Team:Marburg/Notebook:March

From 2013.igem.org

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{{:Team:Marburg/Template:ContentTitle}}
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{{:Team:Marburg/Template:ContentTitleNav}}
-
Notebook
+
Notebook: March <html><a href="https://2013.igem.org/Team:Marburg/Notebook:April"><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:Ptricornutum"><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>
-
<html>
 
-
 
-
<!-- Beispiel Allgemein -->
 
<div class="notebooky-entry">
<div class="notebooky-entry">
<h2 class="title">
<h2 class="title">
-
<a name="11-11-2012">11.11.2011</a>
+
<a name="20-03-2013">20.03.2013</a>
</h2>
</h2>
-
<fieldset class="experiment general">
+
 
-
     <legend><a name="title">Überschrift</a></legend>
+
<!-- Transformation-->
 +
<fieldset class="experiment transformation">
 +
     <legend>Transformation</legend>
     <div class="investigator">
     <div class="investigator">
<span class="inv">Investigator:</span>
<span class="inv">Investigator:</span>
-
<span class="inv-names">X,Y</span>
+
<span class="inv-names">Christian</span>
</div>
</div>
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">We attempt to get results.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Transformation into <i>E. coli</i> DH5&alpha;.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Wichtig ist, dass immer die verwendete Materialien (Plasmide, genomische DNA [aus welchen Stamm], Primer) genau benannt und/oder ggf. irgendwo nummeriert werden. Es sollte alles nachvollziehbar sein, wann ihr mit welchen Proben was gemacht habt.</p>
+
<p>Transformation of 1 µl pPha-NR, 1 µl pPha-T1 and 1 µl pCAT (diluted 1:3 with bidest. H2O) in <i>E. coli</i> DH5&alpha; (30 min ice, 90 sec 42°C, 1 min ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, overnight at 37°C.</p>
-
<p>Auf den folgenden Seiten sind Beispiele, wie wir uns das Notebook vom Aufbau und Detailgrad vorstellen.</p>
+
</div>
</div>
</fieldset>
</fieldset>
 +
</div>
</div>
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="21-03-2013">21.03.2013</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">Christian</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Preparation of BioBrick templates &rarr; Inoculation of transformants for miniprep.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>2 colonies of pPha-NR and pPha-T1 inoculated in 5 ml LB<sub>amp</sub>, overnight at 37°C.</p>
 +
<p>pCAT is a cosmid, transformation failed.</p>
 +
</div>
 +
</fieldset>
 +
 +
</div>
-
<!-- Beispiel Ligation, Verdau -->
 
<div class="notebooky-entry">
<div class="notebooky-entry">
<h2 class="title">
<h2 class="title">
-
<a name="17-04-2013">17.04.2013</a>
+
<a name="22-03-2013">22.03.2013</a>
</h2>
</h2>
-
<fieldset class="experiment ligation">
+
 
-
     <legend><a name="lig">Ligation</a></legend>
+
<!-- Miniprep -->
 +
<fieldset class="experiment miniprep">
 +
     <legend><a name="min">Miniprep</a></legend>
     <div class="investigator">
     <div class="investigator">
<span class="inv">Investigator:</span>
<span class="inv">Investigator:</span>
-
<span class="inv-names">Franzi, Lucas</span>
+
<span class="inv-names">Christian</span>
</div>
</div>
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Assemble the Bricks 1, 3, 4, 5, 6, 7 and 8 into the vector pSB1C3.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Miniprep of the template-plasmids.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p><ul class="lig">
+
<p>Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.</p>
-
<li>5 µl vector DNA (pSB1C3)</li>
+
-
<li>20 µl insert DNA (1, 3, 4, 5, 6, 7 or 8)</li>
+
-
<li>3 µl 10x T4 DNA ligase buffer</li>
+
-
<li>2 µl T4 DNA ligase</li>
+
-
</ul>
+
-
<p>The samples were incubated for 14 h at 18° C.</p>
+
</div>
</div>
</fieldset>
</fieldset>
-
</div>
+
 
