insert</li>
 +
<li>1 µl 10x T4 DNA ligase buffer</li>
 +
<li>1 µl T4 DNA ligase</li>
 +
<li>ad 10 µl bidest. H2O</li>
 +
</ul>
 +
<p>Samples (all <i>EcoR</i>I/<i>Pst</i>I-cut):</p>
 +
<p>120 ng eGFP (iBB9)</p>
 +
<p>60 ng SPTP1 (iBB10)</p>
 +
<p>50 ng SPTP2 (iBB11)</p>
 +
<p>55 ng SP1 (iBB12)</p>
 +
<p>50 ng SP2 (iBB13)</p>
 +
<p>The samples were incubated for 3 h at RT.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Transformation-->
 +
<fieldset class="experiment transformation">
 +
    <legend>Transformation</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of BioBricks &rarr; Transformation of single BioBricks in pSB1C3 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Complete ligation samples were transformed into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB<sub>CM</sub>, overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Inoculation -->
 +
<fieldset class="experiment inoculation">
 +
    <legend><a name="ino">Inoculation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Inoculation of transformants for miniprep.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>3 colonies of pSB1A3 iBB1+6 inoculated in 5 ml LB<sub>amp</sub>, 4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LB<sub>CM</sub>, overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
-
<html>
 
<div class="notebooky-entry">
<div class="notebooky-entry">
<h2 class="title">
<h2 class="title">
-
<a name="25-06-2013">25.06.2013</a>
+
<a name="04-06-2013">04.06.2013</a>
</h2>
</h2>
 +
<!-- Miniprep -->
 +
<fieldset class="experiment miniprep">
 +
    <legend><a name="min">Miniprep</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Miniprep of pSB1A3 iBB1+6 and pSB1C3 iBB4864.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O. 2 Cultures of pSB1C3 iBB4864 discarded (red colour).</p>
 +
</div>
 +
</fieldset>
 +
 +
<!-- Digest -->
<fieldset class="experiment digest">
<fieldset class="experiment digest">
     <legend><a name="dig">Digest</a></legend>
     <legend><a name="dig">Digest</a></legend>
     <div class="investigator">
     <div class="investigator">
<span class="inv">Investigator:</span>
<span class="inv">Investigator:</span>
-
<span class="inv-names">Dominik</span>
+
<span class="inv-names">Franzi</span>
</div>
</div>
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with <i>Eco</i>RI and <i>Pst</i>I</span>
+
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Control digestion of pSB1A3 iBB1+6 and pSB1C3 iBB4864.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p><ul class="digest">
+
<ul class="digest">
-
<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
+
<li>1 µl DNA template</li>
-
<li>0.3 µl EcoRI</li>
+
<li>0,5 µl <i>EcoR</i>I-HF</li>
-
<li>0.3 µl PstI</li>
+
<li>0,5 µl <i>Pst</i>I-HF</li>
-
<li>1 µl CutSmart</li>
+
<li>1 µl Cut Smart buffer</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 2 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 43: Line 193:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/5/5f/Gel_2013-06-04.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 50: Line 200:
<ul class="gel-sub">
<ul class="gel-sub">
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
-
<li>10 µl RedSafe in 50 ml gel</li>
+
<li>10 µl RedSafe per 50 ml gel</li>
-
<li>x µl Hyper Ladder</li>
+
<li>6 µl 2-Log DNA Ladder (NEB)</li>
 +
<li>6x loading buffer (for samples)</li>
</ul>
</ul>
</p>
</p>
<p>
<p>
-
<span class="exp">Expactations</span>
+
<table class="gel">
-
<ul class="exp">
+
<colgroup>
-
<li>Lane 1: 5300 kbp</li>
+
<col width="10%" />
-
<li>Lane 2: 3300 kbp</li>
+
<col width="2%" />
-
<li>Lane 3: 1300 kbp</li>
+
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2+3</td>
 +
<th>&nbsp;</th>
 +
<td>iBB4864 K1-2</td>
 +
<th>&nbsp;</th>
 +
<td>2580 bp</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<th>&nbsp;</th>
 +
<td>empty</td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
<tr>
 +
<td>5-7</td>
 +
<th>&nbsp;</th>
 +
<td>iBB16 K3-5</td>
 +
<th>&nbsp;</th>
 +
<td>650 bp</td>
 +
</tr>
 +
</table>
 +
</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Digest -->
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Digestion of combined 2 BioBricks in pSB1A3 and combined 4 BioBricks in pSB1C3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>10 µl DNA template</li>
 +
<li>0,5 µl enzyme 1</li>
 +
<li>0,5 µl enzyme 2</li>
 +
<li>2 µl Cut Smart buffer</li>
 +
<li>7 µl bidest. H2O</li>
 +
</ul>
 +
<p>Samples:</p>
 +
<p>Digestion of pSB1C3 iBB7631 K4 and pSB1A3 iBB16 K4 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 +
<p>Digestion of pSB1C3 iBB4864 K1 and pSB1A3 iBB54 K2 with <i>EcoR</i>I-HF and <i>Spe</i>I-HF.</p>
 +
<p>The samples were incubated for 1 h at 37 °C.</p>
 +
<p>They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Ligation -->
 +
<fieldset class="experiment ligation">
 +
    <legend><a name="lig">Ligation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Ligation of two combined 2 BioBricks into pSB1C3 and two combined 4 BioBricks into pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Antibody:</p>
 +
<ul class="lig">
 +
<li>80 ng vector DNA (pSB1A3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>160 ng iBB7631</li>
 +
<li>190 ng iBB4864 </li>
 +
<li>1 µl 10x T4 DNA ligase buffer</li>
 +
<li>1 µl T4 DNA ligase</li>
 +
<li>ad 10 µl bidest. H2O</li>
 +
</ul>
 +
<p>Test-vector:</p>
 +
<ul class="lig">
 +
<li>45 ng vector DNA (pSB1C3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>150 ng iBB54</li>
 +
<li>120 ng iBB16</li>
 +
<li>1 µl 10x T4 DNA ligase buffer</li>
 +
<li>1 µl T4 DNA ligase</li>
 +
<li>ad 10 µl bidest. H2O</li>
 +
</ul>
 +
<p>Both samples were incubated overnight at 16 °C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Inoculation -->
 +
<fieldset class="experiment inoculation">
 +
    <legend><a name="ino">Inoculation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector and BioBricks &rarr; Inoculation of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Transformations (03.06.13) inoculated in 5 ml LB<sub>CM</sub>, overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="05-06-2013">05.06.2013</a>
 +
</h2>
 +
 
