Team:Goettingen/NoteBook w1

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<span class="date">07th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
<div class="cont">
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<p class="timeline-title">Plasmid mini-prep for Part1-7</p>
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<p class="timeline-title goe-rt" title="Click to fold/extend">Plasmid mini-prep for Part1-7</p>
<div class="timeline-cont">
<div class="timeline-cont">
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<b>Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):</b>
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  <p class="MsoNormal"><b><span lang="EN-US">Cryo-Stocks of <i style="mso-bidi-font-style:
-
<ul>
+
  normal">E. coli </i>transformants (C1, parts 1 – 7):<o:p></o:p></span></b></p>
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<li>900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)</li>
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  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
-
<li>vortex</li>
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  l4 level1 lfo1"><!--[if !supportLists]--><span lang="FR" style="font-family:Symbol;
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<li>store at – 70 °C (red box)</li>
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  mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-ansi-language:
-
</ul>
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  FR;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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<b>Plasmid Mini-Preparation of parts 1 - 7:</b>
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  </span></span></span><!--[endif]--><span lang="FR" style="mso-ansi-language:FR;
-
<p>ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids</p>
+
  mso-bidi-font-weight:bold">900 μl <i style="mso-bidi-font-style:normal">E.coli</i>
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<p>Step 5: recommended washing of silica membrane with buffer AW was performed</p>
+
  ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)<o:p></o:p></span></p>
-
<p>Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H2O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)</p>
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  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
-
<br / >
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  l4 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
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<p>NanoDrop – Plasmid concentrations</p>
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  Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-bidi-font-weight:
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<table cellspacing="0">
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  bold"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
<tr><th>Part Number</th><th>c(DNA)[ng/μl]</th><th>A<sub>260</sub>/A<sub>280</sub></th><th>A<sub>260</sub>/A<sub>230</sub></th>
+
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-
<tr><td>1</td><td>84.6</td><td>1.94</td><td>2.18</td></tr>
+
  bold">vortex<o:p></o:p></span></p>
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<tr><td>2</td><td>79.8</td><td>1.94</td><td>2.10</td></tr>
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  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
-
<tr><td>3</td><td>151.0</td><td>1.89</td><td>2.24</td></tr>
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  l4 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
-
<tr><td>4</td><td>29.5</td><td>1.91</td><td>2.07</td></tr>
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  Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-bidi-font-weight:
-
<tr><td>5</td><td>154.9</td><td>1.88</td><td>2.25</td></tr>
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  bold"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
<tr><td>6</td><td>88.9</td><td>1.92</td><td>2.08</td></tr>
+
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-
<tr><td>7</td><td>5.9</td><td>14.08</td><td>1.87</td></tr>
+
  bold">store at – 70 °C (red box)<o:p></o:p></span></p>
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</table>
+
  <p class="MsoNormal"><b><span lang="EN-US">Backup plates:<o:p></o:p></span></b></p>
-
<p>Stored in red box at - 20°C</p>
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  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Storage
-
<b>Primers arrived!</b>
+
  at 4 °C<o:p></o:p></span></p>
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<p>iGEM_32 ~ 37: dissolved in HPLC water, stored at -20oC in our box</p>
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  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
-
<p>100uM stock , for PCR dilute 1:20 in HPLC water.</p>
+
  <p class="MsoNormal"><b><span lang="EN-US">Plasmid Mini-Preparation of parts 1 -
-
<p>Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR.</p>
+
  7:<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">ON
 +
  cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von
 +
  Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol
 +
  for purification of high copy plasmids)<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><u><span lang="EN-US" style="mso-bidi-font-weight:bold">Step
 +
  5:</span></u><span lang="EN-US" style="mso-bidi-font-weight:bold"> recommended
 +
  washing of silica membrane with buffer AW was performed<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><u><span lang="EN-US" style="mso-bidi-font-weight:bold">Step
 +
  7:</span></u><span lang="EN-US" style="mso-bidi-font-weight:bold"> <b><u><span style="color:#92D050">Elution</span></u></b> with buffer AE pre-heated to 50
 +
  – 60 °C <span style="color:red">(in future: DON’T elute with this buffer, </span><b><u><span style="color:#92D050">use pre-heated HPLC-H<sub>2</sub>O</span></u></b><span style="color:red"> instead! No one knows what’s inside the buffer and its
 +
  components (EDTA?) could interfere with sequencing and other reactions)<o:p></o:p></span></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">NanoDrop
 +
  – Plasmid concentrations<o:p></o:p></span></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid windowtext .5pt;
 +
  mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;
 +
  mso-border-insidev:.5pt solid windowtext">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Part no.<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">c(DNA) </span></b><b><span lang="EN-US" style="font-family:Calibri">[</span><span lang="EN-US">ng/μl]<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">A<sub>260</sub>/A<sub>280</sub><o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">A<sub>260</sub>/A<sub>230</sub><o:p></o:p></span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">84.6<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.94<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.18<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">79.8<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.94<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.10<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">3<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">151.0<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.89<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.24<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">4<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">29.5<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.91<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.07<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:5">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">5<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">154.9<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
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    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
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 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.88<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.25<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:6">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">6<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">88.9<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.92<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.08<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:7;mso-yfti-lastrow:yes">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">7<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">5.9<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">14.08<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.87<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
 