-
<!-- Beispiel Verdau -->
+
<!-- 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">Christian, Patrick</span>
+
<span class="inv-names">Christian</span>
</div>
</div>
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Digest of pSB1C3-J04450 with <i>Mlu</i>I and <i>Hind</i>III.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Control digestion of the template plasmids.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p><ul class="digest">
+
<ul class="digest">
-
<li>4 µl DNA (pSB1C3-J04450)</li>
+
<li>3 µl DNA template</li>
-
<li>1 µl MluI</li>
+
<li>1 µl enzyme 1</li>
-
<li>1 µl HindIII</li>
+
<li>1 µl enzyme 2</li>
-
<li>1,5 µl 10x red buffer</li>
+
<li>4 µl buffer 2</li>
-
<li>1,5 µl 10x orange g loading buffer</li>
+
<li>31 µl bidest. H2O</li>
-
<li>6 µl H2O</li>
+
</ul>
</ul>
-
<p>The samples were incubated for 4 h at 37° C.</p>
+
<p>Samples:</p>
 +
<p>Digestion of pPha-NR with <i>EcoR</i>I and <i>Nco</i>I.</p>
 +
<p>Digestion of pPha-T1 with <i>EcoR</i>I and <i>Nde</i>I.</p>
 +
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 92: Line 111:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/d/d9/Gel_2013-03-22.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>pPha-NR</td>
 +
<th>&nbsp;</th>
 +
<td>2868 bp + 992 bp</td>
 +
</tr>
 +
<tr>
 +
<td>4+5</td>
 +
<th>&nbsp;</th>
 +
<td>pPha-T1</td>
 +
<th>&nbsp;</th>
 +
<td>3554 bp + 541 bp</td>
 +
</tr>
 +
</table>
 +
</p>
 +
<p>All positive.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- PCR -->
 +
<fieldset class="experiment pcr">
 +
    <legend><a name="pcr">PCR</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 BioBricks &rarr; Deletion of restriction sites in iBB3, iBB4 and iBB5 via mutagenesis PCR.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Mutagenesis PCR of Pf<sub>cpB</sub> (iBB3), P<sub>NR</sub> (iBB4), and T<sub>fcpA</sub> (iBB5).</p>
 +
<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>Phusion polymerase</td>
 +
<td>&nbsp;</td>
 +
<td>95</td>
 +
<td colspan="2">3 min</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>Primer fw (100 pmol/µl)</td>
 +
<td>&nbsp;</td>
 +
<td>95</td>
 +
<td colspan="2">30 sec</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>Primer rv (100 pmol/µl) </td>
 +
<td>&nbsp;</td>
 +
<td>65</td>
 +
<td>45 sec</td>
 +
<td rowspan="3">x19</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>Template pPha-NR (diluted 1:100)</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>4:10 min</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>dNTPs</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>6 min</td>
 +
</tr>
 +
<tr>
 +
<td>5 µl</td>
 +
<td>10x buffer</td>
 +
<td>&nbsp;</td>
 +
<td>4</td>
 +
<td colspan="2"><span class="hold">Hold</span></td>
 +
</tr>
 +
<tr>
 +
<td>35 µl</td>
 +
<td>bidest. H2O</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
</table>
 +
<br />
 +
<!-- Gel-Bild-->
 +
<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/e/e6/Gel_2013-03-25.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 100: Line 264:
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
-
<li>x µl Hyper Ladder</li>
+
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</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>&nbsp;</th>
 +
</thead>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub></td>
 +
<th>&nbsp;</th>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub></td>
 +
<th>&nbsp;</th>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpA</sub></td>
 +
<th>&nbsp;</th>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub></td>
 +
<th>&nbsp;</th>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub></td>
 +
<th>&nbsp;</th>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpA</sub></td>
 +
<th>&nbsp;</th>
 +
<td>&nbsp;</td>
 +
</tr>
 +
</table>
</p>
</p>
-
<p>We don’t receive all expected fragments. The expected fragment in lane 1 is missing.</p>
 
</td>
</td>
</tr>
</tr>
Line 119: Line 335:
</fieldset>
</fieldset>
-
<!-- Beispiel Transformation, PCR -->
+
<!-- Digest -->
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</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 BioBricks &rarr; Digestion of the template plasmid.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Digestion of the template with 1 µl <i>Dpp</i>I.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
<!-- Transformation-->
 +
<fieldset class="experiment transformation">
 +
    <legend>Transformation</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 BioBricks &rarr; Transformation of mutagenised plasmids.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Transformation of 1 µl PCR-product into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 1 min ice, 30 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, overnight at 37°C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
<div class="notebooky-entry">
<div class="notebooky-entry">
<h2 class="title">
<h2 class="title">
-
<a name="18-04-2012">18.04.2012</a>
+
<a name="26-03-2013">26.03.2013</a>
</h2>
</h2>
-
<fieldset class="experiment transformation">
+
 