 +
<!-- Miniprep -->
 +
<fieldset class="experiment miniprep">
 +
    <legend><a name="min">Miniprep</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector and BioBricks &rarr; Miniprep of single BioBricks and pSB1C3 iBB6315.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Digest -->
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector and BioBricks &rarr; Control digestion of single BioBricks and pSB1C3 iBB6315.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA template</li>
 +
<li>0,3 µl <i>EcoR</i>I-HF</li>
 +
<li>0,3 µl <i>Pst</i>I-HF</li>
 +
<li>1 µl Cut Smart buffer</li>
 +
<li>7,4 µl bidest. H2O</li>
 +
</ul>
 +
<p>The samples were incubated for 1,5 h at 37 °C.</p>
 +
<table class="gel digest">
 +
<colgroup>
 +
<col width="50%" />
 +
<col width="50%" />
 +
</colgroup>
 +
<thead>
 +
<tr>
 +
<th colspan="2" class="title">Gel electrophoresis</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/f/fd/Gel_2013-06-05_1.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<span class="gel-elc">Gel substances</span>
 +
<ul class="gel-sub">
 +
<li>1% Agarose gel</li>
 +
<li>10 µl RedSafe per 50 ml gel</li>
 +
<li>6 µl 2-Log DNA Ladder (NEB)</li>
 +
<li>6x loading buffer (for samples)</li>
</ul>
</ul>
</p>
</p>
-
<p>Worked as expected.</p>
+
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2-4</td>
 +
<th>&nbsp;</th>
 +
<td>iBB9 K1-3</td>
 +
<th>&nbsp;</th>
 +
<td>780 bp</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<th>&nbsp;</th>
 +
<td>empty</td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>6-8</td>
 +
<th>&nbsp;</th>
 +
<td>iBB10 K1-3</td>
 +
<th>&nbsp;</th>
 +
<td>228 bp</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td>pSB1C3: 2 kb</td>
 +
</tr>
 +
</table>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/f/f2/Gel_2013-06-05_2.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2-4</td>
 +
<th>&nbsp;</th>
 +
<td>iBB11 K1-3</td>
 +
<th>&nbsp;</th>
 +
<td>147 bp</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<th>&nbsp;</th>
 +
<td>empty</td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>6-8</td>
 +
<th>&nbsp;</th>
 +
<td>iBB12 K1-3</td>
 +
<th>&nbsp;</th>
 +
<td>123 bp</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td>pSB1C3: 2 kb</td>
 +
</tr>
 +
</table>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/5/5a/Gel_2013-06-05_3.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2-4</td>
 +
<th>&nbsp;</th>
 +
<td>iBB13 K1-3</td>
 +
<th>&nbsp;</th>
 +
<td>108 bp</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<th>&nbsp;</th>
 +
<td>empty</td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>6-8</td>
 +
<th>&nbsp;</th>
 +
<td>iBB6315 K1-3</td>
 +
<th>&nbsp;</th>
 +
<td>1200 bp</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td>pSB1C3: 2 kb</td>
 +
</tr>
 +
</table>
 +
</p>
</td>
</td>
</tr>
</tr>
Line 69: Line 554:
</div>
</div>
</fieldset>
</fieldset>
 +
 +
<!-- Transformation-->
 +
<fieldset class="experiment transformation">
 +
    <legend>Transformation</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Transformation of combined 4 BioBricks in pSB1C3 and combined 8 BioBricks in pSB1A3 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Ligations (04.06.13) were transformed into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 60 min (1C3)/40 min (1A3) 37 °C + 900 µl LB), plated on LB<sub>CM</sub> (1C3)/LB<sub>amp</sub> (1A3), overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 +
<!-- Sequencing -->
 +
<fieldset class="experiment sequencing">
 +
    <legend>Sequencing</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of BioBricks &rarr; Examination of sequencing results of various constructs.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>iBB9 K1 fw - AGB000K 263</p>
 +
<p>iBB9 K1 rv - AGB000K 264</p>
 +
<p>iBB9 K2 fw - AGB000K 265</p>
 +
<p>iBB9 K2 rv - AGB000K 266</p>
 +
<p>iBB10 K1 fw - AGB000K 267</p>
 +
<p>iBB10 K2 fw - AGB000K 268</p>
 +
<p>iBB11 K1 fw - AGB000K 269</p>
 +
<p>iBB11 K2 fw - AGB000K 270</p>
 +
<p>iBB12 K1 fw - AGB000K 271</p>
 +
<p>iBB12 K2 fw - AGB000K 272</p>
 +
<p>iBB13 K1 fw - AGB000K 273</p>
 +
<p>iBB13 K2 fw - AGB000K 274</p>
 +
<p> </p>
 +
<p>all correct, except iBB10 G&rarr;T pos.30, iBB11 T&rarr;C pos.24. Probably template mutated.</p>
 +
</div>
 +
</fieldset>
 +
</div>
</div>
 +
<div class="notebooky-entry">
<div class="notebooky-entry">
<h2 class="title">
<h2 class="title">
-
<a name="26-06-2013">26.06.2013</a>
+
<a name="06-06-2013">06.06.2013</a>
</h2>
</h2>
 +
<!-- Inoculation -->
 +
<fieldset class="experiment inoculation">
 +
    <legend><a name="ino">Inoculation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Inoculation of pSB1C3 iBB5416 and pSB1A3 iBB48647631 for miniprep.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>3 colonies of pSB1C3 iBB5416 inoculated in 5 ml LB<sub>CM</sub>, 4 colonies of pSB1A3 iBB48647631 inoculated in 5 ml LB<sub>amp</sub>, overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="07-06-2013">07.06.2013</a>
 +
</h2>
 +
 +
<!-- Miniprep -->
 +
<fieldset class="experiment miniprep">
 +
    <legend><a name="min">Miniprep</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Patrick</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Miniprep of pSB1C3 iBB5416 and pSB1A3 iBB48647631.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.</p>
 +
</div>
 +
</fieldset>
 +
 +
<!-- Digest -->
<fieldset class="experiment digest">
<fieldset class="experiment digest">
     <legend><a name="dig">Digest</a></legend>
     <legend><a name="dig">Digest</a></legend>
     <div class="investigator">
     <div class="investigator">
<span class="inv">Investigator:</span>
<span class="inv">Investigator:</span>
-
<span class="inv-names">Dominik</span>
+
<span class="inv-names">Patrick</span>
</div>
</div>
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Construction of the plasmids pSB1A3-iBB10+9, pSB1A3-iBB11+9, pSB1A3-iBB12+9 and pSB1A3-iBB13+9</span>
+
<span class="aim-desc">Construction of P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Control digestion of pSB1C3 iBB5416 and pSB1A3 iBB48647631.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p><ul class="digest">
+
<ul class="digest">
-
<li>1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)</li>
+
<li>1 µl DNA template</li>
-
<li>0.5 µl EcoRI</li>
+
<li>0,3 µl <i>EcoR</i>I-HF</li>
-
<li>0.5 µl SpeI</li>
+
<li>0,3 µl <i>Pst</i>I-HF</li>
-
<li>2 µl CutSmart</li>
+
<li>1 µl Cut Smart buffer</li>
-
<li>12 µl ddH2O</li>
+
<li>7,4 µl bidest. H2O</li>
</ul>
</ul>
-
+
<p>The samples were incubated for 1 h at 37 °C.</p>
-
<p><ul class="digest">
+
<table class="gel digest">
-
<li>1 µl Plasmid (pSB1C3-iBB9)</li>
+
<colgroup>
-
<li>0.5 µl XbaI</li>
+
<col width="50%" />
-
<li>0.5 µl PstI</li>
+
<col width="50%" />
-
<li>2 µl CutSmart</li>
+
</colgroup>
-
<li>12 µl ddH2O</li>
+
<thead>
 +
<tr>
 +
<th colspan="2" class="title">Gel electrophoresis</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/8/8b/Gel_2013-06-07.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<span class="gel-elc">Gel substances</span>
 +
<ul class="gel-sub">
 +
<li>1% Agarose gel</li>
 +
<li>10 µl RedSafe per 50 ml gel</li>
 +
<li>6 µl 2-Log DNA Ladder (NEB)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2-4</td>
 +
<th>&nbsp;</th>
 +
<td>iBB5416 K1-3</td>
 +
<th>&nbsp;</th>
 +
<td>
 +
<p>pSB1C3: 2070 bp</p>
 +
<p>iBB5416: 1500 bp</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>5-8</td>
 +
<th>&nbsp;</th>
 +
<td>iBB48647631 K1-4</td>
 +
<th>&nbsp;</th>
 +
<td>
 +
<p>pSB1A3: 2150 bp</p>
 +
<p>iBB48647631: 4157 bp</p>
 +
</td>
 +
</tr>
 +
</table>
 +
</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="10-06-2013">10.06.2013</a>
 +
</h2>
 +
 
