Team:Marburg:Project:Notebook:Methods

From 2014.igem.org

(Difference between revisions)
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{{Team:Marburg/Template:Submenu:Notebook:Methods}}
{{Team:Marburg/Template:Submenu:Notebook:Methods}}
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...
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<html>
 +
 
 +
<!-- Miniprep -->
 +
 
 +
<div class="method">
 +
<fieldset class="Miniprep">
 +
    <legend><a name="miniprep">Miniprep (Plasmid DNA Miniprep Kit II, Omega)</a></legend>
 +
    <div class="exp-content">
 +
  <ul class="miniprep">
 +
  <li>centrifuge at 10.000g for 1min</li>
 +
  <li>add 250&micro;l Resuspension Buffer P1 per tube (incl. RNase), vortex</li>
 +
  <li>transfer suspension into new 1,5 ml tube</li>
 +
  <li>add 250&micro;l Lysis Buffer P2 per tube, invert until lysate is clear (2-3min)</li>
 +
  <li>add 350&micro;l Neutralisation Buffer N3 per tube</li>
 +
          <li>invert immediately (precipitate forms)</li>
 +
          <li>centrifuge 10min at 13.000 rpm</li>
 +
          <li>transfer supernatant onto column</li>
 +
          <li>centrifuge for 1min
 +
          <li>add 500&micro;l Washing Buffer PE (HBC Buffer) per tube</li>
 +
          <li>centrifugation for 1min throw filrate away</li>
 +
          <li>add 700&micro;l DNA Wash Buffer per tube</li>
 +
          <li>centrifugation for 1min throw filrate away</li>
 +
          <li>add 30&micro;l milipore per tube – let column sit for 2min</li>
 +
          <li>centrifuge for 2min</li>
 +
</ul>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- Gel-Ex -->
 +
 
 +
<div class="method">
 +
<fieldset class="Gel Extraction">
 +
    <legend><a name="gelex">Gel Extraction (QIAquick Gel Extraction Kit)</a></legend>
 +
<div class="exp-content">
 +
<ul class="gelex">
 +
<li>band is cut out of the gel under UV light and transferred into a 1,5ml tube</li>
 +
<li>weigh the gel with the tube</li>
 +
<li>add QG (100mg gel &rarr; 100&micro;l)</li>
 +
<li>incubate for approx. 10min at 50-60°C, vortex to dissolve the gel</li>
 +
            <li>put spin colum into 2mlcollection tube</li>
 +
            <li>add dissolved gel onto column &rarr; centrifuge for 1min</li>
 +
            <li>discard flow-through</li>
 +
            <li>wash with 750µl Buffer PE &rarr;
 +
centrifuge for 1min          <li>discard flow-through</li>
 +
            <li>place column in 1,5ml tube</li>
 +
            <li>elute with 50µl milipore water: incubate 5min at 37°C &rarr;
 +
centrifuge for 1min</li>
 +
</ul>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR Purification -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR Purification">
 +
    <legend><a name="purification">Purification of PCR Products (QIAquick Gel Extraction Kit) short protocol</a></legend>
 +
<div class="exp-content">
 +
    <p>short protocol without gel extraction</p>
 +
<ul class="gelex">
 +
<li>add 3 volumes or QG Buffer to tubes  and 1 volume isopropanol</li>
 +
<li>place QIAquick spin column in 2ml collection tube</li>
 +
<li>centrifuge for 1min &rarr;discard flow-through</li>
 +
<li>add 750µl Buffer PE</li>
 +
<li>centrifuge for 1min  &rarr;discard flow-through</li>
 +
<li>place column in 1,5 ml tube</li>
 +
<li>eluate with 50µl milipore water</li>
 +
<li>incubate for 5min at 37°C, centrifuge for 1min</li>
 +
</ul>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR 1 -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR1">PCR1</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Step</th>
 +
    <th scope="col">Temperature &deg;C</th>
 +
    <th scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>98</td>
 +
    <td>3 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>98</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>55</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>1 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>30x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>4min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>8</td>
 +
    <td>infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR 2 -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR2">PCR2</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Step</th>
 +
    <th scope="col">Temperature &deg;C</th>
 +
    <th scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>2 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>58</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>68</td>
 +
    <td>2,5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>40x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>68</td>
 +
    <td>4min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>8</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR 3 -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR3">PCR3</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Step</th>
 +
    <th scope="col">Temperature &deg;C</th>
 +
    <th scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>3 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>58</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>1 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>35x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>5min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR 4 -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR4">PCR4</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Step</th>
 +
    <th scope="col">Temperature &deg;C</th>
 +
    <th scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>55-65</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>2,5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>35x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>5min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR 5 -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR5">PCR5</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Step</th>
 +
    <th scope="col">Temperature &deg;C</th>
 +
    <th scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>55</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>35x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>5min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR Q5 -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR_Q5">PCRQ5</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Step</th>
 +
    <th scope="col">Temperature &deg;C</th>
 +
    <th scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>3 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>55</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>1 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>30x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>5min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR Q5-2-->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR_Q5_2">PCRQ5-2</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Step</th>
 +
    <th scope="col">Temperature &deg;C</th>
 +
    <th scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>58</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>50 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>35x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>5min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- PCR Q5 2kb fragments -->
 +
 
