Team:Goettingen/NoteBook w2

From 2013.igem.org

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<span class="date">08th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
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<span class="date">14th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
<div class="cont">
<div class="cont">
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<p class="timeline-title">Cloning DarR ORF from g-DNA</p>
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<p class="timeline-title goe-rt">Observation of Back-up plates and preparation of media</p>
<div class="timeline-cont">
<div class="timeline-cont">
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<b>Colonies on the plates: parts 8, 7 C1</b>
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<p>10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep</p>
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  <p class="MsoNormal"><b><span lang="EN-US">Observation of Back-up plates<o:p></o:p></span></b></p>
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<p>backup plates: C1, C2, C3 for part 8</p>
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  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
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<b>Colonies on the plates: parts 8, 7 C1</b>
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  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
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<p>Preparation of 250 ml LB broth media (stored on shelf above bench)</p>
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  &quot;Times New Roman&quot;;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;&nbsp;
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<p>10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep</p>
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  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-
<p>backup plates: C1, C2, C3 for part 8</p>
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  bold">Part 1, C1 and C2 probably very light pink<o:p></o:p></span></p>
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<b>Re-streak of E.coli clones from prtas 6 and 7 on new LBChloram plates</b>
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  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
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<b>Preparation of new LBChloram plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf above bench)</b>
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  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
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<b>PCR with DarR primers with different enzymes and different buffers</b>
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  &quot;Times New Roman&quot;;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;&nbsp;
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<ul>
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  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
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<li>dilution of primer stocks 1:20 (100 pmol -> 5 pmol):95 μl HPLC water + 5 μl primer 100 pmol</li>
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  bold">Part 2, C2 and C3 became light pink, but C1 stayed white (compare with
-
<li>preparation of dNTP mix -> dilute stocks 1:8 (100 mM ? 12.5 mM):50μl dNTP + 200μl dH<sub>2</sub>O</li>
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  restriction analysis <st1:chsdate isrocdate="False" islunardate="False" day="30" month="12" year="1899" w:st="on">13.6.13</st1:chsdate>)<o:p></o:p></span></p>
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<li>diluted primers and dNTP mix stored in red box at -20 °C</li>
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  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
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</ul>
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  <p class="MsoNormal"><b><span lang="EN-US">Preparation of media for inoculation
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<p>1x reaction(50μl)</p>
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  on Sunday (by Katrin Gunka)<o:p></o:p></span></b></p>
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<table cellspacing="0">
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  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
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<tr><th>Component</th><th>Volume(μl)</th></tr>
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  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
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<tr><td>Buffer (5x GC buffer or 5x HF buffer)</td><td>10</td></tr>
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  &quot;Times New Roman&quot;;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;&nbsp;
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<tr><td>dNTP mix (12.5 mM each)</td><td>2</td></tr>
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  </span></span></span><!--[endif]--><u><span lang="EN-US" style="mso-bidi-font-weight:
-
<tr><td>Primer fwd iGEM_34 (5 pmol)</td><td>2</td></tr>
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  bold">For Plate Reader assay:</span></u><span lang="EN-US" style="mso-bidi-font-weight:
-
<tr><td>Primer rev iGEM_35 (5 pmol)</td><td>2</td></tr>
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  bold"> 4 ml LB + 4 μl Cm or Amp for C1, C2 and C3 of parts 1 – 4 and part 6,
-
<tr><td>Chromosomal DNA M. smegmatis</td><td>2</td></tr>
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  negative control DH5α in 4 ml LB<o:p></o:p></span></p>
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<tr><td>DNA-Polymerase (Phusion or PhuS)<br /> or dH2O for water control</td><td>1</td></tr>
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  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
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<tr><td>dH2O</td><td>31</td></tr>
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  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
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</table>
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  &quot;Times New Roman&quot;;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;&nbsp;
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<p>Master Mix for 6 reactions</p>
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  </span></span></span><!--[endif]--><u><span lang="EN-US" style="mso-bidi-font-weight:
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<table>
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  bold">For Plasmid Mini-Preparation:</span></u><span lang="EN-US" style="mso-bidi-font-weight:bold"> 10 ml LB + 10 μl Amp for inoculation of C2
-
<tr><th>Component</th><th>Volume(μl)</th></tr>
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  Part 2<b><o:p></o:p></b></span></p>
-
<tr><td>dNTP mix (12.5 mM each)</td><td>12</td></tr>
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-
<tr><td>Primer fwd iGEM_34 (5 pmol)</td><td>12</td></tr>
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<div class="fbutton">Fold ↑</div>
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<tr><td>Primer rev iGEM_35 (5 pmol)</td><td>12</td></tr>
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<tr><td>dH2O</td><td>186</td></tr>
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-
<table>
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-
<p>addition of template (chrom. DNA/dH2O), polymerase and buffer individually, then addition of 37 μl master mix</p>
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-
<p><b>For tested combinations of buffer and Pol: see table for gel loading</b></p>
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-
<p>PCR protocol (cycler 7; folder “Katrin” > “iGEM” > “DarR seq”)</p>
+
-
<table>
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-
<tr><th>Step</th><th>Temperature</th><th>Time</th></tr>
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-
<tr><td>Initial denaturation</td><td>98.5 °C</td><td>5 min</td></tr>
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-
<tr><td>Denaturation</td><td>98.5 °C</td><td>30 s</td></tr>
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-
<tr><td>Annealing</td><td>60 °C<br />(TA = TM (≈66 °C) – 6 °C)
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-
</td><td>35 s</td></tr>
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-
<tr><td>Elongation</td><td>72 °C</td><td>2 min (Phu
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-
needs more time than Phusion!)</td></tr>
+
-
<tr><td>Final elongation</td><td>72 °C</td><td>10 min</td></tr>
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-
<tr><td>Hold</td><td>15 °C</td><td>∞</td></tr>
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</table>
+
-
<ul>
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-
<li>Protocol of 1 % Agarose-1xTAE gel</li>
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-
<li>4 μl PCR reaction + 1 μl 5x DNA Loading Dye</li>
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-
<li>3 μl 1 kb ladder (Quick Load)</li>
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-
<li>Run at 100 V in 1xTAE buffer</li>
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-
<li>Staining in EtBr and destaining in water</li>
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-
<li>UV detection</li>
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</ul>
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<p>Loading scheme</p>
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-
<table>
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-
<tr><th>M</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>M</th></tr>
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-
<tr><td>1kb ladder<br />(QuickLoad)</td><td>HF</td><td>GC</td><td>HF</td><td>GC</td><td>Water control <br />with HF</td><td>1 kb ladder<br />(QuickLoad)</td>
+
-
</table>
+
-
<img src="https://static.igem.org/mediawiki/2013/3/30/Goe-10.6.13-RT.png" />
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-
<p>Reactions stored at - 20°C in 50 ml Falcon</p>
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</div>
</div>
</div>
</div>
</div>
</div>
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<div class="tlob" id="tl_0613">
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<span class="date">13th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
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<div class="cont">
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<p class="timeline-title goe-rt">Test restriction of plasmids containing parts 1 – 8</p>
 +
<div class="timeline-cont">
 +
 +
  <p class="MsoNormal"><b><span lang="EN-US">Test restriction of plasmids
 +
  containing parts 1 – 8<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
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  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Reactions/Master Mixes: see Excel-sheet “dropbox &gt; iGEM &gt;
 +
  Reporter-Team &gt; digestion system 2hour”<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
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  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Plasmids: parts 1 – 6 (plasmids purified on <st1:chsdate isrocdate="False" islunardate="False" day="30" month="12" year="1899" w:st="on">7.6.13</st1:chsdate>)
 +
  and part 7, 8 (plasmids purified on 11.6.13)<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
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  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">1x reaction with single enzyme:<o:p></o:p></span></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:36.0pt;border-collapse:collapse;border:none;mso-border-alt:
 +
  solid black .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:
 +
  .5pt solid black;mso-border-insidev:.5pt solid black">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
 +
    mso-border-alt:solid black .5pt;background:#17365D;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Component<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    background:#17365D;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Volume<o:p></o:p></span></b></p>
 +
    </td>
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  </tr>
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  <tr style="mso-yfti-irow:1">
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    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Enzyme<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">0.5 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2">
 +
    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Buffer 10x<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3">
 +
    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Plasmid DNA<o:p></o:p></span></b></p>
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    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">200 ng<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4;mso-yfti-lastrow:yes">
 +
    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    background:#8DB3E2;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Total volume<o:p></o:p></span></b></p>
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    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;background:#8DB3E2;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">10 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">For double digestion:<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
  </span>0.5 µl of each enzyme in case of EcoRI and PstI;<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
  </span>for SpeI and PstI, ratio 1:2 is recommended </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
 +
  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> 0.5 µl SpeI and 1 µl PstI used<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
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  bold">Incubation at 37°C for1.5 hours<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Additon of 5xDNA-Loading Dye to whole reaction<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Loading of 8 µl on agarose gel (~1.5 %, 1xTAE), QuickLoad 1 kb ladder
 +