 +
<!-- Digest -->
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Lukas</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector, P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Digestion of pSB1A3 iBB48647631 and single BioBricks.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>X µl DNA template</li>
 +
<li>0,5 µl enzyme 1</li>
 +
<li>0,5 µl enzyme 2</li>
 +
<li>2,2 µl Cut Smart buffer</li>
 +
<li>20 µl bidest. H2O</li>
</ul>
</ul>
-
+
<p>Samples:</p>
-
<p><ul class="digest">
+
<p>Digestion of 1,5 µl pSB1A3 iBB48647631 with <i>Spe</i>I-HF and <i>Pst</i>I-HF</p>
-
<li>1 µl Plasmid (pSB1A3)</li>
+
<p>Digestion of 5 µl pSB1C3 iBB5 and iBB9 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
-
<li>0.5 µl EcoRI</li>
+
<p>Digestion of 4 µl pSB1C3 iBB3 and iBB4 with <i>EcoR</i>I-HF and <i>Spe</i>I-HF.</p>
-
<li>0.5 µl PstI</li>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
-
<li>2 µl CutSmart</li>
+
<p>They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.</p>
-
<li>12 µl ddH2O</li>
+
</div>
 +
</fieldset>
 +
 
 +
<!-- Ligation -->
 +
<fieldset class="experiment ligation">
 +
    <legend><a name="lig">Ligation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Christian</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector, P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Ligation of pSB1A3 iBB48647631 and single BioBricks in pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Ligation of pSB1A3 iBB48647631 with iBB5, pSB1A3 iBB48647631 with iBB3 and iBB9, pSB1A3 iBB48647631 with iBB4 and iBB9; overnight at 16°C</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="11-06-2013">11.06.2013</a>
 +
</h2>
 +
 
 +
<!-- Transformation-->
 +
<fieldset class="experiment transformation">
 +
    <legend>Transformation</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector, P<sub>fcpB</sub>-test-vector and antibody-vector %rarr; Transformation of ligations.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Transformation of the ligations into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 25 min 37 °C + 900 µl LB), plated on LB<sub>amp</sub>, overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="12-06-2013">12.06.2013</a>
 +
</h2>
 +
 
 +
<!-- Inoculation -->
 +
<fieldset class="experiment inoculation">
 +
    <legend><a name="ino">Inoculation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector, P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Inoculation of transformants for miniprep.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>4 colonies of each transformation inoculated in 4 ml LB<sub>amp</sub>, overnight at 37 °C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="13-06-2013">13.06.2013</a>
 +
</h2>
 +
 
