Team:Marburg/Notebook:Mai

From 2013.igem.org

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Notebook: Mai
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Notebook: MaY
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{{:Team:Marburg/Template:ContentStart}}
<html>
<html>
-
<!-- HIER -->
+
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="02-05-2013">02.05.2013</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">TBA Inoculation of colonies for plasmid preparation.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>5 colonies of pSB1C3 iBB8 inoculated in 5 ml LB<sub>cm</sub> each, 2 colonies of 2xNR HC + LC inoculated in 5 ml LB<sub>amp</sub> each. Incubation overnight at 37° C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="02-05-2013">03.05.2013</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">TBA Preparation of plasmids.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>5 samples of pSB1C3 iBB8 and 2 samples of 2xNR HC + LC were isolated via Miniprep (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)).
 +
</p>
 +
</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">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">TBA Test-PCR of 2xNR HC + LC</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>0,5 µl</td>
 +
<td>Phusion-Polymerase</td>
 +
<td>&nbsp;</td>
 +
<td>95</td>
 +
<td colspan="2">3 min</td>
 +
</tr>
 +
<tr>
 +
<td>0,5 µl</td>
 +
<td>Primer Cl4mA_l fw</td>
 +
<td>&nbsp;</td>
 +
<td>95</td>
 +
<td colspan="2">30 sec</td>
 +
</tr>
 +
<tr>
 +
<td>0,5 µl</td>
 +
<td>Primer Cl4mA_l rv </td>
 +
<td>&nbsp;</td>
 +
<td>65</td>
 +
<td>30 sec</td>
 +
<td rowspan="3">x30</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>Template 2xNR HC + LC (9,2 ng/µl)</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>45 sec</td>
 +
</tr>
 +
<tr>
 +
<td>0,5 µl</td>
 +
<td>dNTPs</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>7 min</td>
 +
</tr>
 +
<tr>
 +
<td>5 µl</td>
 +
<td>5xGC buffer</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td>17 µl</td>
 +
<td>ddH20</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
</table>
 +
<br />
 +
<!-- Das Gel-Bild ist bei der Testrestriktion mit drauf -->
 +
<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="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 in 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>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">TBA Testrestriction of pSB1C3 iBB8 K1-5</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA template</li>
 +
<li>0,5 µl <i>EcoR</i>I</li>
 +
<li>0,5 µl <i>Pst</i>I</li>
 +
<li>1 µl buffer O</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>iBB8 K1 + 2 chosen for further work.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="06-05-2013">06.05.2013</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">Preparation of pSB1A3 &rarr; Transformation of pSB1A3 J04450.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>3 µl of pSB1A3 J04450 (2012) were transformed into <i>E. coli</i> DH5&alpha; as described previously (30 min ice, 60 sec 42°C, 60 min 37°C with 900 µl LB), concentrated and plated on LB<sub>amp</sub>, overnight 37°C.
 +
</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="07-05-2013">07.05.2013</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">Preparation of pSB1A3 &rarr; Inoculation of pSB1A3 J04450 for miniprep.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Two red colonies were picked and inoculated in 5 ml LB<sub>amp</sub> each. Incubation 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 iBB2 (cat) &rarr; Examination of sequencing results.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>1C3 iBB1 K1 + K2 correct</p>
 +
<p>iBB2 K2 + 4: both positive</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="08-05-2013">08.05.2013</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">Preparation of pSB1A3 &rarr; Miniprep of pSB1A3 J04450 </span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of the cultures inoculated the previous day (“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">Preparation of pSB1A3 &rarr; Digestion of pSB1A3 J04450 with <i>EcoR</i>I and <i>Pst</i>I.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA template</li>
 +
<li>1 µl <i>EcoR</i>I-HF</li>
 +
<li>1 µl <i>Pst</i>I-HF</li>
 +
<li>3,6 µl Cut Smart buffer</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>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl H2O.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="13-05-2013">13.05.2013</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 antibody-vector &rarr; Digestion of various BioBricks.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>5 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>12 µl H2O</li>
 +
</ul>
 +
<p>Samples:</p>
 +
<p>IBB4 K1, iBB6 K1, iBB7 K3 and iBB3 K1 were digested with <i>EcoR</i>I-HF and <i>Spe</i>I-HF.</p>
 +
<p>iBB8K1, iBB4K1, iBB6K1 and iBB1K1 were digested with <i>Xba</i>I and <i>Pst</i>I-HF.
 +
<p>The samples were incubated for 1 h at 37° C. Then 1 µl CIP was addend and the samples were incubated for another 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 30 µl elution buffer.</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 antibody-vector &rarr; Combination of two BioBricks into vector pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="lig">