  as marker<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Run at 200 V for 1.5 h in 1xTAE<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">EtBr staining ca. 45 min, destaining in water ca. 30 min<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">UV detection<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Expected Fragments:<o:p></o:p></span></b></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid black .5pt;
 +
  mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid black;
 +
  mso-border-insidev:.5pt solid black">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;mso-border-alt:
 +
    solid black .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
 +
    12.0pt">Part no.<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="144" style="width:107.85pt;border:solid black 1.0pt;border-left:
 +
    none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
 +
    12.0pt">Fragments for PstI and SpeI (bp)<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="193" style="width:144.7pt;border:solid black 1.0pt;border-left:
 +
    none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
 +
    12.0pt">Plasmid size (linearization with PstI or SpeI)(bp)<o:p></o:p></span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;page-break-inside:avoid">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">1<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="144" rowspan="4" style="width:107.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .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-size:12.0pt">~900 + 2000 <o:p></o:p></span></p>
 +
    </td>
 +
    <td width="193" rowspan="4" style="width:144.7pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .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-size:12.0pt">2948<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2;page-break-inside:avoid">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">2<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3;page-break-inside:avoid">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">3<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4;page-break-inside:avoid">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">4<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:5">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
 +
    12.0pt">Part no.</span></b><span lang="EN-US" style="mso-bidi-font-size:12.0pt"><o:p></o:p></span></p>
 +
    </td>
 +
    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
 +
    12.0pt">Fragments for PstI and EcoRI(bp)</span></b><span lang="EN-US" style="mso-bidi-font-size:12.0pt"><o:p></o:p></span></p>
 +
    </td>
 +
    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
 +
    12.0pt">Plasmid size (linearization with PstI or EcoRI)(bp)</span></b><span lang="EN-US" style="mso-bidi-font-size:12.0pt"><o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:6">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">5<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="337" colspan="2" style="width:252.55pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .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-size:12.0pt">???<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">(if RFP gene </span><span lang="EN-US" style="mso-bidi-font-size:12.0pt;font-family:Wingdings;mso-ascii-font-family:
 +
    &quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:
 +
    symbol;mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;
 +
    mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-size:12.0pt"> same pattern as expected for parts 1 –
 +
    4; if no RPF gene </span><span lang="EN-US" style="mso-bidi-font-size:12.0pt;
 +
    font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:
 +
    &quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-size:12.0pt"> same pattern as expected for
 +
    part 8)<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:7">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">6<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">917<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2029<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">2946<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:8">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">7<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">140<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2050<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">2199<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:9;mso-yfti-lastrow:yes">
 +
    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
 +
    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">8<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">53<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2038<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
 +
    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
 +
    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .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-size:12.0pt">2091<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Gel: </span></b><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:bold">See ppt-file “dropbox
 +
  &gt; iGEM &gt; Reporter-Team &gt; Geldoc“<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-fareast-language:
 +
  DE"><!--[if gte vml 1]><v:shape id="Objekt_x0020_2" o:spid="_x0000_i1026"
 +
  type="#_x0000_t75" style='width:282.75pt;height:204pt;
 +
  mso-position-horizontal-relative:page;mso-position-vertical-relative:page'>
 +
  <v:imagedata src="part1.files/image003.png" o:title="" croptop="-1230f"
 +
    cropbottom="-168f" cropleft="-598f"/>
 +
  </v:shape><![endif]--><!--[if !vml]--> <img src="https://static.igem.org/mediawiki/2013/8/8f/Goe-13.6.13-RT.png" width="500px" v:shapes="Objekt_x0020_2"><!--[endif]--></span><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Conclusions:<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><b><span lang="EN-US">Partial digestion</span></b><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold"> (next time: longer incubation time, less plasmid…)<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">For <b>parts 1, 3, 4, 6, 7 and 8</b>,
 +
  the expected bands were observed (<b>ok</b>)<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">Band pattern of <b>part 5 </b>resembles
 +
  that expected for parts 1 – 4 </span><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
 +
  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
 +
  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> this plasmid <b>contains RFP
 +
  gene</b><o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">In case of <b>part 2, SpeI was
 +
  unable to cut the plasmid</b> </span><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
 +
  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
 +
  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> no cleavage for SpeI single
 +
  digest + linearization for SpeI/PstI double digest </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
 +
  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> plasmid contains probably something else
 +
  (SpeI/XbaI scar at actual SpeI site?)<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><b><span lang="EN-US">For cloning: terminator (part 7) and strong RBS (part 8) might be
 +
  difficult to extract from the gel, since they are very short </span></b><b><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
  Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> think of other cloning strategy without gel extraction (e.g.
 +
  “play” with resistances of plasmid backbones to digest backbone after cutting
 +
  out part…)<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Preparation of DarR PCR samples for
 +
  sequencing<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">4 µl of DarR seq PCR product of reaction 1 + 1 µl of iGEM_34 (fwd) or
 +
  iGEM_35 (rev)<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:
 +
  bold">Tubes:<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:
 +
  bold">kgun_1 </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:
 +
  &quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;
 +
  mso-symbol-font-family:Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:bold"> iGEM_34<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:
 +
  bold">kgun_2 </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:
 +
  &quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;
 +
  mso-symbol-font-family:Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:bold"> iGEM_35<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Sequencing at G2L<b><o:p></o:p></b></span></p>
 +
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">SDS-PAGE of overexpressing cells (#811, 814, 1013) containing 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:11.5pt">SDS-PAGE of overexpressing cells (#811,
 +
  814, 1013) containing pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)</span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.5pt">&nbsp;</span></b><span lang="EN-US" style="font-size:11.5pt">- take pelletized cells - add 5 μl of 5x SDS loading
 +
  dye</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">&nbsp;- add 10 μl ofZAP buffer - incubate
 +
  for 10 min at 96°C</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">&nbsp;→ load samples on SDS PAGE and run
 +
  gels at first at 80V, then turn to 100V </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">&nbsp;- after running: cover gels with
 +
  Coomassie Brilliant Blue staining solution and incubae for 30 min</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">&nbsp;- wash the gels afterwards twiche
 +
  with deionized H</span><span lang="EN-US" style="font-size:7.0pt">2</span><span lang="EN-US" style="font-size:11.5pt">O</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">&nbsp;- destain gels overnight in 10%
 +
  acetic acid with agitation </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">Gel: </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">first pre-experiment Marker (10 - 170 kDa
 +
  ladder) | control (0 h) | control (1 h) | control (2 h) | 811 (0 h) | 811 (1
 +
  h) | 811 (2 h) | 814 (0 h) | 814 (1 h) | 814 (2 h) </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt"><img border="0" width="354" height="243" id="图片 25" src="https://static.igem.org/mediawiki/2013/0/09/Image013.gif"></span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">Gel: first pre-experiment 1013 (0 h) |
 +
  1013 (1 h) | Marker (10 – 170 kDa ladder) | 1013 (2 h) | 1013 (4.5 h) </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt"><img border="0" width="360" height="269" id="图片 26" src="https://static.igem.org/mediawiki/2013/e/e7/Image014.gif"></span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">The T7 polymerase has a molecular weight
 +
  of approximately 99 kDa. The full length cdaA protein from L. monocytogenes
 +
  has a molecular weight of approximately 30.5 kDa, fused to a Strep-tag it has
 +
  31.5 kDa. The DAC domain of L. monocytogenes has a molecular weight of
 +
  approximately 18.8 kDa, fused to a Strep-tag it has 19.8 kDa. </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">For the first 2 h after gene induction by
 +
  IPTG no gene expression could be detected! </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">&nbsp;</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">Gel: repeat pre-experiment control (0 h)
 +
  | control (1 h) | 811 (0 h) | 811 (14 h) | 811 (2 h) | 814 (0 h) | 814 (14 h)
 +
  | 1013 ( 0 h) | 1013 (14 h) | Marker (10 – 170 kDa ladder) </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt"><img border="0" width="368" height="290" id="图片 27" src="https://static.igem.org/mediawiki/2013/a/a8/Goe-dac-Image015.gif"></span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">After 14 h could be gene expression for
 +
  both T7 polymerase and DAC domain detected. For cdaA gene expression no
 +
  signal could be detected. Therefore in further experiments cells should be
 +
  incubated at least for 14 h to obtain high expression levels! </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 class="tlob" id="tl_0612">
 +
<span class="date">12th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 +
<div class="cont">
 +
<p class="timeline-title goe-dac">Continue: Repeat: Pre-experiment:
 +
  Overexpression of cdaA and DAC domain by IPTG induction </span></b><span lang="EN-US" style="font-size:11.5pt">-measure OD600 of induced cultures </p>
 +
<div class="timeline-cont">
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.5pt">Continue: Repeat: Pre-experiment:
 +
  Overexpression of cdaA and DAC domain by IPTG induction </span></b><span lang="EN-US" style="font-size:11.5pt">-measure OD600 of induced cultures </span></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="165" valign="top" style="width:124.05pt;border: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:11.5pt">Culture </span></b></p>
 +
    <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">&nbsp;</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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"><b><span lang="EN-US" style="font-size:11.5pt">OD600</span></b></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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"><b><span lang="EN-US" style="font-size:11.5pt">Saved cells [μl] at t</span></b><b><span lang="EN-US" style="font-size:7.0pt">0</span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">#811</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">0.69</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">145</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">#814</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">0.79</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">127</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">#1013</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">1.82</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">55</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">control</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">2.20</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">46</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US">- pelletize cells and store at - 20?C </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>
 +
 +
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
</div>
 +
</div>
 +
 