 +
<!-- Miniprep -->
 +
<fieldset class="experiment miniprep">
 +
    <legend><a name="min">Miniprep</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector, P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Miniprep of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Digest -->
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector, P<sub>fcpB</sub>-test-vector and antibody-vector &rarr; Control digestion the minipreps and pSB1A3 iBB48647631 K3 (comparison of size).</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA template</li>
 +
<li>0,3 µl <i>EcoR</i>I-HF</li>
 +
<li>0,3 µl <i>Pst</i>I-HF</li>
 +
<li>1 µl Cut Smart buffer</li>
 +
<li>7,4 µl bidest. H2O</li>
</ul>
</ul>
-
+
<p>The samples were incubated for 1 h at 37 °C.</p>
-
<p>The samples were incubated for 1.5 h at 37° C.</p>
+
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 125: Line 895:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/2/22/Gel_2013-06-13_1.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 132: Line 902:
<ul class="gel-sub">
<ul class="gel-sub">
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
-
<li>10 µl RedSafe in 50 ml gel</li>
+
<li>10 µl RedSafe per 50 ml gel</li>
-
<li>x µl Hyper Ladder</li>
+
<li>6 µl 2-Log DNA Ladder (NEB)</li>
 +
<li>6x loading buffer (for samples)</li>
</ul>
</ul>
</p>
</p>
<p>
<p>
-
<span class="exp">Expactations</span>
+
<table class="gel">
-
<ul class="exp">
+
<colgroup>
-
<li>Lane 1: 5300 kbp</li>
+
<col width="10%" />
-
<li>Lane 2: 3300 kbp</li>
+
<col width="2%" />
-
<li>Lane 3: 1300 kbp</li>
+
<col width="50%" />
-
</ul>
+
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>upper:</td>
 +
</tr>
 +
<tr>
 +
<td>2+3</td>
 +
<th>&nbsp;</th>
 +
<td>iBB49 K3-4</td>
 +
<th>&nbsp;</th>
 +
<td>1260 bp</td>
 +
</tr>
 +
<tr>
 +
<td>4-7</td>
 +
<th>&nbsp;</th>
 +
<td>iBB39 K1-4</td>
 +
<th>&nbsp;</th>
 +
<td>1030 bp</td>
 +
</tr>
 +
<tr>
 +
<td>lower:</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>iBB49647631</td>
 +
<th>&nbsp;</th>
 +
<td>4157 bp</td>
 +
</tr>
 +
<tr>
 +
<td>3-6</td>
 +
<th>&nbsp;</th>
 +
<td>iBB496476315 K1-4</td>
 +
<th>&nbsp;</th>
 +
<td>4404 bp</td>
 +
</tr>
 +
<tr>
 +
<td>7+8</td>
 +
<th>&nbsp;</th>
 +
<td>iBB49 K1-2</td>
 +
<th>&nbsp;</th>
 +
<td>1260 bp</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td>Vector pSB1A3: 2150 bp</td>
 +
</tr>
 +
</table>
</p>
</p>
-
<p>All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).</p>
+
<p>IBB48647631 and iBB486476315 probably positive, but better repeat or do a Colony-PCR.</p>
</td>
</td>
</tr>
</tr>
Line 151: Line 980:
</div>
</div>
</fieldset>
</fieldset>
 +
</div>
</div>
 +
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="16-06-2013">16.06.2013</a>
 +
</h2>
 +
 +
<!-- Digest -->
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Lukas</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of antibody-vector &rarr; Repeat of control digestion.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA template</li>
 +
<li>0,3 µl <i>EcoR</i>I-HF</li>
 +
<li>0,3 µl <i>Pst</i>I-HF</li>
 +
<li>1 µl Cut Smart buffer</li>
 +
<li>7,4 µl bidest. H2O</li>
 +
</ul>
 +
<p>Templates: PSB1A3 iBB486476315 and pSB1A3 iBB48647631 K3 (comparison of size).</p>
 +
<p>The samples were incubated for 1 h at 37 °C.</p>
 +
<table class="gel digest">
 +
<colgroup>
 +
<col width="50%" />
 +
<col width="50%" />
 +
</colgroup>
 +
<thead>
 +
<tr>
 +
<th colspan="2" class="title">Gel electrophoresis</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/3/38/Gel_2013-06-16.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<span class="gel-elc">Gel substances</span>
 +
<ul class="gel-sub">
 +
<li>1% Agarose gel</li>
 +
<li>10 µl RedSafe per 50 ml gel</li>
 +
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2+3</td>
 +
<th>&nbsp;</th>
 +
<td>iBB48647631</td>
 +
<th>&nbsp;</th>
 +
<td>4157 bp</td>
 +
</tr>
 +
<tr>
 +
<td>4-7</td>
 +
<th>&nbsp;</th>
 +
<td>iBB486476315</td>
 +
<th>&nbsp;</th>
 +
<td>TBA bp</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td></td>
 +
<th>&nbsp;</th>
 +
<td>Vector pSB1A3: 2150 bp</td>
 +
</tr>
 +
</table>
 +
</p>
 +
<p>Too much DNA used, will be repeated as PCR.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="17-06-2013">17.06.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Alex</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Digest of pSB1A3_3+9 and pSB1A3_4+9 with <i>Spe</i>I and <i>Pst</i>I, and corresponding inserts pSB1C3_5+4+1+6 and pSB1C3_6+3+1+5 with <i>EcoR</i>I and <i>Pst</i>I.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA</li>
 +
<li>0.3 µl Enzyme 1 (<i>Spe</i>I or <i>EcoR</i>I, resp.)</li>
 +
<li>0.3 µl Enzyme 2 (<i>Pst</i>I)</li>
 +
<li>1 µl Cut Smart buffer</li>
 +
<li>7.4 µl ddbidest. H2O</li>
 +
</ul>
 +
<p>The samples were incubated for 1.5 h at 37 °C.</p>
 +
<p>Approaches were unsewed by means of gel electrophoresis, corresponding bands were picked under UV, DNA extraction from gel was performed using DNA Gel Extraction Kit (<i>QIAGEN</i>).</p>
 +
</fieldset>
 +
 +
<fieldset class="experiment ligation">
 +
    <legend><a name="lig">Ligation, Transformation, Plasmid prep</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Alex</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Assemble the combined bricks 5416 and 6315 into their appropriate vectors pSB1A3_39 and pSB1A3_49, resp.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="lig">
 +
<li>Insert und plasmid were transformed using a 1:2 ratio. Samples were incubated ON at RT.</li>
 +
<li>Ligation product were transformed into chemical competent DH5α cells (30 min on ice, 90 sec 42 °C, 45 min 37 °C + 900 µl LB).</li>
 +
<li>Transformed cells were spread out on LB Amp culture plates.</li>
 +
<li>Incubation ON at RT.</li>
 +
                        <li>4 clones were picked for overnight at cultures.</li>
 +
                        <li>A plasmid preparation was carried out the next day.</li>
 +
</ul>
 +
</div>
 +
</fieldset>
 +
</div>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="22-06-2013">22.06.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Test digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Alex</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Digest of pSB1A3_395416 and pSB1A3_496315 with <i>EcoR</i>I and <i>Pst</i>I.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl Plasmid DNA</li>
 +
<li>0.3 µl EcoRI</li>
 +
<li>0.3 µL PstI</li>
 +
<li>1 µl Cut Smart</li>
 +
<li>7.4 µl ddbidest. H2O</li>
 +
</ul>
 +
<p>The samples were incubated for 2 h at 37 °C.</p>
 +
<table class="gel digest">
 +
<colgroup>
 +
<col width="50%" />
 +
<col width="50%" />
 +
</colgroup>
 +
<thead>
 +
<tr>
 +
<th colspan="2" class="title">Gel electrophoresis</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/5/53/Gel_2013-06-17_2.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<span class="gel-elc">Gel substances</span>
 +
<ul class="gel-sub">
 +
<li>1% Agarose gel</li>
 +
<li>10 µl Ethidium bromide 50 ml gel</li>
 +
<li>4 µl 2-log DNA ladder (New England Biolabs)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>upper:</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>iBB6315</td>
 +
<th>&nbsp;</th>
 +
<td>1200 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<th>&nbsp;</th>
 +
<td>iBB49</td>
 +
<th>&nbsp;</th>
 +
<td>1200 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<tr>
 +
<td>4-7</td>
 +
<th>&nbsp;</th>
 +
<td>iBB496315 K4-1</td>
 +
<th>&nbsp;</th>
 +
<td>2400 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<td>lower:</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>iBB5416</td>
 +
<th>&nbsp;</th>
 +
<td>1500 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<th>&nbsp;</th>
 +
<td>iBB39</td>
 +
<th>&nbsp;</th>
 +
<td>1000 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<td>4-7</td>
 +
<th>&nbsp;</th>
 +
<td>iBB395416 K1-4</td>
 +
<th>&nbsp;</th>
 +
<td>2400 + 2000 bp</td>
 +
</tr>
 +
</table>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2" class="gel-fazit">