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Stored
 +
  in red box at - 20°C<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Primers arrived!<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US">iGEM_32 ~ 37: dissolved in HPLC water,
 +
  stored at -20<sup>o</sup>C in our box<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US">100uM stock , for PCR dilute 1:20 in HPLC
 +
  water.<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><i><span lang="EN-US">Primer 32 and 33 are strong in forming
 +
  2<sup>nd</sup> structures, increase the amount in PCR<o:p></o:p></span></i></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">Preparation of cryo-cultures of #811,
 +
  814, 1011, 1012 and 1013</p>
 +
<div class="timeline-cont">
 +
 
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.0pt">Preparation of cryo-cultures of #811,
 +
  814, 1011, 1012 and 1013 </span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">preparation of cryo-cultures: </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">- take 900 μl of overnight culture </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="DE" style="font-size:11.0pt">- add 100 </span><span lang="EN-US" style="font-size:11.0pt">μ</span><span lang="DE" style="font-size:11.0pt">l
 +
  100% DMSO (filter sterile) </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">→ store at - 80°C </span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
</div>
</div>
</div>
</div>
-
 
+
<br />
-
 
+
-
 
+
<div class="tlob" id="tl_0606">
<div class="tlob" id="tl_0606">
-
<span class="date">06th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
+
<span class="date">06th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
<div class="cont">
<div class="cont">
-
<p class="timeline-title">Pick the colonies of part1-7</p>
+
<p class="timeline-title goe-rt" title="Click to fold/extend">Pick the colonies of part1-7</p>
<div class="timeline-cont">
<div class="timeline-cont">
-
<b>Media preparation</b>
+
-
<p>1000ml LB+Amp Solid medium => about 50 Plates(with black code)</p>
+
  <p class="MsoNormal"><b><span lang="EN-US">Media preparation:<o:p></o:p></span></b></p>
-
<b>Transformation</b>
+
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1000ml
-
<table cellspacing="0">
+
  LB+Ampicillin Agar =&gt; ca. 50 Plates (Black Code)<o:p></o:p></span></p>
-
<tr><th>Entry Numer</th><th>Location on the kit</th></tr>
+
  <p class="MsoNormal"><b><span lang="EN-US">Transformation:<o:p></o:p></span></b></p>
-
<tr><td>BBa_B0034</td><td>P5 2 M</td></tr>
+
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid windowtext .5pt;
-
</table>
+
  mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid windowtext;
-
<b>Pick the colonies of Part1-7</b>
+
  mso-border-insidev:.5pt solid windowtext">
-
<p>4ml LB with antibiotics, overnight culture for mini-prep[marked with C1]</p>
+
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
-
<p>Backup plates:marked with C1,C2,C3 for each part.</p>
+
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Number<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">whereabout<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;mso-yfti-lastrow:yes">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">B0034<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">P5 2
 +
    M<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
 
 +
  <p class="MsoNormal"><b><span lang="EN-US">Colonies on the plate: parts 1- 7:<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US">4ml LB with antibiotics, overnight
 +
  culture for mini-prep[C1]<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US">Backup plates: C1,C2,C3 for each part<span style="mso-bidi-font-weight:bold"><o:p></o:p></span></span></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
 +
<p class="timeline-title goe-dac">Continue of transformation of competent <i>E.
 +
  coli </i>cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)</p>
 +
<div class="timeline-cont">
 +
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.0pt">Continue of transformation of competent <i>E.
 +
  coli </i>cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101) </span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">only clones #811 and #814 (expressing pGP172
 +
  + cdaA) grew overnight </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">→ inoculate them in liquid LB media </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">→ grow normally in LB media clones #
 +
  1011, 1012, 1013 were directly inoculated in liquid LB medium </span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
 +
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
</div>
</div>
 +
</div>
 +
<br />
<div class="tlob" id="tl_0605">
<div class="tlob" id="tl_0605">
<span class="date">05th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
<span class="date">05th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
<div class="cont">
<div class="cont">
-
<p class="timeline-title">Preparation of the medium, antibioticks, Transformation.</p>
+
<p class="timeline-title goe-rt">Preparation of the medium, antibioticks, Transformation.</p>
<div class="timeline-cont">
<div class="timeline-cont">
-
<b>Preparation of Antibiotic Stocks</b>
+
 