 +
  <div class="tlob" id="tl_0611">
 +
<span class="date">11th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
 +
<div class="cont">
 +
<p class="timeline-title goe-rt">Miniprep of Part7,8 and DarR sequencing PCR clean-up</p>
 +
<div class="timeline-cont">
 +
 +
  <p class="MsoNormal"><b><span lang="EN-US">Cryo-Stocks of <i style="mso-bidi-font-style:
 +
  normal">E. coli</i> transfomants (8, 7 C1)<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Stored
 +
  in -70 °C (red box)<o:p></o:p></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"><b><span lang="EN-US">Plasmid Mini-Preparation of parts 8, 7
 +
  C1<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l5 level1 lfo6"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid
 +
  Mini-Preparation </span><span lang="EN-US" style="font-family:Wingdings;
 +
  mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
 +
  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
 +
  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> stored at – 20 °C in red box<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l5 level1 lfo6"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">harvesting of remaining culture for today’s prep<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l1 level1 lfo7"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">Performed as on <st1:chsdate year="1899" month="12" day="30" islunardate="False" isrocdate="False" w:st="on">7.6.13</st1:chsdate><o:p></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 black .5pt;
 +
  mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid black;
 +
  mso-border-insidev:.5pt solid black">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid black 1.0pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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 black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">c(DNA)<o:p></o:p></span></b></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><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 black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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 black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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 black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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 black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">67.0<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 black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.98<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 black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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:2;mso-yfti-lastrow:yes">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">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 black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">62.7<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 black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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 black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">DarR seq PCR purification<o:p></o:p></span></b></p>
 +
 
