Team:Marburg/Notebook:June

From 2013.igem.org

(Difference between revisions)
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<html>
<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 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>
 +
<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="beispiel.png" width="50%" 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(0.1-10.0 kb, New England Bioloabs)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</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 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 ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB<sub>CM</sub>, oN 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 H2O</li>
 +
</ul>
 +
<p>Samples (all <i>EcoR</i>I/<i>Pst</i>I-cut):</p>
 +
<p>120 ng eGFP</p>
 +
<p>60 ng SPTP1</p>
 +
<p>50 ng SPTP2</p>
 +
<p>55 ng SP1</p>
 +
<p>50 ng SP2</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 ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB<sub>CM</sub>, oN 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>, oN 37°C.</p>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="04-06-2013">04.06.2013</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>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 H2O. 2 Cultures of pSB1C3 iBB4864 discarded (red colour).</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>fcpB</sub>-test-vector and antibody-vector &rarr; Control digestion of pSB1A3 iBB1+6 and pSB1C3 iBB4864.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA template</li>
 +
<li>0,5 µl <i>EcoR</i>I-HF</li>
 +
<li>0,5 µl <i>Pst</i>I-HF</li>
 +
<li>1 µl Cut Smart buffer</li>
 +
<li>7 µl 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="beispiel.png" width="50%" 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(0.1-10.0 kb, New England Bioloabs)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<span class="exp">Expectations</span>
 +
<ul class="exp">
 +
<li>Vector: 2 kb</li>
 +
<li>iBB4864: 2580 bp</li>
 +
<li>iBB1+6: 650 bp</li>
 +
</ul>
 +
</p>
 +
<p>All positive.</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 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>
 +
<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="beispiel.png" width="50%" 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(0.1-10.0 kb, New England Bioloabs)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</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 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 H2O</li>
 +
</ul>
 +
<p>Both samples were incubated oN 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>, oN 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 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 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="beispiel.png" width="50%" 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(0.1-10.0 kb, New England Bioloabs)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<span class="exp">Expectations</span>
 +
<ul class="exp">
 +
<li>pSB1C3: 2 kb</li>
 +
<li>iBB9: 780 bp</li>
 +
<li>iBB10: 228 bp</li>
 +
<li>iBB11: 147 bp</li>
 +
<li>iBB12: 123 bp</li>
 +
<li>iBB13: 108 bp</li>
 +
<li>iBB6315: 1200 bp</li>
 +
</ul>
 +
</p>
 +
<p>Problems with the TBE-buffer %rarr; Longer time with lower volt.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</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>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 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), oN 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 class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="06-06-2013">06.06.2013</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>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>, oN 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 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">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; Control digestion of pSB1C3 iBB5416 and pSB1A3 iBB48647631.</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 H2O</li>
 +
</ul>
 +
<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="beispiel.png" width="50%" 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(0.1-10.0 kb, New England Bioloabs)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<span class="exp">Expectations</span>
 +
<ul class="exp">
 +
<li>pSB1C3: 2070 bp</li>
 +
<li>iBB5416: 1500 bp</li>
 +
<li>pSB1A3: 2150 bp</li>
 +
<li>iBB48647631: 4157 bp</li>
 +
</ul>
 +
</p>
 +
<p>All positive.</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 H2O</li>
 +
</ul>
 +
<p>Samples:</p>
 +
<p>Digestion of 1,5 µl pSB1A3 iBB48647631 with <i>Spe</i>I-HF and <i>Pst</i>I-HF</p>
 +
<p>Digestion of 5 µl pSB1C3 iBB5 and iBB9 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 +
<p>Digestion of 4 µl pSB1C3 iBB3 and iBB4 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>
 +
<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="beispiel.png" width="50%" 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(0.1-10.0 kb, New England Bioloabs)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</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; oN 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 ice, 60 sec 42°C, 25 min 37°C + 900 µl LB), plated on LB<sub>amp</sub>, oN 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>, oN 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 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 H2O</li>
 +
</ul>
 +
<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="beispiel.png" width="50%" 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(0.1-10.0 kb, New England Bioloabs)</li>
 +
<li>6x loading buffer (for samples)</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<span class="exp">Expectations</span>
 +
<ul class="exp">
 +
<li>pSB1A3: 2150 bp</li>
 +
<li>iBB48647631: 4157 bp</li>
 +
<li>iBB486476315: 4404 bp</li>
 +
<li>iBB49: 1260 bp</li>
 +
<li>iBB39: 1030 bp</li>
 +
</ul>
 +
</p>
 +
<p>For better separation another 30 min.</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>Probably positive, but better repeat or do a Colony-PCR.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 +
</div>
 +
<div class="notebooky-entry">
<div class="notebooky-entry">