-
<ul>
+
  <p class="MsoNormal"><b><span lang="EN-US">Preparation of Antibiotic Stocks<o:p></o:p></span></b></p>
-
<li>1000x Ampicillin 10 1mL Stocks (EPs in the red box in -20 freezer)</li>
+
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1000x
-
<li>1000x Chloramphenicol 10mL Stock (Falcon in Freezer)</li>
+
  Ampicillin 10 1mL Stocks (Freezer red box)<o:p></o:p></span></p>
-
</ul>
+
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1000x
-
<b>Media preparation</b>
+
  Chloramphenicol 10mL Stock (Falcon in Freezer)<o:p></o:p></span></p>
-
<ul><li>250ml*4 LB media with Cm </li>
+
 
-
<li>250ml*1 LB media with Amp </li>
+
  <p class="MsoNormal"><b><span lang="EN-US">Media preparation:<o:p></o:p></span></b></p>
-
</ul>
+
  <p class="MsoNormal"><b><span lang="EN-US"><span style="mso-spacerun:yes">&nbsp;</span></span></b><span lang="EN-US">250ml *4
-
<b>Primer design</b>
+
  Chl</span></p>
-
<table cellspacing="0">
+
  <p class="MsoNormal"><span lang="EN-US"><span style="mso-spacerun:yes">&nbsp;</span><i>[<span class="GramE">use</span> the
-
<tr><th>Code Name</th><th>   Description</th></tr>
+
  ones marked with “5<sup>th.</sup>.6.13 LB<sup>chl</sup>” on EVERY
-
<tr><td>iGEM_36</td><td>   DarR operator sequence + biobrick prefix</td></tr>
+
  plate first. ]*</i></span></p>
-
<tr><td>iGEM_37</td><td>   DarR operator sequence + biobrick sufix</td></tr>
+
  <p class="MsoNormal"><span lang="EN-US"><span style="mso-spacerun:yes">&nbsp;</span>250ml *1 Amp<span style="mso-spacerun:yes">&nbsp; </span>(marked with black) </span></p>
-
</table><br />
+
 
-
<b>Transformation</b>
+
  <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">primer
-
<table cellspacing="0">
+
  design</span></b><span lang="EN-US">&lt;ordered&gt;</span></p>
-
<tr><th>Biobrick entry number</th><th>Mark</th></tr>
+
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;mso-table-layout-alt:fixed;border:none;
-
<tr><td>BBa_J23117</td><td>   part 1</td></tr>
+
  mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;
-
<tr><td>BBa_J23116</td><td>   part 2</td></tr>
+
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
-
<tr><td>BBa_J23110</td><td>   part 3</td></tr>
+
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes;height:15.0pt">
-
<tr><td>BBa_J23118</td><td>   part 4</td></tr>
+
    <td width="104" valign="bottom" style="width:78.0pt;border:solid windowtext 1.0pt;
-
<tr><td>BBa_J61101</td><td>   part 5</td></tr>
+
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
-
<tr><td>BBa_BE0240-Cm</td><td>   part 6 -- Cm</td></tr>
+
    height:15.0pt">
-
<tr><td>BBa_B0015</td><td>   part 7 -- Cm</td></tr><tr>
+
    <p class="MsoNormal" align="left" style="text-align:left;mso-pagination:widow-orphan"><span lang="DE" style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:
-
<td>BBa_B0034</td><td>   part 8</td></tr>
+
    &quot;Times New Roman&quot;;color:black;mso-font-kerning:0pt;mso-ansi-language:DE;
-
</table>
+
    mso-fareast-language:DE">iGEM_36<o:p></o:p></span></p>
-
Resuspended but not transformed
+
    </td>
-
<table>
+
    <td width="522" valign="bottom" style="width:391.6pt;border:solid windowtext 1.0pt;
-
<tr><th>Entry number</th><th>   location on kit</th></tr>
+
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
-
<tr><td>BBa_E0204-Amp</td><td>   P5 12 M</td></tr>
+
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
-
<tr><td>BBa_QO3121</td><td>   P5 20 N</td></tr>
+
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
-
</table>
+
    &quot;Courier New&quot;;color:black">DarR operator sequence + prefix<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;mso-yfti-lastrow:yes;height:15.0pt">
 +
    <td width="104" valign="bottom" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal" align="left" style="text-align:left;mso-pagination:widow-orphan"><span lang="DE" style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:
 +
    &quot;Times New Roman&quot;;color:black;mso-font-kerning:0pt;mso-ansi-language:DE;
 +
    mso-fareast-language:DE">iGEM_37<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="522" valign="bottom" style="width:391.6pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">DarR operator sequence + suffix<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p></o:p></span></b></p>
 +
 