 +
<p>We didn't receive all expected fragments. All expected fragments in lanes 4-7 were missing. Ligation failed &#x2639;</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="24-06-2013">24.06.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment pcr">
 +
    <legend><a name="pcr">PCR</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Alex</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Check right assembly of BioBrick BBa_K1071005 in the plasmid for antibody production</span>
 +
</div>
 +
<div class="exp-content">
 +
<table class="pcr">
 +
<colgroup>
 +
<col width="15%" />
 +
<col width="30%" />
 +
<col width="5%" />
 +
<col width="20%" />
 +
<col width="20%" />
 +
<col width="10%" />
 +
</colgroup>
 +
<thead>
 +
<th>Volume</th>
 +
<th>Reagent</th>
 +
<th>&nbsp;</th>
 +
<th>Temp (°C)</th>
 +
<th colspan="2">Time</th>
 +
</thead>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>pSB1A3_486476315</td>
 +
<td>&nbsp;</td>
 +
<td>95</td>
 +
<td colspan="2">3 min</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 µl</td>
 +
<td>Primer fwd</td>
 +
<td>&nbsp;</td>
 +
<td>95</td>
 +
<td colspan="2">30 sec</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 µl</td>
 +
<td>Primer rev</td>
 +
<td>&nbsp;</td>
 +
<td>60</td>
 +
<td>30 sec</td>
 +
<td rowspan="3">x30</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 µl</td>
 +
<td>dNTPs</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>42 sec</td>
 +
</tr>
 +
<tr>
 +
<td>0.5 µl</td>
 +
<td>Phusion polymerase</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>5 min</td>
 +
</tr>
 +
<tr>
 +
<td>5 µl</td>
 +
<td>Phusion buffer 5x</td>
 +
<td>&nbsp;</td>
 +
<td>4</td>
 +
<td colspan="2"><span class="hold">Hold</span></td>
 +
</tr>
 +
                        <tr>
 +
<td>add 25 µL</td>
 +
<td>ddbidest. H2O</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
</table>
 +
<br />
 +
<!-- Das Gel-Bild fast gleich wie beim Verdau -->
 +
<table class="gel pcr">
 +
<colgroup>
 +
<col width="50%" />
 +
<col width="50%" />
 +
</colgroup>
 +
<thead>
 +
<tr>
 +
<th colspan="2" class="title">Gel electrophoresis</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/5/58/Gel_2013-06-24.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<span class="gel-elc">Gel substances</span>
 +
<ul class="gel-sub">
 +
<li>1% Agarose gel</li>
 +
<li>10 µl Ethidium bromide 50 ml gel</li>
 +
<li>4 µl 2-log DNA ladder (New England Biolabs)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2-5</td>
 +
<th>&nbsp;</th>
 +
<td>pSB1A3 iBB486476315 K1-4</td>
 +
<th>&nbsp;</th>
 +
<td>
 +
<p>~ 6,000 bp for pSB1A3_486476315</p>
 +
<p>280 bp for BBa_K1071005</p>
 +
</td>
 +
</tr>
 +
</table>
 +
</p>
 +
<p>Each sample contained 2 bands, that means we got all expected bands and it could be assumed that the insertion of BBa_K1071005 was successful.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="25-06-2013">25.06.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with <i>EcoR</i>I and <i>Pst</i>I</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><ul class="digest">
 +
<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
 +
<li>0.3 µl <i>EcoR</i>I</li>
 +
<li>0.3 µl <i>Pst</i>I</li>
 +
<li>1 µl CutSmart</li>
 +
<li>7.4 µl ddbidest. H2O</li>
 +
</ul>
 +
<p>The samples were incubated for 1 h at 37 °C.</p>
 +
<p>Worked as expected.</p>
 +
</div>
 +
</fieldset>
 +
</div>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="26-06-2013">26.06.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of the plasmids pSB1A3-iBB10+9, pSB1A3-iBB11+9, pSB1A3-iBB12+9 and pSB1A3-iBB13+9</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><ul class="digest">
 +
<li>1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)</li>
 +
<li>0.5 µl <i>EcoR</i>I</li>
 +
<li>0.5 µl <i>Spe</i>I</li>
 +
<li>2 µl CutSmart</li>
 +
<li>12 µl ddbidest. H2O</li>
 +
</ul>
 +
 +
<p><ul class="digest">
 +
<li>1 µl Plasmid (pSB1C3-iBB9)</li>
 +
<li>0.5 µl <i>Xba</i>I</li>
 +
<li>0.5 µl <i>Pst/</i>I</li>
 +
<li>2 µl CutSmart</li>
 +
<li>12 µl ddbidest. H2O</li>
 +
</ul>
 +
 +
<p><ul class="digest">
 +
<li>1 µl Plasmid (pSB1A3)</li>
 +
<li>0.5 µl <i>EcoR</i>I</li>
 +
<li>0.5 µl <i>Pst</i>I</li>
 +
<li>2 µl CutSmart</li>
 +
<li>12 µl ddbidest. H2O</li>
 +
</ul>
 +
 +
<p>The samples were incubated for 1.5 h at 37 °C.</p>
 +
<p>All expected fragments were present. The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).</p>
 +
</div>
 +
</fieldset>
 +
<fieldset class="experiment ligation">
<fieldset class="experiment ligation">
     <legend><a name="lig">Ligation</a></legend>
     <legend><a name="lig">Ligation</a></legend>
Line 169: Line 1,500:
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl T4 DNA ligase</li>
<li>2 µl T4 DNA ligase</li>
-
<li>2 µl ddH2O</li>
+
<li>2 µl ddbidest. H2O</li>
</ul>
</ul>
<p>The samples were incubated overnight at room temperature.</p>
<p>The samples were incubated overnight at room temperature.</p>
</div>
</div>
</fieldset>
</fieldset>
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</div>
<div class="notebooky-entry">
<div class="notebooky-entry">
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</div>
<div class="exp-content">
<div class="exp-content">
-
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates. <br />
+
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates.<br />
-
The plates were incubated over night at 37° C.</p>
+
The plates were incubated over night at 37 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
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<fieldset class="experiment inoculation">
 +
    <legend><a name="ino">Inoculation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">inoculation of colonies for plasmid preparation</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>4 colonies of pSB1A3-iBB395146 and 4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.</p>
 +
</div>
 +
</fieldset>
-
{{:Team:Marburg/Template:ContentEnd}}
+
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Alex</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Digest of pSB1A3-iBB395416 and pSB1A3-iBB496315 with <i>EcoR</i>I and <i>Pst</i>I.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl Plasmid DNA</li>
 +
<li>0.3 µl <i>EcoR</i>I</li>
 +
<li>0.3 µl <i>Pst</i>I</li>
 +
<li>1 µl Cut Smart</li>
 +
<li>7.4 µl ddbidest. H2O</li>
 +
</ul>
 +
<p>The samples were incubated for 2 h at 37 °C.</p>
 +
<table class="gel digest">
 +
<colgroup>
 +
<col width="50%" />
 +
<col width="50%" />
 +
</colgroup>
 +
<thead>
 +
<tr>
 +
<th colspan="2" class="title">Gel electrophoresis</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/5/52/Gel_2013-06-27.png" width="100%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<span class="gel-elc">Gel substances</span>
 +
<ul class="gel-sub">
 +
<li>1% Agarose gel</li>
 +
<li>10 µl Ethidium bromide in 50 ml gel</li>
 +
<li>4 µl 2-log DNA ladder (New England Biolabs)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>upper:</td>
 +
</tr>
 +
<tr>
 +
<td>2-5</td>
 +
<th>&nbsp;</th>
 +
<td>iBB395416 K1-4</td>
 +
<th>&nbsp;</th>
 +
<td>2400 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<th>&nbsp;</th>
 +
<td>iBB39</td>
 +
<th>&nbsp;</th>
 +
<td>1000 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<tr>
 +
<td>7</td>
 +
<th>&nbsp;</th>
 +
<td>iBB6416</td>
 +
<th>&nbsp;</th>
 +
<td>1400 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<td>lower:</td>
 +
</tr>
 +
<tr>
 +
<td>2-6</td>
 +
<th>&nbsp;</th>
 +
<td>iBB496315 K1-5</td>
 +
<th>&nbsp;</th>
 +
<td>2400 + 2000 bp</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<th>&nbsp;</th>
 +
<td>iBB49</td>
 +
<th>&nbsp;</th>
 +
<td>1200 + 2000 bp</td>
 +
</tr>
 +
</table>
 +
</p>
 +
<p>All clones show a double band above 2000 bp.</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2" class="gel-fazit">
 +
<p>We received all expected fragments. The success of the ligation should be double-checked by means of PCR.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
</div>
 +
 