Revision as of 19:35, 1 October 2013

Notebook: June

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 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.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
  • 6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl 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 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 ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LBCM, oN 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 H2O

Samples (all EcoRI/PstI-cut):

120 ng eGFP

60 ng SPTP1

50 ng SPTP2

55 ng SP1

50 ng SP2

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 ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LBCM, oN 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, oN 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 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 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(0.1-10.0 kb, New England Bioloabs)
  • 6x loading buffer (for samples)

Expectations

  • Vector: 2 kb
  • iBB4864: 2580 bp
  • iBB1+6: 650 bp

All positive.
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 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.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
  • 6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl 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 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 H2O

Both samples were incubated oN 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, oN 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 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 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(0.1-10.0 kb, New England Bioloabs)
  • 6x loading buffer (for samples)

Expectations

  • pSB1C3: 2 kb
  • iBB9: 780 bp
  • iBB10: 228 bp
  • iBB11: 147 bp
  • iBB12: 123 bp
  • iBB13: 108 bp
  • iBB6315: 1200 bp

Problems with the TBE-buffer %rarr; Longer time with lower volt.
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 ice, 60 sec 42°C, 60 min (1C3)/40 min (1A3) 37°C + 900 µl LB), plated on LBCM (1C3)/LBamp (1A3), oN 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, oN 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 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 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(0.1-10.0 kb, New England Bioloabs)
  • 6x loading buffer (for samples)

Expectations

  • pSB1C3: 2070 bp
  • iBB5416: 1500 bp
  • pSB1A3: 2150 bp
  • iBB48647631: 4157 bp

All positive.

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 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.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)
  • 6x loading buffer (for samples)

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl 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; oN 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 ice, 60 sec 42°C, 25 min 37°C + 900 µl LB), plated on LBamp, oN 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, oN 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 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 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(0.1-10.0 kb, New England Bioloabs)
  • 6x loading buffer (for samples)

Expectations

  • pSB1A3: 2150 bp
  • iBB48647631: 4157 bp
  • iBB486476315: 4404 bp
  • iBB49: 1260 bp
  • iBB39: 1030 bp

For better separation another 30 min.
gel-electrophoresis-image

Probably positive, but better repeat or do a Colony-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
  • 7.4 µl ddH2O

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 in 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.
  • Transformed cells were spread out on LB Amp culture plates.
  • Incubation ON at RT.
  • 4 clones were picked for overnight 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 ddH2O

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

Expectations (upper gel)

  • Lane 2: 1,200 + 2,000 bp
  • Lane 3: 1,200 + 2,000 bp
  • Lane 4-7: 2,400 + 2,000 bp

Expectations (lower gel)

  • Lane 2: 1,500 + 2,000 bp
  • Lane 3: 1,000 + 2,000 bp
  • Lane 4-7: 2,400 + 2,000 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 ddH2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide 50 ml gel
  • 4 µl 2-log DNA ladder

Expactations

  • Lane 1: ~ 6,000 bp for pSB1A3_486476315
  • Lane 2: 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 ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

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 ddH2O

  • 1 µl Plasmid (pSB1C3-iBB9)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • 12 µl ddH2O

  • 1 µl Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • 12 µl ddH2O

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

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
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 ddH2O

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 ddH2O

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

Expectations (upper gel)

  • Lane 2-5: 2,400 + 2,000 bp
  • Lane 6: 1,000 + 2,000 bp
  • Lane 7: 1,400 + 2,000 bp

Expectations (lower gel)

  • Lane 2-7: 2,400 + 2,000 bp
  • Lane 8: 1,200 + 2,000 bp

All clones show a double band above 2,000 bp.

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

02.07.2013

Miniprep
Investigator: Dominik
Aim: preparation of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Worked as expected.

23.07.2013

Digest
Investigator: Dominik
Aim: Test digest of the plasmids pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB4+iBB11+iBB9 and pSB1C3-iBB4+iBB12+iBB9)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Worked as expected.

24.07.2013

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

4 colonies of pSB1C3-iBB4+iBB10+iBB9 and 4 colonies of pSB1C3-iBB4+iBB13+iBB9 were inoculated in 5 ml LB medium containing chloramphenicol.

Digest
Investigator: Dominik
Aim: Digest of pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9, pSB1C3-iBB6315 and pSB1A3.

  • 1000 ng Plasmid (pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9)
  • 0.5 µl EcoRI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
Ligation
Investigator: Dominik
Aim: Assemble the biobricks iBB4+iBB11+iBB96315 and iBB4+iBB12+iBB96315 into the vector pSB1A3.