 +
  <p class="MsoNormal"><b><span lang="EN-US">Transformation:<o:p></o:p></span></b></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;mso-table-layout-alt:fixed;border:none;
 +
  mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
 +
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">Number</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">Mark</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23117</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">1</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23116</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">2</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23110</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">3</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23118</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">4</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:5">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J61101</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">5</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:6">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">BBa_BE0240-Chl</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">6 ---Chl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:7">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">BBa_B0015</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">7----Chl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:8;mso-yfti-lastrow:yes">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">B0034</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">8</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Resuspended DNA from iGEM kit </span></b><span lang="EN-US" style="mso-bidi-font-weight:bold">(already stored in red box in
 +
  -20 frigde)<b>:<o:p></o:p></b></span></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;mso-table-layout-alt:fixed;border:none;
 +
  mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;
 +
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Number<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">whereabout<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">BBa_E0204-Amp<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">P5 12
 +
    M<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2;mso-yfti-lastrow:yes">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">BBa_QO3121<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">P5 20
 +
    N<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p></o:p></span></b></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-array">Transformation of GP 911</p>
 +
<div class="timeline-cont">
 +
 
 +
  <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;;text-decoration:none">&nbsp;</span></h2>
 +
  <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;;text-decoration:none">Transformation of GP 911</span></h2>
 +
  <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">According
 +
  to the protocoll of AG Stülke</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent:
 +
  -18.0pt"><span lang="EN-GB" style="font-family:&quot;Courier New&quot;">o<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp; </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Started at 8.00; OD<sub>600</sub>
 +
  = 1.4 at 11.00h</span></p>
 +
  <p class="MsoListParagraphCxSpLast" style="margin-left:72.0pt;"><span lang="EN-GB" style="font-family:&quot;Courier New&quot;">o<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">For
 +
  the expression mix, the CSE supernatant was used instead of water.</span></p>
 +
  <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Results:</span></p>
 +
  <table class="MsoTableGrid" border="0" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr style="height:129.75pt">
 +
    <td width="247" valign="top" style="width:185.45pt;padding:0cm 5.4pt 0cm 5.4pt;
 +
    height:129.75pt">
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    <p class="MsoNormal"><img width="257" height="132" src="https://static.igem.org/mediawiki/2013/6/66/Goe-arr-Image002.gif" align="left" hspace="12" alt="Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130605_Ergenisse erste Trafe:130605_Feeding_1.tif"></p>
 +
    </td>
 +
    <td width="247" valign="top" style="width:185.5pt;padding:0cm 5.4pt 0cm 5.4pt;
 +
    height:129.75pt">
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">controls on LB medium, containig the appropriate AB.<br>
 +
    left: neg. control: 991 + water<br>
 +
    right: pos. Control: 991 + BP121 (another cat cassette)</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="height:133.75pt">
 +
    <td width="247" valign="top" style="width:185.45pt;padding:0cm 5.4pt 0cm 5.4pt;
 +
    height:133.75pt">
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    <p class="MsoNormal"><img width="257" height="132" src="https://static.igem.org/mediawiki/2013/0/0d/Goe-arr-Image003.gif" align="left" hspace="12" alt="Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130605_Ergenisse erste Trafe:130605_Feeding_2.tif"></p>
 +
    </td>
 +
    <td width="247" valign="top" style="width:185.5pt;padding:0cm 5.4pt 0cm 5.4pt;
 +
    height:133.75pt">
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">the pos. control on LB medium with sterile filtrated
 +
    supernatant</span><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;"> </span></p>
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="247" valign="top" style="width:185.45pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    <p class="MsoNormal"><img width="259" height="132" src="https://static.igem.org/mediawiki/2013/e/e5/Goe-arr-Image004.gif" align="left" hspace="12" alt="Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130605_Ergenisse erste Trafe:130605_Feeding_3.tif"></p>
 +
    </td>
 +
    <td width="247" valign="top" style="width:185.5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">the pos. control on LB medium with cooked and sterile
 +
    filtrated supernatant</span><span lang="EN-GB" style="font-size:11.0pt;
 +
    font-family:&quot;Arial&quot;,&quot;sans-serif&quot;"> </span></p>
 +
    <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">No
 +
  growth of the actual experiment on these plates (GP 991 + GP 997)</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">This
 +
  might be due to the fact that the expression mix, used during the trafo,
 +
  contained the supernatant containing all three antibiotics (E/L, tet, cat)</span></p>
 +
  <p class="MsoListParagraphCxSpLast" style="margin-left:72.0pt;"><span lang="EN-US" style="font-family:&quot;Courier New&quot;">o<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Redo
 +
  this next week with supernatant lacking the AB’s</span></p>
 +
 