 +
</html>
 +
{{:Team:Marburg/Template:ContentEndNav}}
{{:Team:Marburg/Template:Footer}}
{{:Team:Marburg/Template:Footer}}

Latest revision as of 11:14, 27 October 2013

Notebook: June Next Previous

03.06.2013

Digest
Investigator: Franzi
Aim: Construction of PNR-test-vector → Digestion of combined 2 BioBricks.
  • 10 µl DNA template
  • 0,5 µl enzyme 1
  • 0,5 µl enzyme 2
  • 2 µl Cut Smart buffer
  • 7 µl bidest. H2O

Samples:

Digestion of pSB1A3 iBB1+5 with XbaI and PstI-HF

Digestion of pSB1A3 iBB6+3 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.

Ligation
Investigator: Franzi
Aim: Construction of PNR-test-vector → Ligation of two combined 2 BioBricks in pSB1C3.
  • 45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 140 ng iBB6+3 (EcoRI/PstI-cut)
  • 150 ng iBB1+5 (EcoRI/PstI-cut)
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated for 1 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of PNR-test-vector → Transformation of combined 4 BioBricks in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

Ligation
Investigator: Franzi
Aim: Construction of BioBricks → Ligation of single BioBricks into pSB1C3.
  • 45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • X ng insert
  • 1 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 10 µl bidest. H2O

Samples (all EcoRI/PstI-cut):

120 ng eGFP (iBB9)

60 ng SPTP1 (iBB10)

50 ng SPTP2 (iBB11)

55 ng SP1 (iBB12)

50 ng SP2 (iBB13)

The samples were incubated for 3 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of BioBricks → Transformation of single BioBricks in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

Inoculation
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Inoculation of transformants for miniprep.

3 colonies of pSB1A3 iBB1+6 inoculated in 5 ml LBamp, 4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LBCM, overnight at 37 °C.

04.06.2013

Miniprep
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Miniprep of pSB1A3 iBB1+6 and pSB1C3 iBB4864.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O. 2 Cultures of pSB1C3 iBB4864 discarded (red colour).

Digest
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Control digestion of pSB1A3 iBB1+6 and pSB1C3 iBB4864.
  • 1 µl DNA template
  • 0,5 µl EcoRI-HF
  • 0,5 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 7 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl 2-Log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2+3   iBB4864 K1-2   2580 bp
4   empty  
5-7   iBB16 K3-5   650 bp

Digest
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Digestion of combined 2 BioBricks in pSB1A3 and combined 4 BioBricks in pSB1C3.
  • 10 µl DNA template
  • 0,5 µl enzyme 1
  • 0,5 µl enzyme 2
  • 2 µl Cut Smart buffer
  • 7 µl bidest. H2O

Samples:

Digestion of pSB1C3 iBB7631 K4 and pSB1A3 iBB16 K4 with XbaI and PstI-HF

Digestion of pSB1C3 iBB4864 K1 and pSB1A3 iBB54 K2 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.