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">for DarR seq PCR reactions 1 - 4<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">with QIAquick PCR purification Kit (Qiagen), according to quick start
 +
  manual<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">ca. 50 µl of PCR reaction + 250 µl of PB buffer<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US" style="mso-bidi-font-weight:bold">reactions 2 and 4 turned violet </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
 +
  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> addition of 10 µl NaAc 3.3 M, pH = 5.0<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">elution: 30 µl pre-warmed HPLC water directly applied to membrane,
 +
  incubation for 2 min at RT, centrifugation<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">purified PCR products stored at – 20 °C in red box<o:p></o:p></span></p>
 +
 
 +
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-array">Preparation of c-di-AMP supernatant
 +
  without AB</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">Preparation of c-di-AMP supernatant
 +
  without AB</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;">168
 +
  innoculation of 1L CSE to an OD<sub>600</sub> = 0.1 at 9.00h (used preculture
 +
  that grew over night [50 ml CSE at ])<br>
 +
  11.00h: 0.384<br>
 +
  12.00h: 0.69<br>
 +
  12.20h: 0.82<br>
 +
  12.50h: 1.035</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;">centrifuge
 +
  (5000 rpm, 10 min), cook for 30 min (preheat in microwave), centrifuge (5000
 +
  rpm, 5 min), sterile filtration</span></p>
 +
  <p class="MsoListParagraphCxSpLast" 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;">store
 +
  in fridge</span></p>
 +
  <h1 align="left" style="margin-left:36.0pt;text-align:left;;
 +
  line-height:normal;page-break-after:auto"><span lang="EN-GB" style="font-size:
 +
  11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Microarray in Groningen</span></h1>
 +
 +
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">Repeat: Pre-experiment: Overexpression of
 +
  cdaA and DAC domain by IPTG induction </p>
 +
<div class="timeline-cont">
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><b><span lang="EN-US" style="font-size:11.5pt">Repeat: Pre-experiment: Overexpression of
 +
  cdaA and DAC domain by IPTG induction </span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">- take over night cultures of #811, 814,
 +
  1011, 1012 and 1013 and measure OD600: </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">-inoculate afterwards 4 ml liquid media
 +
  to an OD600 = 0.1 </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">- add IPTG [1mM] to a final concentration
 +
  of 1 μM to the liquid cultures</span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.5pt">- incubate at 16°C for 12 – 14 h </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 class="tlob" id="tl_0610">
 +
<span class="date">10th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
 +
<div class="cont">
 +
<p class="timeline-title goe-rt">Cloning DarR ORF from g-DNA</p>
 +
<div class="timeline-cont">
 +
 +
  <p class="MsoNormal"><b><span lang="EN-US">Colonies on the plates: parts 8, 7 C1</span></b><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
 +
 
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">10 ml
 +
  LB medium with 10</span><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
  bold"> </span><span lang="EN-US" style="font-size:10.0pt;color:#262626;
 +
  mso-font-kerning:0pt;mso-fareast-language:DE">µl</span><span lang="EN-US" style="font-size:12.0pt;font-family:Verdana;mso-bidi-font-family:Verdana;
 +
  color:#262626;mso-font-kerning:0pt;mso-fareast-language:DE"> </span><span lang="EN-US" style="mso-bidi-font-weight:bold">antibiotics, overnight culture
 +
  for mini-prep<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">backup
 +
  plates: C1, C2, C3 for part 8<o:p></o:p></span></p>
 +
 