 +
<li>40 ng vector DNA (pSB1A3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>120 ng insert DNA (variable)</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>Samples:</p>
 +
<p>iBB4 <i>EcoR</i>I/<i>Spe</i>I with iBB8 <i>Xba</i>I/<i>Pst</i>I</p>
 +
<p>iBB6 <i>EcoR</i>I/<i>Spe</i>I with iBB4 <i>Xba</i>I/<i>Pst</i>I</p>
 +
<p>iBB7 <i>EcoR</i>I/<i>Spe</i>I with <i>Xba</i>I/<i>Pst</i>I</p>
 +
<p>iBB3 <i>EcoR</i>I/<i>Spe</i>I with <i>Xba</i>I/<i>Pst</i>I</p>
 +
<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 Antibody-vector &rarr; Transformation of combined 2 BioBricks in pSB1A3 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Complete ligation samples (see above) were transformed into <i>E. coli</i> DH5&alpha; as described previously (30 min ice, 60 sec 42°C, 20 min 37°C with 900 µl LB), concentrated and plated on LB<sub>amp</sub>, overnight 37°C.
 +
</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="14-05-2013">14.05.2013</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 antibody-vector &rarr; Inoculation of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Colonies from iBB4+8, iBB6+4 and iBB3+1 were picked and inoculated in 5 ml LB<sub>amp</sub>, respectively.</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 antibody-vector &rarr; Ligation of iBB7 and iBB6 into pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Ligation of iBB7 and iBB6 was repeated.</p>
 +
<ul class="lig">
 +
<li>40 ng vector DNA (pSB1A3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>120 ng insert DNA </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 Antibody-vector &rarr; Transformation of combined 2 BioBricks in pSB1A3 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Complete ligation samples (see above) were transformed into <i>E. coli</i> DH5&alpha; as described previously (30 min ice, 60 sec 42°C, 20 min 37°C with 900 µl LB), concentrated and plated on LB<sub>amp</sub>, overnight 37°C.
 +
</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="15-05-2013">15.05.2013</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 antibody-vector &rarr; Repeat of digestion of various BioBricks (13.05.13).</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Liquid cultures and transformation samples from previous day were discarded because of red colonies (Insert J04450). Digestion of BioBricks repeated like 13.5.13, but without CIP.</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 elution buffer.</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 antibody-vector &rarr; Repeat of combination of two BioBricks into vector pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Ligation like 13.5.13.</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 Antibody-vector &rarr; Repeat of transformation of combined 2 BioBricks in pSB1A3 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Transformation like 13.5.13.</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">Preparation of pSB1A3 &rarr; Inoculation of pSB1A3 J04450.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><i>E. coli</i> pSB1A3 J04450 was inoculated in 5 ml LB<sub>amp</sub>, overnight 37°C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="16-05-2013">16.05.2013</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">Preparation of pSB1A3 &rarr; Miniprep of pSB1A3 J04450.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>PSB1A3 J04450 (see 15.05.13) was isolated via Miniprep (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)).</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 antibody-vector &rarr; Inoculation of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>4 colonies of each transformation inoculated in 5 ml LB<sub>amp</sub>, respectively. overnight 37°C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="17-05-2013">17.05.2013</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 antibody-vector &rarr; Miniprep of combined 2 BioBricks into pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 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 antibody-vector &rarr; Control digestion of combined 2 BioBricks.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA template</li>
 +
<li>1 µl <i>EcoR</i>I</li>
 +
<li>1 µl <i>Pst</i>I</li>
 +
<li>2 µl buffer O</li>
 +
<li>0,15 µl H2O</li>
 +
</ul>
 +
<p>Samples:</p>
 +
<p>iBB4 + iBB8</p>
 +
<p>iBB6 + iBB4</p>
 +
<p>iBB7 + iBB6</p>
 +
<p>iBB3 + iBB1</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>
 +
<span class="exp">Expactations</span>
 +
<ul class="exp">
 +
<li>6+4: 700 bp</li>
 +
<li>7+6: 1000 bp</li>
 +
<li>3+1: 700 bp</li>
 +
<li>4+8: 1900 bp</li>
 +
</ul>
 +
</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 antibody-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>Digest of pSB1A3 iBB6+4 K1+2 and pSB1A3 iBB3+1 K1+4 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 +
<p>PSB1A3 iBB4+8 K1+4 and pSB1A3 iBB7+6 K2+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>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="21-05-2013">21.05.2013</a>
 +
</h2>
 +
 