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">Continue of transformation of competent <i>E.coli </i>cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)</p>
 +
<div class="timeline-cont">
 +
<td width="643" valign="top" style="width:17.0cm;border-top:none;border-left:
 +
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.0pt">Continue of transformation of competent <i>E.
 +
  coli </i>cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101) </span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><i><span lang="EN-US" style="font-size:11.0pt">E. coli</span></i><span lang="EN-US" style="font-size:11.0pt"> cells expressing pGP172 + cdaA (from #81) are
 +
  growing very slowly and have to be incubated longer </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">→ chose four clones and name them: #811,
 +
  812, 813 and 814 </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">→ re-streak the om LB Amp100 plates </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><i><span lang="EN-US" style="font-size:11.0pt">E. coli</span></i><span lang="EN-US" style="font-size:11.0pt"> cells expressing pGP172 + DAC (from #101) are
 +
  growing normally </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">→ chose three clones and name them:
 +
  #1011, 1012 and 1013 </span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  </td>
 +
 
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
</div>
</div>
</div>
</div>
 +
<br />
-
<div class="tlob start" id="tl_0604">
+
<div class="tlob" id="tl_0604">
<span class="date">04th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
<span class="date">04th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
<div class="cont">
<div class="cont">
-
<p class="timeline-title">Find the correct DNA sequence of DarR and primer design</p>
+
<p class="timeline-title goe-rt">Find the correct DNA sequence of DarR and primer design</p>
<div class="timeline-cont">
<div class="timeline-cont">
-
<table cellspacing="0">
+
  <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">primer
-
<tr><th>Code Name</th><th>  Description</th></tr>
+
  design</span></b><span lang="EN-US">&lt;ordered&gt;, found the correct sequence
-
<tr><td>iGEM_32</td><td>   Forward primer for DarR ORF amplification, with biobrick prefix</td></tr>
+
   for DarR </span></p>
-
<tr><td>iGEM_33</td><td>   Reverse primer for DarR ORF amplification, with biobrick sufix</td></tr>
+
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:2.55pt;border-collapse:collapse;mso-table-layout-alt:
-
<tr><td>iGEM_34</td><td>   Forward primer for DarR ORF PCR amplification sequencing.</td></tr>
+
  fixed;border:none;mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;
-
<tr><td>iGEM_35</td><td>   Reverse primer for DarR ORF PCR amplification sequencing.</td></tr>
+
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
-
</table>
+
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_32<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR + Prefix forw.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_33<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR + Suffix rev.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_34<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR sequencing forw.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3;mso-yfti-lastrow:yes;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_35<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR sequencing rev.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US"><o:p></o:p></span></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
 +
<p class="timeline-title goe-array">Creation of c-di-AMP supernatant,
 +
  which is used for the plates</p>
 +
<div class="timeline-cont">
 +
  <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;;text-decoration:none">Creation of c-di-AMP supernatant,
 +
  which is used for the plates</span></h2>
 +
  <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoListParagraphCxSpFirst" style=";line-height:
 +
  normal"><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-
 +
  growth curve of 168 in CSE:<br>
 +
  9.00h: &nbsp; &nbsp; 0,1<br>
 +
  11.00h: &nbsp; 0.37/0.384<br>
 +
  12.00h: &nbsp; 0.72/0.71<br>
 +
  12.30h: &nbsp; 0.99/0.95</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">continued
 +
  as described on 29.05.13</span></p>
 +
  <p class="MsoListParagraphCxSpLast" style="margin-left:72.0pt;"><span lang="EN-GB" style="font-family:&quot;Courier New&quot;">o<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;
 +
  </span></span><span lang="EN-GB" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Addition:
 +
  the prepared falcons containing the 25 ml supernatant were preheated in the
 +
  37 °C room. That’s makes pouring the plates easier</span></p>
 +
  <p class="MsoNormal"><span lang="DE" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
 +
<p class="timeline-title goe-dac">Sequencing of plasmids from clone #81 and #101<br />Transformation of competent <i>E. coli</i>cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)</p>
 +
<div class="timeline-cont">
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.0pt">Sequencing of plasmids from clone #81 and
 +
  #101 </span></b></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="137" valign="top" style="width:102.55pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">Component</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.55pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">81F</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">81R</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">101f</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">101R</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="137" valign="top" style="width:102.55pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">DNA</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.55pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">10</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">10</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">10</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">10</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="137" valign="top" style="width:102.55pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">Forward primer</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.55pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">4</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">-</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">4</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">-</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="137" valign="top" style="width:102.55pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">Reverse primer</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.55pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">-</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">4</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">-</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">4</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="137" valign="top" style="width:102.55pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">Total</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.55pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">14</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">14</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">14</span></p>