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Colonies on the plates: parts 8, 7 C1</span></b><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Preparation
 +
  of 250 ml LB broth media (stored on shelf above bench)<o:p></o:p></span></p>
 +
 
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">10 ml
 +
  LB medium with 10</span><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
  bold"> </span><span lang="EN-US" style="font-size:10.0pt;color:#262626;
 +
  mso-font-kerning:0pt;mso-fareast-language:DE">µl</span><span lang="EN-US" style="font-size:12.0pt;font-family:Verdana;mso-bidi-font-family:Verdana;
 +
  color:#262626;mso-font-kerning:0pt;mso-fareast-language:DE"> </span><span lang="EN-US" style="mso-bidi-font-weight:bold">antibiotics, overnight culture
 +
  for mini-prep<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">backup
 +
  plates: C1, C2, C3 for part 8<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Re-streak of <i style="mso-bidi-font-style:
 +
  normal">E.coli</i> clones from prtas 6 and 7 on new LB<sup>Chloram</sup>
 +
  plates<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Preparation of new LB<sup>Chloram</sup>
 +
  plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf
 +
  above bench)</span></b><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">PCR with DarR primers with different
 +
  enzymes and different buffers<o:p></o:p></span></b></p>
 +
 
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l6 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">dilution of primer stocks 1:20 (100 pmol </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
 +
  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> 5 pmol):<o:p></o:p></span></p>
 +
  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">95 µl HPLC water + 5 µl primer
 +
  100 pmol<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l6 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">preparation of dNTP mix </span><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
 +
  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
 +
  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> dilute stocks 1:8 (100 mM </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
 +
  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> 12.5 mM):<o:p></o:p></span></p>
 +
  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">50 µl of dATP 100 mM<o:p></o:p></span></p>
 +
  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 50 µl of dGTP 100 mM<o:p></o:p></span></p>
 +
  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 50 µl of dTTP 100 mM<o:p></o:p></span></p>
 +
  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 50 µl of dCTP 100 mM<o:p></o:p></span></p>
 +
  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 200 µl dH<sub>2</sub>O<o:p></o:p></span></p>
 +
 
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l8 level1 lfo3"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">diluted primers and dNTP mix
 +
  stored in red box at -20 °C<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1x
 +
  reaction (50 µl)<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 black .5pt;
 +
  mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid black;
 +
  mso-border-insidev:.5pt solid black">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt">Component<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Volume<o:p></o:p></span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
    bold">Buffer (5x GC buffer or 5x HF buffer)<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">10 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
    bold">dNTP mix (12.5 mM each)<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
    bold">Primer fwd iGEM_34 (5 pmol)<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
    bold">Primer rev iGEM_35 (5 pmol)<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:5;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
    bold">Chromosomal DNA <i style="mso-bidi-font-style:normal">M. smegmatis</i><o:p></o:p></span></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:6;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
    bold">DNA-Polymerase (Phusion or PhuS) or dH<sub>2</sub>O for water control<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">1 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:7;mso-yfti-lastrow:yes;height:14.2pt">
 +
    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
 +
    bold">dH<sub>2</sub>O<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">31 µl<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">preparation of master mix for 6 reactions containing<o:p></o:p></span></p>
 +
 
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:36.0pt;border-collapse:collapse;border:none;mso-border-alt:
 +
  solid black .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:
 +
  .5pt solid black;mso-border-insidev:.5pt solid black">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><b><span lang="EN-US" style="font-size:10.0pt">Component<o:p></o:p></span></b></p>
 +
    </div>
 +
    </td>
 +
    <td width="66" valign="top" style="width:49.6pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><b><span lang="EN-US" style="font-size:10.0pt">Volume<o:p></o:p></span></b></p>
 +
    </div>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">dNTP mix (12.5 mM each)<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">12 µl<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2">
 +
    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Primer fwd iGEM_34 (5
 +
    pmol)<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">12 µl<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3">
 +
    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Primer rev iGEM_35 (5
 +
    pmol)<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">12 µl<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4;mso-yfti-lastrow:yes">
 +
    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">dH<sub>2</sub>O<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
 +
    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
 +
    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
 +
    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">186 µl<o:p></o:p></span></p>
 +
    </div>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
 
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">addition of template (chrom. DNA/dH<sub>2</sub>O), polymerase and
 +
  buffer individually, then addition of 37 µl master mix<b><o:p></o:p></b></span></p>
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l7 level1 lfo5"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><b><span lang="EN-US">For tested combinations of buffer and Pol: see table for gel
 +
  loading<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="DE" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;mso-ansi-language:DE;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="DE" style="mso-ansi-language:DE;
 +
  mso-bidi-font-weight:bold">PCR protocol (cycler 7; folder “Katrin” &gt;
 +
  “iGEM” &gt; “DarR seq”)<b><o:p></o:p></b></span></p>
 +
 
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:36.0pt;border-collapse:collapse;border:none;mso-border-alt:
 +
  solid black .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:
 +
  .5pt solid black;mso-border-insidev:.5pt solid black">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Step<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="142" valign="top" style="width:106.3pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Temperature<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Time<o:p></o:p></span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;height:38.3pt">
 +
    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt;height:38.3pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Initial denaturation<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:38.3pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">98.5 °C<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:38.3pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">5 min<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2">
 +
    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Denaturation<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">98.5 °C<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">30 s<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3">
 +
    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Annealing<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">60 °C<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt;mso-bidi-font-weight:bold">(T<sub>A</sub> = T<sub>M</sub>
 +
    (≈66 °C) – 6 °C)<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">35 s<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4">
 +
    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Elongation<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">72 °C<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 min (Phu<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold"><span style="mso-spacerun:yes">&nbsp;</span>needs more time than Phusion!)<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:5">
 +
    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Final elongation<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">72 °C<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">10 min<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:6;mso-yfti-lastrow:yes">
 +
    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Hold<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">15 °C<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">∞<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
 