 +
<!-- 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">Contruction of antibody-vector &rarr; ligation of two combined 2 BioBricks into pSB1C3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="lig">
 +
<li>30 ng vector DNA (pSB1C3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>100 ng insert 1 </li>
 +
<li>100 ng insert 2 </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>Samples:</p>
 +
<p>iBB4+8 K4 + iBB6+4 K2</p>
 +
<p>iBB7+6 K2 + iBB3+1 K1</p>
 +
<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 Antibody-vector &rarr; Repeat of transformation of combined 4 BioBricks in pSB1C3 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Complete ligation samples (see above) were transformed into <i>E. coli</i> DH5&alpha; as described previously (30 min ice, 60 sec 42°C, 60 min 37°C with 900 µl LB), concentrated and plated on LB<sub>cm</sub>, overnight 37°C.
 +
</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 iBB4 &rarr; Retransformation into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Retransformation of pSB1C3 iBB4 K1 as described above.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="22-05-2013">22.05.2013</a>
 +
</h2>
 +
 
 +
<!-- 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 antibody-vector &rarr; Repeat of ligation of two combined 2 BioBricks into pSB1C3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Ligation of pSB1C3 iBB 4+8+6+4 repeated like 21.5.13.</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 iBB4 &rarr; Inoculation of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>2 colonies of pSB1C3 iBB4 K1 were inoculated in 5 ml LB<sub>cm</sub>, overnight 37°C.</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="23-05-2013">23.05.2013</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 iBB4 &rarr; Miniprep.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of pSB1C3 iBB4 K1 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 20 µl 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">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of iBB4 &rarr; Inoculation of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>4 colonies of pSB1C3 iBB7631 inoculated in 5 ml LB<sub>cm</sub>, oN 37°C.</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 iBB4 &rarr; Transformation of pSB1C3 iBB4864 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Ligation of pSB1C3 iBB4864 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>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="24-05-2013">24.05.2013</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 antibody-vector &rarr; Miniprep of pSB1C3 iBB7631.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of pSB1C3 iBB7631 K1-4 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 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 antibody-vector &rarr; Control digestion of pSB1C3 iBB7631.</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 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">Expactations</span>
 +
<ul class="exp">
 +
<li>Vector: 2150 bp</li>
 +
<li>insert (iBB7631): 1610 bp</li>
 +
</ul>
 +
</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</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 antibody-vector &rarr; Inoculation of transformants.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>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="27-05-2013">27.05.2013</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 and P<sub>fcpB</sub>-test-vector &rarr; digestion of single BioBricks.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>5 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>12 µl H2O</li>
 +
</ul>
 +
<p>Samples:</p>
 +
<p>Digestion of pSB1C3 iBB6, iBB3, iBB5 and iBB4 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 +
<p>Digestion of pSB1C3 iBB6 and iBB1 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 and P<sub>fcpB</sub>-test-vector &rarr; Combination of two BioBricks into pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="lig">
 +
<li>40 ng vector DNA (pSB1A3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
 +
<li>120 ng insert 1 </li>
 +
<li>120 ng insert 2 </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>Samples:</p>
 +
<p>iBB5 E/S + iBB4 X/P</p>
 +
<p>iBB1 E/S + iBB6 X/P</p>
 +
<p>iBB6 E/S + iBB3 X/P</p>
 +
<p>iBB1 E/S + iBB5 X/P</p>
 +
<p>The samples were incubated for 2,5 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 and P<sub>fcpB</sub>-test-vector &rarr; transformation of combined 2 BioBricks 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, 20 min 37°C + 900 µl LB), plated on LB<sub>amp</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 antibody-vector &rarr; Repeat of ligation of two combined 2 BioBricks into pSB1C3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="lig">
 +
<li>30 ng vector DNA</li>
 +
<li>40 ng iBB48 </li>
 +
<li>40 ng iBB64 </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>The samples were incubated for oN at 16° 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">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Preparation of pSB1C3 &rarr; digestion with <i>EcoR</i>I and <i>Pst</i>I.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>10 µl DNA template (pSB1C3 J04450)</li>
 +
<li>0,5 µl <i>EcoR</i>I-HF</li>
 +
<li>0,5 µl <i>Pst</i>I-HF</li>
 +
<li>2 µl Cut Smart buffer</li>
 +
<li>6 µl H2O</li>
 +
</ul>
 +
<p>The samples were incubated oN 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 (done 28.05.2013)</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 30 µl H2O.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="28-05-2013">28.05.2013</a>
 +
</h2>
 +
 