-
     <legend>Transformation</legend>
+
<!-- Inoculation -->
 +
<fieldset class="experiment inoculation">
 +
     <legend><a name="ino">Inoculation</a></legend>
     <div class="investigator">
     <div class="investigator">
<span class="inv">Investigator:</span>
<span class="inv">Investigator:</span>
-
<span class="inv-names">Franzi, Christian</span>
+
<span class="inv-names">Christian</span>
</div>
</div>
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Transformation of the plasmid DNA pB201-40JO in <i>E. coli</i> for amplification.</span>
+
<span class="aim-desc">Construction of BioBricks &rarr; Inoculation of transformants for miniprep.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>The chemo competent <i>E. coli</i> DH5a cells were transformed with pB201-40JO DNA and plated on dYT-Amp-plates. <br />
+
<p>Transformation of iBB5 failed, new mutagenesis primers ordered.</p>
-
The plates were incubated over night at 37° C .</p>
+
<p>3 colonies of iBB3 and iBB4 inoculated in 5 ml LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</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">Christian</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Production of chemo-competent <i>E. coli</i> cells &rarr; Inoculation of <i>E. coli</i> DH5&alpha; pre-culture.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Inoculation of Ca-competent <i>E. coli</i> DH5&alpha; in 2x5 ml LB, 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">Christian</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Preparation of BioBrick templates &rarr; Inoculation for glycerol stocks.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>PPha-NR and pPha-T1 were inoculated in 5 ml LB, overnight at 37°C (for glycerol stocks).</p>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="27-03-2013">27.03.2013</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">Christian</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of BioBricks &rarr; Miniprep of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of of iBB3 and iBB4 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 30 µl bidest. H2O.</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">Christian</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Production of chemo-competent <i>E. coli</i> cells &rarr; Inoculation of <i>E. coli</i> DH5&alpha; pre-culture.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>3 ml <i>E. coli</i> DH5&alpha; pre-culture inoculated in 100 ml LB.</p>
 +
</div>
 +
</fieldset>
 +
<!-- PCR -->
<!-- PCR -->
<fieldset class="experiment pcr">
<fieldset class="experiment pcr">
-
     <legend>PCR</legend>
+
     <legend><a name="pcr">PCR</a></legend>
     <div class="investigator">
     <div class="investigator">
<span class="inv">Investigator:</span>
<span class="inv">Investigator:</span>
-
<span class="inv-names">Patrick, Lucas</span>
+
<span class="inv-names">Christian</span>
</div>
</div>
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Receive <i>Pvu</i>II point-mutation on colonies 3 and 5 of E. coli pB201-40JO.</span>
+
<span class="aim-desc">Construction of BioBricks &rarr; Amplification of iBB2 via PCR.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
 +
<p>PCR on CAT-cassette (iBB2): </p>
<table class="pcr">
<table class="pcr">
<colgroup>
<colgroup>
Line 169: Line 494:
</thead>
</thead>
<tr>
<tr>
-
<td>10 µl</td>
+
<td>1 µl</td>
-
<td>5x Buffer</td>
+
<td>Q5 Polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 176: Line 501:
</tr>
</tr>
<tr>
<tr>
-
<td>1,5 µl</td>
+
<td>1 µl</td>
-
<td>Primer fwd 13</td>
+
<td>Primer fw (1pmol/µl)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
-
<td colspan="2">3 sec</td>
+
<td colspan="2">30 sec</td>
</tr>
</tr>
<tr>
<tr>
-
<td>1,5 µl</td>
+
<td>1 µl</td>
-
<td>Primer rev 14</td>
+
<td>Primer rv (1pmol/µl) </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
-
<td>58</td>
+
<td>65</td>
<td>30 sec</td>
<td>30 sec</td>
-
<td rowspan="3">x17</td>
+
<td rowspan="3">x26</td>
</tr>
</tr>
<tr>
<tr>
Line 198: Line 523:
</tr>
</tr>
<tr>
<tr>
-
<td>36 µl</td>
+
<td>1 µl</td>
-
<td>H2O</td>
+
<td>dNTPs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
 +
<td>72</td>
<td>7 min</td>
<td>7 min</td>
-
<td>3 min</td>
 
</tr>
</tr>
<tr>
<tr>
-
<td>1 µl Phusion</td>
+
<td>10 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>5x Q5-buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4</td>
<td>4</td>
<td colspan="2"><span class="hold">Hold</span></td>
<td colspan="2"><span class="hold">Hold</span></td>
 +
</tr>
 +
<tr>
 +
<td>36 µl</td>
 +
<td>bidest. H2O</td>
 +
<td>&nbsp;</td>
</tr>
</tr>
</table>
</table>
-
<br />
+
<p>The PCR was cleaned via 1% agarose gel: Relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.</p>
-
<!-- Das Gel-Bild fast gleich wie beim Verdau -->
+
</div>
-
<table class="gel pcr">
+
</fieldset>
 +
 