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">1 % Agarose-1xTAE gel<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">4 µl PCR reaction + 1 µl 5x DNA Loading Dye<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">3 µl 1 kb ladder (Quick Load)<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Run at 100 V in 1xTAE buffer<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">Staining in EtBr and destaining in water<o:p></o:p></span></p>
 +
  <p class="MsoNormal" style="margin-left:36.0pt;;mso-list:
 +
  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
 +
  &quot;Times New Roman&quot;;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;&nbsp;
 +
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
 +
  bold">UV detection<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 black .5pt;
 +
  mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid black;
 +
  mso-border-insidev:.5pt solid black">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">M<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <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="57" valign="top" style="width:42.75pt;border:solid black 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">M<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1 kb ladder<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">(QuickLoad)<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">HF<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">GC<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">HF<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">GC<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">Water controlHF<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1 kb ladder<o:p></o:p></span></p>
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">(QuickLoad)<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2;mso-yfti-lastrow:yes">
 +
    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
 +
    padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
 +
    </td>
 +
    <td width="114" colspan="2" valign="top" style="width:85.4pt;border-top:none;
 +
    border-left:none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">Phusion<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="171" colspan="3" valign="top" style="width:128.1pt;border-top:none;
 +
    border-left:none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">PhuS<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="57" valign="top" style="width:42.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
 +
    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><!--[if gte vml 1]><v:shapetype id="_x0000_t75"
 +
  coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe"
 +
  filled="f" stroked="f">
 +
  <v:stroke joinstyle="miter"/>
 +
  <v:formulas>
 +
    <v:f eqn="if lineDrawn pixelLineWidth 0"/>
 +
    <v:f eqn="sum @0 1 0"/>
 +
    <v:f eqn="sum 0 0 @1"/>
 +
    <v:f eqn="prod @2 1 2"/>
 +
    <v:f eqn="prod @3 21600 pixelWidth"/>
 +
    <v:f eqn="prod @3 21600 pixelHeight"/>
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    <v:f eqn="sum @0 0 1"/>
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    <v:f eqn="prod @6 1 2"/>
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    <v:f eqn="prod @7 21600 pixelWidth"/>
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    <v:f eqn="sum @8 21600 0"/>
 +
    <v:f eqn="prod @7 21600 pixelHeight"/>
 +
    <v:f eqn="sum @10 21600 0"/>
 +
  </v:formulas>
 +
  <v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
 +
  <o:lock v:ext="edit" aspectratio="t"/>
 +
  </v:shapetype><v:shape id="_x0000_i1025" type="#_x0000_t75" style='width:300pt;
 +
  height:223.5pt;mso-position-horizontal-relative:page;
 +
  mso-position-vertical-relative:page'>
 +
  <v:imagedata src="part1.files/image001.png" o:title="DarR different enzymes"/>
 +
  </v:shape><![endif]--><!--[if !vml]--><img src="https://static.igem.org/mediawiki/2013/3/30/Goe-10.6.13-RT.png" v:shapes="_x0000_i1025"><!--[endif]--><o:p></o:p></span></b></p>
 +
 
 +
  <p class="MsoNormal" style="margin-left:54.0pt;;mso-list:
 +
  l7 level1 lfo5"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
 +
  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
 +
  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">Reactions stored at - 20°C in 50
 +
  ml Falcon<b><o:p></o:p></b></span></p>
 +
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction</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">Pre-experiment: Overexpression of cdaA
 +
  and DAC domain by IPTG induction </span></b></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">- take over night cultures of #811, 814,
 +
  1011, 1012 and 1013 and measure OD600: </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">-inoculate afterwards 4 ml liquid media
 +
  to an OD600 = 0.1 </span></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="165" valign="top" style="width:123.85pt;border: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">Culture</span></b></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.85pt;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"><b><span lang="EN-US" style="font-size:11.0pt">OD600</span></b></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.9pt;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"><b><span lang="EN-US" style="font-size:11.0pt">Used μl for inoculation</span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:123.85pt;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">#811</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.85pt;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">0.67</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.9pt;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">600</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:123.85pt;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">#814</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.85pt;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">1.39</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.9pt;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">290</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:123.85pt;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">#1011</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.85pt;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">1.29</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.9pt;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">310</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:123.85pt;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">#1012</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.85pt;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">1.18</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.9pt;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">340</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:123.85pt;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">#1013</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.85pt;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">0.95</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.9pt;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">420</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:123.85pt;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">control</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.85pt;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">1.54</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:123.9pt;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">220</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">- add IPTG [1mM] to a final concentration
 +
  of 1 μM to the liquid cultures for </span></p>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US" style="font-size:11.0pt">induction - incubate at 16°C for 0 – 2 h
 +
  - measure OD600 of induced cultures → take equivalent amount of cells after 0
 +
  h, 1 h and 2 h: 100 [μl] /OD600 </span></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="165" valign="top" style="width:124.05pt;border: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:11.5pt">Culture</span></b></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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"><b><span lang="EN-US" style="font-size:11.5pt">OD600</span></b></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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"><b><span lang="EN-US" style="font-size:11.5pt">Saved cells [μl] at t</span></b><b><span lang="EN-US" style="font-size:7.0pt">0</span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">#811 </span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">0.60</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">167</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">#814</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">0.64</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">167</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">#1013</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">0.87</span></p>
 +
    </td>
 +
    <td width="165" valign="top" style="width:124.05pt;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:11.5pt">115</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="Default" style="text-align:justify;text-justify:inter-ideograph"><span lang="EN-US">-pelletize cells and store at -20?C </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"><span lang="EN-US" style="font-size:11.0pt">&nbsp;</span></p>
 +
 +
 +
<div class="fbutton">Fold ↑</div>
 +
</div></div>
 +
</div>
 +
 +
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</html>