Ligation
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Ligation of two combined 2 BioBricks into pSB1C3 and two combined 4 BioBricks into pSB1A3.

Antibody:

  • 80 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
  • 160 ng iBB7631
  • 190 ng iBB4864
  • 1 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 10 µl bidest. H2O

Test-vector:

  • 45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 150 ng iBB54
  • 120 ng iBB16
  • 1 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 10 µl bidest. H2O

Both samples were incubated overnight at 16 °C.

Inoculation
Investigator: Franzi
Aim: Construction of PNR-test-vector and BioBricks → Inoculation of transformants.

Transformations (03.06.13) inoculated in 5 ml LBCM, overnight at 37 °C.

05.06.2013

Miniprep
Investigator: Franzi
Aim: Construction of PNR-test-vector and BioBricks → Miniprep of single BioBricks and pSB1C3 iBB6315.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.

Digest
Investigator: Franzi
Aim: Construction of PNR-test-vector and BioBricks → Control digestion of single BioBricks and pSB1C3 iBB6315.
  • 1 µl DNA template
  • 0,3 µl EcoRI-HF
  • 0,3 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 7,4 µl bidest. H2O

The samples were incubated for 1,5 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl 2-Log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2-4   iBB9 K1-3   780 bp
5   empty  
6-8   iBB10 K1-3   228 bp
    pSB1C3: 2 kb

gel-electrophoresis-image

Lane   Content   Expectations
2-4   iBB11 K1-3   147 bp
5   empty  
6-8   iBB12 K1-3   123 bp
    pSB1C3: 2 kb

gel-electrophoresis-image

Lane   Content   Expectations
2-4   iBB13 K1-3   108 bp
5   empty  
6-8   iBB6315 K1-3   1200 bp
    pSB1C3: 2 kb

Transformation
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Transformation of combined 4 BioBricks in pSB1C3 and combined 8 BioBricks in pSB1A3 into E. coli DH5α.

Ligations (04.06.13) were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min (1C3)/40 min (1A3) 37 °C + 900 µl LB), plated on LBCM (1C3)/LBamp (1A3), overnight at 37 °C.

Sequencing
Investigator: Franzi
Aim: Construction of BioBricks → Examination of sequencing results of various constructs.

iBB9 K1 fw - AGB000K 263

iBB9 K1 rv - AGB000K 264

iBB9 K2 fw - AGB000K 265

iBB9 K2 rv - AGB000K 266

iBB10 K1 fw - AGB000K 267

iBB10 K2 fw - AGB000K 268

iBB11 K1 fw - AGB000K 269

iBB11 K2 fw - AGB000K 270

iBB12 K1 fw - AGB000K 271

iBB12 K2 fw - AGB000K 272

iBB13 K1 fw - AGB000K 273

iBB13 K2 fw - AGB000K 274

all correct, except iBB10 G→T pos.30, iBB11 T→C pos.24. Probably template mutated.

06.06.2013

Inoculation
Investigator: Franzi
Aim: Construction of PfcpB-test-vector and antibody-vector → Inoculation of pSB1C3 iBB5416 and pSB1A3 iBB48647631 for miniprep.

3 colonies of pSB1C3 iBB5416 inoculated in 5 ml LBCM, 4 colonies of pSB1A3 iBB48647631 inoculated in 5 ml LBamp, overnight at 37 °C.

07.06.2013

Miniprep
Investigator: Patrick
Aim: Construction of PfcpB-test-vector and antibody-vector → Miniprep of pSB1C3 iBB5416 and pSB1A3 iBB48647631.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.

Digest
Investigator: Patrick
Aim: Construction of PfcpB-test-vector and antibody-vector → Control digestion of pSB1C3 iBB5416 and pSB1A3 iBB48647631.
  • 1 µl DNA template
  • 0,3 µl EcoRI-HF
  • 0,3 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 7,4 µl bidest. H2O

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl 2-Log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2-4   iBB5416 K1-3  

pSB1C3: 2070 bp

iBB5416: 1500 bp
5-8   iBB48647631 K1-4  

pSB1A3: 2150 bp

iBB48647631: 4157 bp

10.06.2013

Digest
Investigator: Lukas
Aim: Construction of PNR-test-vector, PfcpB-test-vector and antibody-vector → Digestion of pSB1A3 iBB48647631 and single BioBricks.
  • X µl DNA template
  • 0,5 µl enzyme 1
  • 0,5 µl enzyme 2
  • 2,2 µl Cut Smart buffer
  • 20 µl bidest. H2O

Samples:

Digestion of 1,5 µl pSB1A3 iBB48647631 with SpeI-HF and PstI-HF

Digestion of 5 µl pSB1C3 iBB5 and iBB9 with XbaI and PstI-HF

Digestion of 4 µl pSB1C3 iBB3 and iBB4 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.

Ligation
Investigator: Christian
Aim: Construction of PNR-test-vector, PfcpB-test-vector and antibody-vector → Ligation of pSB1A3 iBB48647631 and single BioBricks in pSB1A3.

Ligation of pSB1A3 iBB48647631 with iBB5, pSB1A3 iBB48647631 with iBB3 and iBB9, pSB1A3 iBB48647631 with iBB4 and iBB9; overnight at 16°C

11.06.2013

Transformation
Investigator: Franzi
Aim: Construction of PNR-test-vector, PfcpB-test-vector and antibody-vector %rarr; Transformation of ligations.

Transformation of the ligations into E. coli DH5α (30 min on ice, 60 sec 42 °C, 25 min 37 °C + 900 µl LB), plated on LBamp, overnight at 37 °C.

12.06.2013

Inoculation
Investigator: Franzi
Aim: Construction of PNR-test-vector, PfcpB-test-vector and antibody-vector → Inoculation of transformants for miniprep.

4 colonies of each transformation inoculated in 4 ml LBamp, overnight at 37 °C.

13.06.2013

Miniprep
Investigator: Franzi
Aim: Construction of PNR-test-vector, PfcpB-test-vector and antibody-vector → Miniprep of transformants.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.