 +
    </td>
 +
    <td width="137" valign="top" style="width:102.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">14</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;Sent
 +
  for sequencing! </span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.0pt">Transformation of competent <i>E. coli </i>cells
 +
  with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101) </span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Transform competent E. coli cells
 +
  according to the protocoll mentioned above (30.05.2913) with pGP172 + cdaA or
 +
  pGP172 + DAC, respectively. </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">For the transformation were 5 μl of
 +
  plasmid DNA used. The plates were incubated overnight at 37°C. </span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
 +
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
 +
</div>
 +
</div>
 +
<br />
 +
 +
<div class="tlob" id="tl_0603">
 +
<span class="date">03rd<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 +
<div class="cont">
 +
<p class="timeline-title goe-dac">Gel electrophoresis of colony PCR from 31.05.2013<br />Digestion of pGP172 + cdaA (<i>L. monocytogenes</i>) and pGP172 + DAC domain (<i>L. monocytogenes</i>) with SacI and BamHI </p>
 +
<div class="timeline-cont">
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.0pt">Gel electrophoresis of colony PCR from
 +
  31.05.2013 </span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">The obtained PCR products were analyszed
 +
  on 1% agarose gel. Clones #81 – #90 should contain pGP172 + cdaA (<i>L.
 +
  monocytogenes</i>). Clones #101 – #110 should obtain pGP172 + DAC domain (<i>L.
 +
  monocytogenes</i>). Therefore a band of about 900 bp were expected for clones
 +
  #81 – #90 and a band of about 600 bp for clones #101 - #110 </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Gel: Marker (100 bp ladder) | clones #81
 +
  – #90 | clones #101 – #102 | Marker (1kb ladder) </span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;"><img border="0" width="395" height="212" id="图片 22" src="https://static.igem.org/mediawiki/2013/3/37/Goe-dac-Image010.gif"></span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Gel: Marker (100 bp ladder) | clones #103
 +
  – #110 | control (w/o insert) </span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;"><img border="0" width="387" height="277" id="图片 23" src="https://static.igem.org/mediawiki/2013/2/25/Goe-dac-Image011.gif"></span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">For all clones could be a specific band
 +
  and therefore also a specific PCR product were obtained. The plasmids from
 +
  clone #81 and #101 were extracted and purified by </span><span lang="EN-US" style="font-size:11.0pt;color:red">peqlab Miniprep kit </span><span lang="EN-US" style="font-size:11.0pt">according to the manufacturer's
 +
  protocoll. </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">→ The concentration was measured by
 +
  NanoDrop: 81/cdaA (<i>L. monocytogenes</i>): 27 ng/μl 101/DAC domain (<i>L.
 +
  monocytogenes</i>): 27 ng/μl </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">&nbsp;</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.0pt">Digestion of pGP172 + cdaA (<i>L.
 +
  monocytogenes</i>) and pGP172 + DAC domain (<i>L. monocytogenes</i>) with
 +
  SacI and BamHI </span></b></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Components</span></p>
 +
    </td>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">pGP172 + cdaA </span></p>
 +
    </td>
 +
    <td width="183" valign="top" style="width:137.1pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">&nbsp;pGP172 + DAC domain </span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Plasmid (300 ng) </span></p>
 +
    </td>
 +
    <td width="127" valign="top" style="width:95.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">11.1</span></p>
 +
    </td>
 +
    <td width="183" valign="top" style="width:137.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">11.1</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">SacI </span></p>
 +
    </td>
 +
    <td width="127" valign="top" style="width:95.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">1</span></p>
 +
    </td>
 +
    <td width="183" valign="top" style="width:137.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">1</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">BamHI </span></p>
 +
    </td>
 +
    <td width="127" valign="top" style="width:95.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">1</span></p>
 +
    </td>
 +
    <td width="183" valign="top" style="width:137.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">1</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Fast Digest Buffer</span></p>
 +
    </td>
 +
    <td width="127" valign="top" style="width:95.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">1</span></p>
 +
    </td>
 +
    <td width="183" valign="top" style="width:137.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">1</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">HPLC H2O </span></p>
 +
    </td>
 +
    <td width="127" valign="top" style="width:95.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">5.9</span></p>
 +
    </td>
 +
    <td width="183" valign="top" style="width:137.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">5.9</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="127" valign="top" style="width:95.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Total </span></p>
 +
    </td>
 +
    <td width="127" valign="top" style="width:95.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">20</span></p>
 +
    </td>
 +
    <td width="183" valign="top" style="width:137.1pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">20</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">The digestion products were analyzed on
 +
  1% agarose gel. </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">Gel: Marker (100 bp) | undigested pGP172
 +
  | cdaA insert | DAC insert | ligation control | digested pGP172 + cdA |
 +
  digested pGP172 + DAC </span></p>
 +
  <table class="MsoTableGrid" border="0" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="210" valign="top" style="width:157.2pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt"><img border="0" width="188" height="271" id="图片 24" src="https://static.igem.org/mediawiki/2013/8/81/Geo-dac-Image012.gif"></span></p>
 +
    </td>
 +
    <td width="418" valign="top" style="width:313.15pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">The digested plasmids show bands for
 +
    the undigested pGP172, for the corresponding insert as well as a band for
 +
    the undigested pGP172 including the insert. Furthermore could be an
 +
    additional band in the ligation control as well as in the digested pGP172 +
 +
    cdA and the digested pGP172 + DAC observed. This could be due to unspecific
 +
    digestion or the digestion by only one enzyme. Therefore the cloning seems
 +
    to be successful and should be further verified by sequencing! </span></p>
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">&nbsp;</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"></p>
 +
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
</div>
 +
</div>
 +
<br />
 +
</html>
</html>