Latest revision as of 21:22, 30 September 2013

14th

Observation of Back-up plates and preparation of media

Observation of Back-up plates

-          Part 1, C1 and C2 probably very light pink

-          Part 2, C2 and C3 became light pink, but C1 stayed white (compare with restriction analysis 13.6.13)

 

Preparation of media for inoculation on Sunday (by Katrin Gunka)

-          For Plate Reader assay: 4 ml LB + 4 μl Cm or Amp for C1, C2 and C3 of parts 1 – 4 and part 6, negative control DH5α in 4 ml LB

-          For Plasmid Mini-Preparation: 10 ml LB + 10 μl Amp for inoculation of C2 Part 2

Fold ↑
13th

Test restriction of plasmids containing parts 1 – 8

Test restriction of plasmids containing parts 1 – 8

-          Reactions/Master Mixes: see Excel-sheet “dropbox > iGEM > Reporter-Team > digestion system 2hour”

-          Plasmids: parts 1 – 6 (plasmids purified on 7.6.13) and part 7, 8 (plasmids purified on 11.6.13)

-          1x reaction with single enzyme:

Component

Volume

Enzyme

0.5 µl

Buffer 10x

1 µl

Plasmid DNA

200 ng

Total volume

10 µl

è     For double digestion:

           0.5 µl of each enzyme in case of EcoRI and PstI;

          for SpeI and PstI, ratio 1:2 is recommended à 0.5 µl SpeI and 1 µl PstI used

-          Incubation at 37°C for1.5 hours

-          Additon of 5xDNA-Loading Dye to whole reaction

-          Loading of 8 µl on agarose gel (~1.5 %, 1xTAE), QuickLoad 1 kb ladder as marker

-          Run at 200 V for 1.5 h in 1xTAE

-          EtBr staining ca. 45 min, destaining in water ca. 30 min

-          UV detection

 

Expected Fragments:

Part no.

Fragments for PstI and SpeI (bp)

Plasmid size (linearization with PstI or SpeI)(bp)

1

~900 + 2000

2948

2

3

4

Part no.

Fragments for PstI and EcoRI(bp)

Plasmid size (linearization with PstI or EcoRI)(bp)

5

???

(if RFP gene à same pattern as expected for parts 1 – 4; if no RPF gene à same pattern as expected for part 8)

6

917

2029

2946

7

140

2050

2199

8

53

2038

2091

 

Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

Conclusions:

è     Partial digestion (next time: longer incubation time, less plasmid…)

è     For parts 1, 3, 4, 6, 7 and 8, the expected bands were observed (ok)

è     Band pattern of part 5 resembles that expected for parts 1 – 4 à this plasmid contains RFP gene

è     In case of part 2, SpeI was unable to cut the plasmid à no cleavage for SpeI single digest + linearization for SpeI/PstI double digest à plasmid contains probably something else (SpeI/XbaI scar at actual SpeI site?)

è     For cloning: terminator (part 7) and strong RBS (part 8) might be difficult to extract from the gel, since they are very short à think of other cloning strategy without gel extraction (e.g. “play” with resistances of plasmid backbones to digest backbone after cutting out part…)

 

Preparation of DarR PCR samples for sequencing

-          4 µl of DarR seq PCR product of reaction 1 + 1 µl of iGEM_34 (fwd) or iGEM_35 (rev)

Tubes:

kgun_1 à iGEM_34

kgun_2 à iGEM_35

-          Sequencing at G2L

Fold ↑

SDS-PAGE of overexpressing cells (#811, 814, 1013) containing pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

SDS-PAGE of overexpressing cells (#811, 814, 1013) containing pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

 - take pelletized cells - add 5 μl of 5x SDS loading dye

 - add 10 μl ofZAP buffer - incubate for 10 min at 96°C

 → load samples on SDS PAGE and run gels at first at 80V, then turn to 100V

 - after running: cover gels with Coomassie Brilliant Blue staining solution and incubae for 30 min

 - wash the gels afterwards twiche with deionized H2O

 - destain gels overnight in 10% acetic acid with agitation

Gel:

first pre-experiment Marker (10 - 170 kDa ladder) | control (0 h) | control (1 h) | control (2 h) | 811 (0 h) | 811 (1 h) | 811 (2 h) | 814 (0 h) | 814 (1 h) | 814 (2 h)

Gel: first pre-experiment 1013 (0 h) | 1013 (1 h) | Marker (10 – 170 kDa ladder) | 1013 (2 h) | 1013 (4.5 h)

The T7 polymerase has a molecular weight of approximately 99 kDa. The full length cdaA protein from L. monocytogenes has a molecular weight of approximately 30.5 kDa, fused to a Strep-tag it has 31.5 kDa. The DAC domain of L. monocytogenes has a molecular weight of approximately 18.8 kDa, fused to a Strep-tag it has 19.8 kDa.

For the first 2 h after gene induction by IPTG no gene expression could be detected!

 

Gel: repeat pre-experiment control (0 h) | control (1 h) | 811 (0 h) | 811 (14 h) | 811 (2 h) | 814 (0 h) | 814 (14 h) | 1013 ( 0 h) | 1013 (14 h) | Marker (10 – 170 kDa ladder)

After 14 h could be gene expression for both T7 polymerase and DAC domain detected. For cdaA gene expression no signal could be detected. Therefore in further experiments cells should be incubated at least for 14 h to obtain high expression levels!