Digest
Investigator: Franzi
Aim: Construction of PNR-test-vector, PfcpB-test-vector and antibody-vector → Control digestion the minipreps and pSB1A3 iBB48647631 K3 (comparison of size).
  • 1 µl DNA template
  • 0,3 µl EcoRI-HF
  • 0,3 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 7,4 µl bidest. H2O

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl 2-Log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
upper:
2+3   iBB49 K3-4   1260 bp
4-7   iBB39 K1-4   1030 bp
lower:
2   iBB49647631   4157 bp
3-6   iBB496476315 K1-4   4404 bp
7+8   iBB49 K1-2   1260 bp
    Vector pSB1A3: 2150 bp

IBB48647631 and iBB486476315 probably positive, but better repeat or do a Colony-PCR.

16.06.2013

Digest
Investigator: Lukas
Aim: Construction of antibody-vector → Repeat of control digestion.
  • 1 µl DNA template
  • 0,3 µl EcoRI-HF
  • 0,3 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 7,4 µl bidest. H2O

Templates: PSB1A3 iBB486476315 and pSB1A3 iBB48647631 K3 (comparison of size).

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2+3   iBB48647631   4157 bp
4-7   iBB486476315   TBA bp
    Vector pSB1A3: 2150 bp

Too much DNA used, will be repeated as PCR.

17.06.2013

Digest
Investigator: Alex
Aim: Digest of pSB1A3_3+9 and pSB1A3_4+9 with SpeI and PstI, and corresponding inserts pSB1C3_5+4+1+6 and pSB1C3_6+3+1+5 with EcoRI and PstI.
  • 1 µl DNA
  • 0.3 µl Enzyme 1 (SpeI or EcoRI, resp.)
  • 0.3 µl Enzyme 2 (PstI)
  • 1 µl Cut Smart buffer
  • 7.4 µl ddbidest. H2O

The samples were incubated for 1.5 h at 37 °C.

Approaches were unsewed by means of gel electrophoresis, corresponding bands were picked under UV, DNA extraction from gel was performed using DNA Gel Extraction Kit (QIAGEN).

Ligation, Transformation, Plasmid prep
Investigator: Alex
Aim: Assemble the combined bricks 5416 and 6315 into their appropriate vectors pSB1A3_39 and pSB1A3_49, resp.
  • Insert und plasmid were transformed using a 1:2 ratio. Samples were incubated ON at RT.
  • Ligation product were transformed into chemical competent DH5α cells (30 min on ice, 90 sec 42 °C, 45 min 37 °C + 900 µl LB).
  • Transformed cells were spread out on LB Amp culture plates.
  • Incubation ON at RT.
  • 4 clones were picked for overnight at cultures.
  • A plasmid preparation was carried out the next day.

22.06.2013

Test digest
Investigator: Alex
Aim: Digest of pSB1A3_395416 and pSB1A3_496315 with EcoRI and PstI.
  • 1 µl Plasmid DNA
  • 0.3 µl EcoRI
  • 0.3 µL PstI
  • 1 µl Cut Smart
  • 7.4 µl ddbidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide 50 ml gel
  • 4 µl 2-log DNA ladder (New England Biolabs)

Lane   Content   Expectations
upper:
2   iBB6315   1200 + 2000 bp
3   iBB49   1200 + 2000 bp
4-7   iBB496315 K4-1   2400 + 2000 bp
lower:
2   iBB5416   1500 + 2000 bp
3   iBB39   1000 + 2000 bp
4-7   iBB395416 K1-4   2400 + 2000 bp

We didn't receive all expected fragments. All expected fragments in lanes 4-7 were missing. Ligation failed ☹

24.06.2013

PCR
Investigator: Alex
Aim: Check right assembly of BioBrick BBa_K1071005 in the plasmid for antibody production
Volume Reagent   Temp (°C) Time
1 µl pSB1A3_486476315   95 3 min
0.5 µl Primer fwd   95 30 sec
0.5 µl Primer rev   60 30 sec x30
0.5 µl dNTPs   72 42 sec
0.5 µl Phusion polymerase   72 5 min
5 µl Phusion buffer 5x   4 Hold
add 25 µL ddbidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide 50 ml gel
  • 4 µl 2-log DNA ladder (New England Biolabs)

Lane   Content   Expectations
2-5   pSB1A3 iBB486476315 K1-4  

~ 6,000 bp for pSB1A3_486476315

280 bp for BBa_K1071005

Each sample contained 2 bands, that means we got all expected bands and it could be assumed that the insertion of BBa_K1071005 was successful.

25.06.2013

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddbidest. H2O

The samples were incubated for 1 h at 37 °C.

Worked as expected.

26.06.2013

Digest
Investigator: Dominik
Aim: Construction of the plasmids pSB1A3-iBB10+9, pSB1A3-iBB11+9, pSB1A3-iBB12+9 and pSB1A3-iBB13+9

  • 1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)
  • 0.5 µl EcoRI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • 12 µl ddbidest. H2O

  • 1 µl Plasmid (pSB1C3-iBB9)
  • 0.5 µl XbaI
  • 0.5 µl Pst/I
  • 2 µl CutSmart
  • 12 µl ddbidest. H2O

  • 1 µl Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • 12 µl ddbidest. H2O

The samples were incubated for 1.5 h at 37 °C.

All expected fragments were present. The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).

Ligation
Investigator: Dominik
Aim: Assemble the biobricks iBB10+iBB9, iBB11+iBB9, iBB12+iBB9 and iBB13+iBB9 into the vector pSB1A3.

  • 2 µl vector DNA (pSB1A3)
  • 2 µl insert 1 DNA (iBB9)
  • 2 µl insert 2 DNA (iBB10, iBB11, iBB12 or iBB13)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 2 µl ddbidest. H2O

The samples were incubated overnight at room temperature.

27.06.2013

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37 °C.

Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB395146 and 4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

Digest
Investigator: Alex
Aim: Digest of pSB1A3-iBB395416 and pSB1A3-iBB496315 with EcoRI and PstI.
  • 1 µl Plasmid DNA
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl Cut Smart
  • 7.4 µl ddbidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide in 50 ml gel
  • 4 µl 2-log DNA ladder (New England Biolabs)

Lane   Content   Expectations
upper:
2-5   iBB395416 K1-4   2400 + 2000 bp
6   iBB39   1000 + 2000 bp
7   iBB6416   1400 + 2000 bp
lower:
2-6   iBB496315 K1-5   2400 + 2000 bp
7   iBB49   1200 + 2000 bp

All clones show a double band above 2000 bp.

We received all expected fragments. The success of the ligation should be double-checked by means of PCR.

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