 +
<!-- PCR -->
 +
<fieldset class="experiment pcr">
 +
    <legend><a name="pcr">PCR</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; Adding of pre- and suffix via PCR.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>PCR of iBB9 (eGFP) with P40+41, iBB10 (SPTP1) with P42+43, iBB11 (SPTP2) with P44+45, iBB12 (SP1) with P46+47 and iBB13 (SP2) with P48+49.</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 P1 (1pmol/µl)</td>
 +
<td>&nbsp;</td>
 +
<td>95</td>
 +
<td colspan="2">30 sec</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>Primer P2 (1pmol/µl) </td>
 +
<td>&nbsp;</td>
 +
<td>55</td>
 +
<td>30 sec</td>
 +
<td rowspan="3">x33</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>Template</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>45 sec</td>
 +
</tr>
 +
<tr>
 +
<td>1 µl</td>
 +
<td>dNTPs</td>
 +
<td>&nbsp;</td>
 +
<td>72</td>
 +
<td>10 min</td>
 +
</tr>
 +
<tr>
 +
<td>10 µl</td>
 +
<td>5xGC buffer</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td>35 µl</td>
 +
<td>ddH20</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="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 in 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">Expactations</span>
 +
<ul class="exp">
 +
<li>eGFP: 780 bp</li>
 +
<li>SPTP1: 228 bp</li>
 +
<li>SPTP2: 147 bp</li>
 +
<li>SP1: 123 bp</li>
 +
<li>SP2: 108 bp</li>
 +
</ul>
 +
</p>
 +
<p>PCR was repeated (too much template), template diluted 1:50.</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 antibody-vector &rarr; Transformation of pSB1C3 iBB4864 into <i>E. coli</i> DH5&alpha;.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Ligation of pSB1C3 iBB4864 was 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>NR</sub>-test-vector and P<sub>fcpB</sub>-test-vector &rarr; Inoculation of transformants for miniprep.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>2 colonies of each transformation (27.05.13) were inoculated in 5 ml LB<sub>amp</sub>, oN 37°C</p>
 +
</div>
 +
</fieldset>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="29-05-2013">29.05.2013</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 P<sub>fcpB</sub>-test-vector &rarr; Miniprep of combined 2 BioBricks in pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 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 P<sub>fcpB</sub>-test-vector &rarr; Control digestion of combined 2 BioBricks in pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>1 µl DNA templates (see above)</li>
 +
<li>0,5 µl <i>EcoR</i>I</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 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">Expactations</span>
 +
<ul class="exp">
 +
<li>6+3: 500 bp</li>
 +
<li>1+5: 700 bp</li>
 +
<li>5+4: 750 bp</li>
 +
<li>1+6: 650 bp</li>
 +
</ul>
 +
</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">Franzi</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Construction of BioBricks &rarr; Cleaning of PCR-products.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Gel of repeated PCR of iBB9 (eGFP) with P40+41, iBB10 (SPTP1) with P42+43, iBB11 (SPTP2) with P44+45, iBB12 (SP1) with P46+47 and iBB13 (SP2) with P48+49.</p>
 +
<!-- 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="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 in 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">Expactations</span>
 +
<ul class="exp">
 +
<li>eGFP: 780 bp</li>
 +
<li>SPTP1: 228 bp</li>
 +
<li>SPTP2: 147 bp</li>
 +
<li>SP1: 123 bp</li>
 +
<li>SP2: 108 bp</li>
 +
</ul>
 +
</p>
 +
<p>All positive.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
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 +
<!-- Digest -->
 +
<fieldset class="experiment digest">
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    <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 BioBricks &rarr; Digestion with <i>EcoR</i>I and <i>Pst</i>I.</span>
 +
</div>
 +
<div class="exp-content">
 +
<ul class="digest">
 +
<li>29,5 µl DNA templates (PCR-products)</li>
 +
<li>1 µl <i>EcoR</i>I-HF</li>
 +
<li>1 µl <i>Pst</i>I-HF</li>
 +
<li>3,5 µl Cut Smart buffer</li>
 +
<li>0,5 µl H2O</li>
 +
</ul>
 +
<p>The samples were incubated for 1 h at 37° C.</p>
 +
<table class="gel digest">
 +
<colgroup>
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<col width="50%" />
 +
<col width="50%" />
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<thead>
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<tr>
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<th colspan="2" class="title">Gel electrophoresis</th>
 +
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 +
</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>
 +
 