 

Fold ↑
12th

Continue: Repeat: Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction -measure OD600 of induced cultures

Continue: Repeat: Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction -measure OD600 of induced cultures

Culture

 

OD600

Saved cells [μl] at t0

#811

0.69

145

#814

0.79

127

#1013

1.82

55

control

2.20

46

- pelletize cells and store at - 20?C

 

Fold ↑
11th

Miniprep of Part7,8 and DarR sequencing PCR clean-up

Cryo-Stocks of E. coli transfomants (8, 7 C1)

Stored in -70 °C (red box)

 

Plasmid Mini-Preparation of parts 8, 7 C1

-          harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid Mini-Preparation à stored at – 20 °C in red box

-          harvesting of remaining culture for today’s prep

è     Performed as on 7.6.13

NanoDrop – Plasmid concentrations

Part no.

c(DNA)

[ng/µl]

A260/A280

A260/A230

7

67.0

1.98

2.25

8

62.7

1.89

2.08

 

DarR seq PCR purification

-          for DarR seq PCR reactions 1 - 4

-          with QIAquick PCR purification Kit (Qiagen), according to quick start manual

-          ca. 50 µl of PCR reaction + 250 µl of PB buffer

reactions 2 and 4 turned violet à addition of 10 µl NaAc 3.3 M, pH = 5.0

-          elution: 30 µl pre-warmed HPLC water directly applied to membrane, incubation for 2 min at RT, centrifugation

-          purified PCR products stored at – 20 °C in red box

Fold ↑

Preparation of c-di-AMP supernatant without AB

Preparation of c-di-AMP supernatant without AB

·         168 innoculation of 1L CSE to an OD600 = 0.1 at 9.00h (used preculture that grew over night [50 ml CSE at ])
11.00h: 0.384
12.00h: 0.69
12.20h: 0.82
12.50h: 1.035

·         centrifuge (5000 rpm, 10 min), cook for 30 min (preheat in microwave), centrifuge (5000 rpm, 5 min), sterile filtration

·         store in fridge

Microarray in Groningen

Fold ↑

Repeat: Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction

Repeat: Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction

- take over night cultures of #811, 814, 1011, 1012 and 1013 and measure OD600:

-inoculate afterwards 4 ml liquid media to an OD600 = 0.1

- add IPTG [1mM] to a final concentration of 1 μM to the liquid cultures

- incubate at 16°C for 12 – 14 h

 

Fold ↑
10th

Cloning DarR ORF from g-DNA

Colonies on the plates: parts 8, 7 C1

10 ml LB medium with 10 µl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

 

Colonies on the plates: parts 8, 7 C1

Preparation of 250 ml LB broth media (stored on shelf above bench)

10 ml LB medium with 10 µl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

Re-streak of E.coli clones from prtas 6 and 7 on new LBChloram plates

Preparation of new LBChloram plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf above bench)

 

PCR with DarR primers with different enzymes and different buffers

-          dilution of primer stocks 1:20 (100 pmol à 5 pmol):

95 µl HPLC water + 5 µl primer 100 pmol

-          preparation of dNTP mix à dilute stocks 1:8 (100 mM à 12.5 mM):

50 µl of dATP 100 mM

+ 50 µl of dGTP 100 mM

+ 50 µl of dTTP 100 mM

+ 50 µl of dCTP 100 mM

+ 200 µl dH2O

è     diluted primers and dNTP mix stored in red box at -20 °C

 

1x reaction (50 µl)

Component

Volume

Buffer (5x GC buffer or 5x HF buffer)

10 µl

dNTP mix (12.5 mM each)

2 µl

Primer fwd iGEM_34 (5 pmol)

2 µl

Primer rev iGEM_35 (5 pmol)

2 µl

Chromosomal DNA M. smegmatis

2 µl

DNA-Polymerase (Phusion or PhuS) or dH2O for water control

1 µl

dH2O

31 µl

 

-          preparation of master mix for 6 reactions containing

Component

Volume

dNTP mix (12.5 mM each)

12 µl

Primer fwd iGEM_34 (5 pmol)

12 µl

Primer rev iGEM_35 (5 pmol)

12 µl

dH2O

186 µl

-          addition of template (chrom. DNA/dH2O), polymerase and buffer individually, then addition of 37 µl master mix

è     For tested combinations of buffer and Pol: see table for gel loading

 

-          PCR protocol (cycler 7; folder “Katrin” > “iGEM” > “DarR seq”)

Step

Temperature

Time

Initial denaturation

98.5 °C

5 min

Denaturation

98.5 °C

30 s

Annealing

60 °C

(TA = TM (≈66 °C) – 6 °C)

35 s

Elongation

72 °C

2 min (Phu

 needs more time than Phusion!)

Final elongation

72 °C

10 min

Hold

15 °C

∞

-          1 % Agarose-1xTAE gel

-          4 µl PCR reaction + 1 µl 5x DNA Loading Dye

-          3 µl 1 kb ladder (Quick Load)

-          Run at 100 V in 1xTAE buffer

-          Staining in EtBr and destaining in water

-          UV detection

M

1

2

3

4

5

M

1 kb ladder

(QuickLoad)

HF

GC

HF

GC

Water controlHF

1 kb ladder

(QuickLoad)

 

Phusion

PhuS

 

è     Reactions stored at - 20°C in 50 ml Falcon

Fold ↑

Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction

Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction

- take over night cultures of #811, 814, 1011, 1012 and 1013 and measure OD600:

-inoculate afterwards 4 ml liquid media to an OD600 = 0.1

Culture

OD600

Used μl for inoculation

#811

0.67

600

#814

1.39

290

#1011

1.29

310

#1012

1.18

340

#1013

0.95

420

control

1.54

220

- add IPTG [1mM] to a final concentration of 1 μM to the liquid cultures for

induction - incubate at 16°C for 0 – 2 h - measure OD600 of induced cultures → take equivalent amount of cells after 0 h, 1 h and 2 h: 100 [μl] /OD600

Culture

OD600

Saved cells [μl] at t0

#811

0.60

167

#814

0.64

167

#1013

0.87

115

-pelletize cells and store at -20?C

 

 

Fold ↑

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