  • 2 µl vector DNA (pSB1A3)
  • 5 µl insert 1 DNA (iBB6315)
  • 5 µl insert 2 DNA (iBB4+iBB11+iBB9, iBB4+iBB12+iBB9)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 4 µl ddH2O

The samples were incubated for 2h at 16 °C.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

25.07.2013

Sequencing
Investigator: Dominik
Aim: Complete sequencing of pSB1A3-iBB496315.

8 new sequencing samples were sent out.

Miniprep
Investigator: Dominik
Aim: preparation of the plasmids pSB1A3-iBB4+iBB10+iBB9 and pSB1A3-iBB4+iBB13+iBB9.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1C3-iBB4+iBB10+iBB9 and pSB1C3-iBB4+iBB13+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB4+iBB10+iBB9 and pSB1C3-iBB4+iBB13+iBB9)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Two samples of each plasmid preparation showed the expected fragments.
Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB4+iBB11+iBB96315 and 4 colonies of pSB1A3-iBB4+iBB12+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol.

Competent cells
Investigator: Dominik
Aim: Overnight culture for making new aliquots of competent cells.

For making new aliquots of E. coli DH5α cells 50 ml LB medium were inoculated with 500 µl of an E. coli DH5α culture.

26.07.2013

Digest
Investigator: Dominik
Aim: Digest of pSB1C3-iBB4+iBB10+iBB9, pSB1C3-iBB4+iBB13+iBB9, pSB1C3-iBB6315 and pSB1A3.

  • 1000 ng Plasmid (pSB1C3-iBB4+iBB10+iBB9, pSB1C3-iBB4+iBB13+iBB9)
  • 0.5 µl EcoRI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
Ligation
Investigator: Dominik
Aim: Assemble the biobricks iBB4+iBB10+iBB9, iBB4+iBB13+iBB9 and iBB96315 into the vector pSB1A3.

  • 2 µl vector DNA (pSB1A3)
  • 5 µl insert 1 DNA (iBB96315)
  • 5 µl insert 2 DNA (iBB4+iBB10+iBB9,iBB4+iBB13+iBB9)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 4 µl ddH2O

The samples were incubated for 1h at 16 °C.

Resulting in the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

Miniprep
Investigator: Dominik
Aim: preparation of the pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of ppSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 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 ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Most of the preparations resulted in the expected fragments, all others were discarded.
Competent cells
Investigator: Dominik
Aim: New aliquots of competent cells (E. coli DH5α).

About 80 new aliquots were made.

02.07.2013

Sequencing
Investigator: Dominik
Aim: Analysis of the sequence of pSB1A3-iBB496315.

Obviously the wrong plasmid was sent out for sequencing. Due to the lack of enough plasmids for an additional sequence analysis, we will redo a new miniprep.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

30.07.2013

Digest
Investigator: Dominik
Aim: Digest of pSB1A3-iBB49 and pSB1C3-iBB6315.

  • 1000 ng Plasmid (pSB1A3-iBB49)
  • 0.5 µl PstI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB4+iBB10+iBB96315 and the only single colony of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol. Additionally the remaining ligation preparation was transformed in E. coli DH5α?????.

31.07.2013

Ligation
Investigator: Dominik
Aim: Ligation of pSB1A3-iBB49 and iBB6315.

  • 2 µl vector DNA (pSB1A3-iBB49) (40 ng)
  • 5 µl insert DNA (iBB6315) (150 ng)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 9 µl ddH2O

The samples were incubated for 1h at 16 °C.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB49+iBB6315.

The chemo competent E. coli DH5α cells were transformed with the plasmid pSB1A3-iBB49+iBB6315 and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

Miniprep
Investigator: Dominik
Aim: Preparation of the pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

The culture of pSB1A3-iBB4+iBB13+iBB96315 had a pinkish color and therefore was discarded. The plasmid pSB1A3-iBB4+iBB10+iBB96315 was isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB4+iBB10+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB4+iBB10+iBB96315)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Worked as expected.
Sequencing
Investigator: Dominik
Aim: Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.

8 samples were sent out for sequencing.

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

Both colonies of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing ampicillin.

01.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.

2 additional sequence samples were sent out for sequencing.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB4+iBB13+iBB96315 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 ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

After that, a gel electrophoresis was made, but failed and has to be repeated the next day
Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

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

02.07.2013

Miniprep
Investigator: Dominik
Aim: preparation of the plasmid pSB1A3-iBB496315.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB496315 and iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddH2O

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
  • x µl Hyper Ladder

Expactations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Worked as expected.

14.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of sequence analysis of the plasmids pSB1A3-iBB496315, pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

  • pSB1A3-iBB496315: Okay
  • pSB1A3-iBB4+iBB10+iBB96315: mutation in signal peptide that leads to Trp → Leu. Will be ignored
  • pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.

All plasmids are brought to Marian who will transform them in P. tricornutum cells.

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