 +
</div>
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</html>
</html>
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Revision as of 15:18, 1 October 2013

Notebook: MaY

02.05.2013

Inoculation
Investigator: Franzi
Aim: TBA Inoculation of colonies for plasmid preparation.

5 colonies of pSB1C3 iBB8 inoculated in 5 ml LBcm each, 2 colonies of 2xNR HC + LC inoculated in 5 ml LBamp each. Incubation overnight at 37° C.

03.05.2013

Miniprep
Investigator: Franzi
Aim: TBA Preparation of plasmids.

5 samples of pSB1C3 iBB8 and 2 samples of 2xNR HC + LC were isolated via Miniprep (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)).

PCR
Investigator: Franzi
Aim: TBA Test-PCR of 2xNR HC + LC
Volume Reagent   Temp (°C) Time
0,5 µl Phusion-Polymerase   95 3 min
0,5 µl Primer Cl4mA_l fw   95 30 sec
0,5 µl Primer Cl4mA_l rv   65 30 sec x30
1 µl Template 2xNR HC + LC (9,2 ng/µl)   72 45 sec
0,5 µl dNTPs   72 7 min
5 µl 5xGC buffer  
17 µl ddH20  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

All positive.
Digest
Investigator: Franzi
Aim: TBA Testrestriction of pSB1C3 iBB8 K1-5
  • 1 µl DNA template
  • 0,5 µl EcoRI
  • 0,5 µl PstI
  • 1 µl buffer O

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)

iBB8 K1 + 2 chosen for further work.

06.05.2013

Transformation
Investigator: Franzi
Aim: Preparation of pSB1A3 → Transformation of pSB1A3 J04450.

3 µl of pSB1A3 J04450 (2012) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 60 min 37°C with 900 µl LB), concentrated and plated on LBamp, overnight 37°C.

07.05.2013

Inoculation
Investigator: Franzi
Aim: Preparation of pSB1A3 → Inoculation of pSB1A3 J04450 for miniprep.

Two red colonies were picked and inoculated in 5 ml LBamp each. Incubation overnight at 37° C.

Sequencing
Investigator: Franzi
Aim: Construction of iBB2 (cat) → Examination of sequencing results.