Latest revision as of 21:23, 30 September 2013

07th

Plasmid mini-prep for Part1-7

Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):

·         900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)

·         vortex

·         store at – 70 °C (red box)

Backup plates:

Storage at 4 °C

 

Plasmid Mini-Preparation of parts 1 - 7:

ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids)

Step 5: recommended washing of silica membrane with buffer AW was performed

Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H2O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)

 

NanoDrop – Plasmid concentrations

Part no.

c(DNA) [ng/μl]

A260/A280

A260/A230

1

84.6

1.94

2.18

2

79.8

1.94

2.10

3

151.0

1.89

2.24

4

29.5

1.91

2.07

5

154.9

1.88

2.25

6

88.9

1.92

2.08

7

5.9

14.08

1.87

Stored in red box at - 20°C

Primers arrived!

iGEM_32 ~ 37: dissolved in HPLC water, stored at -20oC in our box

100uM stock , for PCR dilute 1:20 in HPLC water.

Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR

Fold ↑

Preparation of cryo-cultures of #811, 814, 1011, 1012 and 1013

Preparation of cryo-cultures of #811, 814, 1011, 1012 and 1013

preparation of cryo-cultures:

- take 900 μl of overnight culture

- add 100 μl 100% DMSO (filter sterile)

→ store at - 80°C

 

Fold ↑

06th

Pick the colonies of part1-7

Media preparation:

1000ml LB+Ampicillin Agar => ca. 50 Plates (Black Code)

Transformation:

Number

whereabout

B0034

P5 2 M

Colonies on the plate: parts 1- 7:

4ml LB with antibiotics, overnight culture for mini-prep[C1]

Backup plates: C1,C2,C3 for each part

Fold ↑

Continue of transformation of competent E. coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

Continue of transformation of competent E. coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

only clones #811 and #814 (expressing pGP172 + cdaA) grew overnight

→ inoculate them in liquid LB media

→ grow normally in LB media clones # 1011, 1012, 1013 were directly inoculated in liquid LB medium

 

Fold ↑

05th

Preparation of the medium, antibioticks, Transformation.

Preparation of Antibiotic Stocks

1000x Ampicillin 10 1mL Stocks (Freezer red box)

1000x Chloramphenicol 10mL Stock (Falcon in Freezer)

Media preparation:

 250ml *4 Chl

 [use the ones marked with “5th..6.13 LBchl” on EVERY plate first. ]*

 250ml *1 Amp  (marked with black)

primer design<ordered>

iGEM_36

DarR operator sequence + prefix

iGEM_37

DarR operator sequence + suffix

Transformation:

Number

Mark

J23117

1

J23116

2

J23110

3

J23118

4

J61101

5

BBa_BE0240-Chl

6 ---Chl

BBa_B0015

7----Chl

B0034

8

Resuspended DNA from iGEM kit (already stored in red box in -20 frigde):

Number

whereabout

BBa_E0204-Amp

P5 12 M

BBa_QO3121

P5 20 N

Fold ↑

Transformation of GP 911

 

 

Transformation of GP 911

·         According to the protocoll of AG Stülke

o   Started at 8.00; OD600 = 1.4 at 11.00h

o   For the expression mix, the CSE supernatant was used instead of water.