 +
<!-- Transformation-->
 +
<fieldset class="experiment transformation">
 +
    <legend>Transformation</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">Production of chemo-competent <i>E. coli</i> &rarr; Control of cells.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Competent cells are harvested and frozen.</p>
 +
<p>Controlled with several Transformations:</p>
 +
<ul class="justalist">
 +
<li>on LB</li>
 +
<li>on LB<sub>amp</sub></li>
 +
<li>on LB<sub>amp</sub> with 1 µl pPha-T1</li>
 +
<li>cells from AG Bange as control</li>
 +
</ul>
 +
<p>30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB; plated, overnight at 37°C.</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">Christian</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of BioBricks &rarr; Control digestion of mutations.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>3 µl DNA template</li>
 +
<li>1 µl enzyme 1</li>
 +
<li>1 µl enzyme 2</li>
 +
<li>1 µl buffer</li>
 +
<li>5 µl bidest. H2O</li>
 +
</ul>
 +
<p>Samples:</p>
 +
<p>Digestion of iBB3 (P<sub>fcpB</sub>) with <i>Pvu</i>II.</p>
 +
<p>Digestion of IBB4 (P<sub>NR</sub>) with <i>Pst</i>I.</p>
 +
<p>The samples were incubated for 1 h at 37 °C.</p>
 +
<!-- Gel-Bild-->
 +
<table class="gel digest">
<colgroup>
<colgroup>
<col width="50%" />
<col width="50%" />
Line 227: Line 607:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/3/3d/Gel_2013-03-27.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 235: Line 615:
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
-
<li>x µl Hyper Ladder</li>
+
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</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>Comment</th>
 +
</thead>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub> 1</td>
 +
<th>&nbsp;</th>
 +
<td rowspan="3">no cut, vector in different forms</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub> 2</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub> 3</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub> 1</td>
 +
<th>&nbsp;</th>
 +
<td rowspan="3">1 cut &rarr; linearized plasmid</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub> 2</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub> 3</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
</table>
</p>
</p>
-
<p>We don’t receive all expected fragments. The expected fragment in lane 1 is missing.</p>
 
</td>
</td>
</tr>
</tr>
Line 253: Line 681:
</div>
</div>
</fieldset>
</fieldset>
 +
</div>
</div>
 +
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="28-03-2013">28.03.2013</a>
 +
</h2>
 +
 +
<!-- Sequencing -->
 +
<fieldset class="experiment sequencing">
 +
    <legend>Sequencing</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 BioBricks &rarr; Sequencing.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>IBB3 and iBB4 with point mutations prepared for sequencing:</p>
 +
<ul class="justalist">
 +
<li>AGBOOOK 128: iBB4 1</li>
 +
<li>AGBOOOK 129: iBB4 2</li>
 +
<li>AGBOOOK 130: iBB4 3</li>
 +
<li>AGBOOOK 131: iBB3 1</li>
 +
<li>AGBOOOK 132: iBB3 2</li>
 +
<li>AGBOOOK 133: iBB3 3</li>
 +
</ul>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
</html>
</html>
-
{{:Team:Marburg/Template:ContentEnd}}
+
{{:Team:Marburg/Template:ContentEndNav}}
{{:Team:Marburg/Template:Footer}}
{{:Team:Marburg/Template:Footer}}

Latest revision as of 11:12, 27 October 2013

Notebook: March Next Previous

20.03.2013

Transformation
Investigator: Christian
Aim: Preparation of BioBrick templates → Transformation into E. coli DH5α.

Transformation of 1 µl pPha-NR, 1 µl pPha-T1 and 1 µl pCAT (diluted 1:3 with bidest. H2O) in E. coli DH5α (30 min ice, 90 sec 42°C, 1 min ice, 45 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

21.03.2013

Inoculation
Investigator: Christian
Aim: Preparation of BioBrick templates → Inoculation of transformants for miniprep.

2 colonies of pPha-NR and pPha-T1 inoculated in 5 ml LBamp, overnight at 37°C.

pCAT is a cosmid, transformation failed.

22.03.2013

Miniprep
Investigator: Christian
Aim: Preparation of BioBrick templates → Miniprep of the template-plasmids.

Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest
Investigator: Christian
Aim: Preparation of BioBrick templates → Control digestion of the template plasmids.
  • 3 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 4 µl buffer 2
  • 31 µl bidest. H2O

Samples:

Digestion of pPha-NR with EcoRI and NcoI.