1C3 iBB1 K1 + K2 correct

iBB2 K2 + 4: both positive

08.05.2013

Miniprep
Investigator: Franzi
Aim: Preparation of pSB1A3 → Miniprep of pSB1A3 J04450

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

Digest
Investigator: Franzi
Aim: Preparation of pSB1A3 → Digestion of pSB1A3 J04450 with EcoRI and PstI.
  • 1 µl DNA template
  • 1 µl EcoRI-HF
  • 1 µl PstI-HF
  • 3,6 µl Cut Smart buffer

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 30 µl H2O.

13.05.2013

Digest
Investigator: Franzi
Aim: Construction of antibody-vector → Digestion of various BioBricks.
  • 5 µl DNA template
  • 0,5 µl enzyme 1
  • 0,5 µl enzyme 2
  • 2 µl Cut Smart buffer
  • 12 µl H2O

Samples:

IBB4 K1, iBB6 K1, iBB7 K3 and iBB3 K1 were digested with EcoRI-HF and SpeI-HF.

iBB8K1, iBB4K1, iBB6K1 and iBB1K1 were digested with XbaI and PstI-HF.

The samples were incubated for 1 h at 37° C. Then 1 µl CIP was addend and the samples were incubated for another 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 30 µl elution buffer.
Ligation
Investigator: Franzi
Aim: Construction of antibody-vector → Combination of two BioBricks into vector pSB1A3.
  • 40 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
  • 120 ng insert DNA (variable)
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl H2O

Samples:

iBB4 EcoRI/SpeI with iBB8 XbaI/PstI

iBB6 EcoRI/SpeI with iBB4 XbaI/PstI

iBB7 EcoRI/SpeI with XbaI/PstI

iBB3 EcoRI/SpeI with XbaI/PstI

The samples were incubated for 1 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of Antibody-vector → Transformation of combined 2 BioBricks in pSB1A3 into E. coli DH5α.

Complete ligation samples (see above) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 20 min 37°C with 900 µl LB), concentrated and plated on LBamp, overnight 37°C.

14.05.2013

Inoculation
Investigator: Franzi
Aim: Construction of antibody-vector → Inoculation of transformants.

Colonies from iBB4+8, iBB6+4 and iBB3+1 were picked and inoculated in 5 ml LBamp, respectively.

Ligation
Investigator: Franzi
Aim: Construction of antibody-vector → Ligation of iBB7 and iBB6 into pSB1A3.

Ligation of iBB7 and iBB6 was repeated.

  • 40 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
  • 120 ng insert DNA
  • 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 Antibody-vector → Transformation of combined 2 BioBricks in pSB1A3 into E. coli DH5α.

Complete ligation samples (see above) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 20 min 37°C with 900 µl LB), concentrated and plated on LBamp, overnight 37°C.

15.05.2013

Digest
Investigator: Franzi
Aim: Construction of antibody-vector → Repeat of digestion of various BioBricks (13.05.13).

Liquid cultures and transformation samples from previous day were discarded because of red colonies (Insert J04450). Digestion of BioBricks repeated like 13.5.13, but without CIP.

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 elution buffer.
Ligation
Investigator: Franzi
Aim: Construction of antibody-vector → Repeat of combination of two BioBricks into vector pSB1A3.

Ligation like 13.5.13.

Transformation
Investigator: Franzi
Aim: Construction of Antibody-vector → Repeat of transformation of combined 2 BioBricks in pSB1A3 into E. coli DH5α.

Transformation like 13.5.13.

Inoculation
Investigator: Franzi
Aim: Preparation of pSB1A3 → Inoculation of pSB1A3 J04450.

E. coli pSB1A3 J04450 was inoculated in 5 ml LBamp, overnight 37°C.

16.05.2013

Miniprep
Investigator: Franzi
Aim: Preparation of pSB1A3 → Miniprep of pSB1A3 J04450.

PSB1A3 J04450 (see 15.05.13) was isolated via Miniprep (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)).

Inoculation
Investigator: Franzi
Aim: Construction of antibody-vector → Inoculation of transformants.

4 colonies of each transformation inoculated in 5 ml LBamp, respectively. overnight 37°C.

17.05.2013

Miniprep
Investigator: Franzi
Aim: Construction of antibody-vector → Miniprep of combined 2 BioBricks into pSB1A3.