Results:

 

Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130605_Ergenisse erste Trafe:130605_Feeding_1.tif

controls on LB medium, containig the appropriate AB.
left: neg. control: 991 + water
right: pos. Control: 991 + BP121 (another cat cassette)

 

Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130605_Ergenisse erste Trafe:130605_Feeding_2.tif

the pos. control on LB medium with sterile filtrated supernatant

 

 

Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130605_Ergenisse erste Trafe:130605_Feeding_3.tif

the pos. control on LB medium with cooked and sterile filtrated supernatant

 

 

·         No growth of the actual experiment on these plates (GP 991 + GP 997)

·         This might be due to the fact that the expression mix, used during the trafo, contained the supernatant containing all three antibiotics (E/L, tet, cat)

o   Redo this next week with supernatant lacking the AB’s

Fold ↑

Continue of transformation of competent E.coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

Continue of transformation of competent E. coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

E. coli cells expressing pGP172 + cdaA (from #81) are growing very slowly and have to be incubated longer

→ chose four clones and name them: #811, 812, 813 and 814

→ re-streak the om LB Amp100 plates

E. coli cells expressing pGP172 + DAC (from #101) are growing normally

→ chose three clones and name them: #1011, 1012 and 1013

 

Fold ↑

04th

Find the correct DNA sequence of DarR and primer design

primer design<ordered>, found the correct sequence for DarR

iGEM_32

Primer DarR + Prefix forw.

iGEM_33

Primer DarR + Suffix rev.

iGEM_34

Primer DarR sequencing forw.

iGEM_35

Primer DarR sequencing rev.

Fold ↑

Creation of c-di-AMP supernatant, which is used for the plates

Creation of c-di-AMP supernatant, which is used for the plates

 

·         - growth curve of 168 in CSE:
9.00h:     0,1
11.00h:   0.37/0.384
12.00h:   0.72/0.71
12.30h:   0.99/0.95

·         continued as described on 29.05.13

o   Addition: the prepared falcons containing the 25 ml supernatant were preheated in the 37 °C room. That’s makes pouring the plates easier

 

Fold ↑

Sequencing of plasmids from clone #81 and #101
Transformation of competent E. colicells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

Sequencing of plasmids from clone #81 and #101

Component

81F

81R

101f

101R

DNA

10

10

10

10

Forward primer

4

-

4

-

Reverse primer

-

4

-

4

Total

14

14

14

14

 Sent for sequencing!

 

Transformation of competent E. coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

Transform competent E. coli cells according to the protocoll mentioned above (30.05.2913) with pGP172 + cdaA or pGP172 + DAC, respectively.

For the transformation were 5 μl of plasmid DNA used. The plates were incubated overnight at 37°C.

 

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03rd

Gel electrophoresis of colony PCR from 31.05.2013
Digestion of pGP172 + cdaA (L. monocytogenes) and pGP172 + DAC domain (L. monocytogenes) with SacI and BamHI

Gel electrophoresis of colony PCR from 31.05.2013

The obtained PCR products were analyszed on 1% agarose gel. Clones #81 – #90 should contain pGP172 + cdaA (L. monocytogenes). Clones #101 – #110 should obtain pGP172 + DAC domain (L. monocytogenes). Therefore a band of about 900 bp were expected for clones #81 – #90 and a band of about 600 bp for clones #101 - #110

Gel: Marker (100 bp ladder) | clones #81 – #90 | clones #101 – #102 | Marker (1kb ladder)

Gel: Marker (100 bp ladder) | clones #103 – #110 | control (w/o insert)

For all clones could be a specific band and therefore also a specific PCR product were obtained. The plasmids from clone #81 and #101 were extracted and purified by peqlab Miniprep kit according to the manufacturer's protocoll.

→ The concentration was measured by NanoDrop: 81/cdaA (L. monocytogenes): 27 ng/μl 101/DAC domain (L. monocytogenes): 27 ng/μl

 

Digestion of pGP172 + cdaA (L. monocytogenes) and pGP172 + DAC domain (L. monocytogenes) with SacI and BamHI

Components

pGP172 + cdaA

 pGP172 + DAC domain

Plasmid (300 ng)

11.1

11.1

SacI

1

1

BamHI

1

1

Fast Digest Buffer

1

1

HPLC H2O

5.9

5.9

Total

20

20

The digestion products were analyzed on 1% agarose gel.

Gel: Marker (100 bp) | undigested pGP172 | cdaA insert | DAC insert | ligation control | digested pGP172 + cdA | digested pGP172 + DAC

The digested plasmids show bands for the undigested pGP172, for the corresponding insert as well as a band for the undigested pGP172 including the insert. Furthermore could be an additional band in the ligation control as well as in the digested pGP172 + cdA and the digested pGP172 + DAC observed. This could be due to unspecific digestion or the digestion by only one enzyme. Therefore the cloning seems to be successful and should be further verified by sequencing!

 

 

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