Digestion of pPha-T1 with EcoRI and NdeI.

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   pPha-NR   2868 bp + 992 bp
4+5   pPha-T1   3554 bp + 541 bp

All positive.
PCR
Investigator: Christian
Aim: Construction of BioBricks → Deletion of restriction sites in iBB3, iBB4 and iBB5 via mutagenesis PCR.

Mutagenesis PCR of PfcpB (iBB3), PNR (iBB4), and TfcpA (iBB5).

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1 µl Primer fw (100 pmol/µl)   95 30 sec
1 µl Primer rv (100 pmol/µl)   65 45 sec x19
1 µl Template pPha-NR (diluted 1:100)   72 4:10 min
1 µl dNTPs   72 6 min
5 µl 10x buffer   4 Hold
35 µl bidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

Lane   Content    
2   PfcpB    
3   PNR    
4   PfcpA    
5   PfcpB    
6   PNR    
7   PfcpA    

Digest
Investigator: Christian
Aim: Construction of BioBricks → Digestion of the template plasmid.

Digestion of the template with 1 µl DppI.

Transformation
Investigator: Christian
Aim: Construction of BioBricks → Transformation of mutagenised plasmids.

Transformation of 1 µl PCR-product into E. coli DH5α (30 min ice, 60 sec 42°C, 1 min ice, 30 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

26.03.2013

Inoculation
Investigator: Christian
Aim: Construction of BioBricks → Inoculation of transformants for miniprep.

Transformation of iBB5 failed, new mutagenesis primers ordered.

3 colonies of iBB3 and iBB4 inoculated in 5 ml LBamp, overnight at 37°C.

Inoculation
Investigator: Christian
Aim: Production of chemo-competent E. coli cells → Inoculation of E. coli DH5α pre-culture.

Inoculation of Ca-competent E. coli DH5α in 2x5 ml LB, overnight at 37°C.

Inoculation
Investigator: Christian
Aim: Preparation of BioBrick templates → Inoculation for glycerol stocks.

PPha-NR and pPha-T1 were inoculated in 5 ml LB, overnight at 37°C (for glycerol stocks).

27.03.2013

Miniprep
Investigator: Christian
Aim: Construction of BioBricks → Miniprep of transformants.

Miniprep of of iBB3 and iBB4 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 30 µl bidest. H2O.

Inoculation
Investigator: Christian
Aim: Production of chemo-competent E. coli cells → Inoculation of E. coli DH5α pre-culture.

3 ml E. coli DH5α pre-culture inoculated in 100 ml LB.

PCR
Investigator: Christian
Aim: Construction of BioBricks → Amplification of iBB2 via PCR.

PCR on CAT-cassette (iBB2):

Volume Reagent   Temp (°C) Time
1 µl Q5 Polymerase   95 3 min
1 µl Primer fw (1pmol/µl)   95 30 sec
1 µl Primer rv (1pmol/µl)   65 30 sec x26
1 µl Template   72 1 min
1 µl dNTPs   72 7 min
10 µl 5x Q5-buffer   4 Hold
36 µl bidest. H2O  

The PCR was cleaned via 1% agarose gel: Relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Transformation
Investigator: Christian
Aim: Production of chemo-competent E. coli → Control of cells.

Competent cells are harvested and frozen.

Controlled with several Transformations:

  • on LB
  • on LBamp
  • on LBamp with 1 µl pPha-T1
  • cells from AG Bange as control

30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB; plated, overnight at 37°C.

Digest
Investigator: Christian
Aim: Construction of BioBricks → Control digestion of mutations.
  • 3 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 1 µl buffer
  • 5 µl bidest. H2O

Samples:

Digestion of iBB3 (PfcpB) with PvuII.

Digestion of IBB4 (PNR) with PstI.

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

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

Lane   Content   Comment
2   PfcpB 1   no cut, vector in different forms
3   PfcpB 2  
4   PfcpB 3  
5   PNR 1   1 cut → linearized plasmid
6   PNR 2  
7   PNR 3  

28.03.2013

Sequencing
Investigator: Christian
Aim: Construction of BioBricks → Sequencing.

IBB3 and iBB4 with point mutations prepared for sequencing:

  • AGBOOOK 128: iBB4 1
  • AGBOOOK 129: iBB4 2
  • AGBOOOK 130: iBB4 3
  • AGBOOOK 131: iBB3 1
  • AGBOOOK 132: iBB3 2
  • AGBOOOK 133: iBB3 3

Retrieved from "http://2013.igem.org/Team:Marburg/Notebook:March"