Miniprep of liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 30 µl H2O.

Digest
Investigator: Franzi
Aim: Construction of antibody-vector → Control digestion of combined 2 BioBricks.
  • 1 µl DNA template
  • 1 µl EcoRI
  • 1 µl PstI
  • 2 µl buffer O
  • 0,15 µl H2O

Samples:

iBB4 + iBB8

iBB6 + iBB4

iBB7 + iBB6

iBB3 + iBB1

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)

Expactations

  • 6+4: 700 bp
  • 7+6: 1000 bp
  • 3+1: 700 bp
  • 4+8: 1900 bp
Digest
Investigator: Franzi
Aim: Construction of antibody-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:

Digest of pSB1A3 iBB6+4 K1+2 and pSB1A3 iBB3+1 K1+4 with XbaI and PstI-HF

PSB1A3 iBB4+8 K1+4 and pSB1A3 iBB7+6 K2+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.

21.05.2013

Ligation
Investigator: Franzi
Aim: Contruction of antibody-vector → ligation of two combined 2 BioBricks into pSB1C3.
  • 30 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 100 ng insert 1
  • 100 ng insert 2
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl H2O

Samples:

iBB4+8 K4 + iBB6+4 K2

iBB7+6 K2 + iBB3+1 K1

The samples were incubated for 1 h at RT.

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

Complete ligation samples (see above) were transformed into E. coli DH5α as described previously (30 min ice, 60 sec 42°C, 60 min 37°C with 900 µl LB), concentrated and plated on LBcm, overnight 37°C.

Transformation
Investigator: Franzi
Aim: Construction of iBB4 → Retransformation into E. coli DH5α.

Retransformation of pSB1C3 iBB4 K1 as described above.

22.05.2013

Ligation
Investigator: Franzi
Aim: Construction of antibody-vector → Repeat of ligation of two combined 2 BioBricks into pSB1C3.

Ligation of pSB1C3 iBB 4+8+6+4 repeated like 21.5.13.

Inoculation
Investigator: Franzi
Aim: Construction of iBB4 → Inoculation of transformants.

2 colonies of pSB1C3 iBB4 K1 were inoculated in 5 ml LBcm, overnight 37°C.

23.05.2013

Miniprep
Investigator: Franzi
Aim: Construction of iBB4 → Miniprep.

Miniprep of pSB1C3 iBB4 K1 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 20 µl H2O.

Inoculation
Investigator: Franzi
Aim: Construction of iBB4 → Inoculation of transformants.

4 colonies of pSB1C3 iBB7631 inoculated in 5 ml LBcm, oN 37°C.

Transformation
Investigator: Franzi
Aim: Construction of iBB4 → Transformation of pSB1C3 iBB4864 into E. coli DH5α.

Ligation of pSB1C3 iBB4864 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.

24.05.2013

Miniprep
Investigator: Franzi
Aim: Construction of antibody-vector → Miniprep of pSB1C3 iBB7631.

Miniprep of pSB1C3 iBB7631 K1-4 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 30 µl H2O.

Digest
Investigator: Franzi
Aim: Construction of antibody-vector → Control digestion of pSB1C3 iBB7631.
  • 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 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)

Expactations

  • Vector: 2150 bp
  • insert (iBB7631): 1610 bp
Inoculation
Investigator: Franzi
Aim: Construction of antibody-vector → Inoculation of transformants.

4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LBcm, oN 37°C.

27.05.2013

Digest
Investigator: Franzi
Aim: Construction of PNR-test-vector and PfcpB-test-vector → digestion of single BioBricks.
  • 5 µl DNA template
  • 0,5 µl enzyme 1
  • 0,5 µl enzyme 2
  • 2 µl Cut Smart buffer
  • 12 µl H2O

Samples:

Digestion of pSB1C3 iBB6, iBB3, iBB5 and iBB4 with XbaI and PstI-HF

Digestion of pSB1C3 iBB6 and iBB1 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 and PfcpB-test-vector → Combination of two BioBricks into pSB1A3.
  • 40 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
  • 120 ng insert 1
  • 120 ng insert 2
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl H2O

Samples:

iBB5 E/S + iBB4 X/P

iBB1 E/S + iBB6 X/P

iBB6 E/S + iBB3 X/P

iBB1 E/S + iBB5 X/P

The samples were incubated for 2,5 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of PNR-test-vector and PfcpB-test-vector → transformation of combined 2 BioBricks into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 20 min 37°C + 900 µl LB), plated on LBamp, oN 37°C.

Ligation
Investigator: Franzi
Aim: Construction of antibody-vector → Repeat of ligation of two combined 2 BioBricks into pSB1C3.
  • 30 ng vector DNA
  • 40 ng iBB48
  • 40 ng iBB64
  • 1 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 10 µl H2O

The samples were incubated for oN at 16° C

Digest
Investigator: Franzi
Aim: Preparation of pSB1C3 → digestion with EcoRI and PstI.
  • 10 µl DNA template (pSB1C3 J04450)
  • 0,5 µl EcoRI-HF
  • 0,5 µl PstI-HF
  • 2 µl Cut Smart buffer
  • 6 µl H2O

The samples were incubated oN at 37° C.

Gel electrophoresis (done 28.05.2013)
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 30 µl H2O.

28.05.2013

PCR
Investigator: Franzi
Aim: Construction of BioBricks → Adding of pre- and suffix via PCR.

PCR of iBB9 (eGFP) with P40+41, iBB10 (SPTP1) with P42+43, iBB11 (SPTP2) with P44+45, iBB12 (SP1) with P46+47 and iBB13 (SP2) with P48+49.

Volume Reagent   Temp (°C) Time
1 µl Phusion-Polymerase   95 3 min
1 µl Primer P1 (1pmol/µl)   95 30 sec
1 µl Primer P2 (1pmol/µl)   55 30 sec x33
1 µl Template   72 45 sec
1 µl dNTPs   72 10 min
10 µl 5xGC buffer  
35 µl ddH20  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

Expactations

  • eGFP: 780 bp
  • SPTP1: 228 bp
  • SPTP2: 147 bp
  • SP1: 123 bp
  • SP2: 108 bp

PCR was repeated (too much template), template diluted 1:50.
Transformation
Investigator: Franzi
Aim: Construction of antibody-vector → Transformation of pSB1C3 iBB4864 into E. coli DH5α.

Ligation of pSB1C3 iBB4864 was 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 PNR-test-vector and PfcpB-test-vector → Inoculation of transformants for miniprep.

2 colonies of each transformation (27.05.13) were inoculated in 5 ml LBamp, oN 37°C

29.05.2013

Miniprep
Investigator: Franzi
Aim: Construction of PNR-test-vector and PfcpB-test-vector → Miniprep of combined 2 BioBricks in pSB1A3.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)). Elution into 30 µl H2O.

Digest
Investigator: Franzi
Aim: Construction of PNR-test-vector and PfcpB-test-vector → Control digestion of combined 2 BioBricks in pSB1A3.
  • 1 µl DNA templates (see above)
  • 0,5 µl EcoRI
  • 0,5 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 7 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)

Expactations

  • 6+3: 500 bp
  • 1+5: 700 bp
  • 5+4: 750 bp
  • 1+6: 650 bp
PCR
Investigator: Franzi
Aim: Construction of BioBricks → Cleaning of PCR-products.

Gel of repeated PCR of iBB9 (eGFP) with P40+41, iBB10 (SPTP1) with P42+43, iBB11 (SPTP2) with P44+45, iBB12 (SP1) with P46+47 and iBB13 (SP2) with P48+49.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

Expactations

  • eGFP: 780 bp
  • SPTP1: 228 bp
  • SPTP2: 147 bp
  • SP1: 123 bp
  • SP2: 108 bp

All positive.
Digest
Investigator: Franzi
Aim: Construction of BioBricks → Digestion with EcoRI and PstI.
  • 29,5 µl DNA templates (PCR-products)
  • 1 µl EcoRI-HF
  • 1 µl PstI-HF
  • 3,5 µl Cut Smart buffer
  • 0,5 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)

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

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