 +
<div class="method">
 +
<fieldset class="PCR">
 +
    <legend><a name="PCR_Q5_2kb_fragments">PCR Q5 elongation for 2kb fragments</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th width="17%" scope="col">Step</th>
 +
    <th width="58%" scope="col">Temperature &deg;C</th>
 +
    <th width="25%" scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>3 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>55</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>2,5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>35x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>5min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- Taq-PCR -->
 +
 
 +
<div class="method">
 +
<fieldset class="Taq PCR">
 +
    <legend><a name="Taq_PCR">Taq PCR</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th width="17%" scope="col">Step</th>
 +
    <th width="58%" scope="col">Temperature &deg;C</th>
 +
    <th width="25%" scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>95</td>
 +
    <td>5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>95</td>
 +
    <td>30 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>58</td>
 +
    <td>20 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>72</td>
 +
    <td>2,5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>Go To 2</td>
 +
    <td>35x</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">6</th>
 +
    <td>72</td>
 +
    <td>5min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">7</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- Annealing with PNK inactivation -->
 +
 
 +
<div class="method">
 +
<fieldset class="Annealing">
 +
    <legend><a name="Annealing_PNK">Annealing with PNK Inactivation</a></legend>
 +
<div class="exp-content">
 +
        <table width="100%" border="1">
 +
  <tr>
 +
    <th width="17%" scope="col">Step</th>
 +
    <th width="58%" scope="col">Temperature &deg;C</th>
 +
    <th width="25%" scope="col">Time</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">1</th>
 +
    <td>37</td>
 +
    <td>1h</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">2</th>
 +
    <td>65</td>
 +
    <td>20 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">3</th>
 +
    <td>95</td>
 +
    <td>5 min</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">4</th>
 +
    <td>95-4</td>
 +
    <td>1 min/1&deg;C</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">5</th>
 +
    <td>4</td>
 +
    <td>Infinite</td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- Medium -->
 +
 
 +
<div class="method">
 +
<fieldset class="Medium">
 +
    <legend><a name="med">Medium</a></legend>
 +
<div class="exp-content">
 +
<table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">LB-medium</th>
 +
    <th scope="col">Concentration</th>
 +
    <th scope="col">Final</th>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">Ampicilin</th>
 +
    <td>Ampicilin (1000x) 100µl:1000ml <br /></td>
 +
    <td>Final: 50&micro;g/ml</td>
 +
  </tr>
 +
  <tr>
 +
    <th scope="row">Canamycin</th>
 +
    <td>Canamycin (1000x) 100µl:100ml</td>
 +
    <td>Final: 50&micro;g/ml</td>
 +
  </tr>
 +
    <tr>
 +
    <th scope="row">Chloramphenicol</th>
 +
    <td>Chloramphenicol (1000x) 100µl:100ml</td>
 +
    <td></td>
 +
  </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 
 +
<!-- Buffer -->
 +
 
 +
<div class="method">
 +
<fieldset class="Buffer">
 +
    <legend><a name="buff">Buffer</a></legend>
 +
<div class="exp-content">
 +
<table width="100%" border="1">
 +
  <tr>
 +
    <th scope="col">Buffer</th>
 +
    <th scope="col">Additions</th>
 +
    </tr>
 +
  <tr>
 +
    <th scope="row">-2.0 Buffer</th>
 +
    <td>2.0 (10x) + BSA (10mg/ml -10) &rarr; BSA end concentration 100&micro;g/ml (0,1&micro;g/&micro;l)</td>
 +
    </tr>
 +
</table>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
</html>
{{Team:Marburg/Template:End}}
{{Team:Marburg/Template:End}}

Revision as of 11:07, 12 October 2014

Notebook: Methods

Miniprep (Plasmid DNA Miniprep Kit II, Omega)
  • centrifuge at 10.000g for 1min
  • add 250µl Resuspension Buffer P1 per tube (incl. RNase), vortex
  • transfer suspension into new 1,5 ml tube
  • add 250µl Lysis Buffer P2 per tube, invert until lysate is clear (2-3min)
  • add 350µl Neutralisation Buffer N3 per tube
  • invert immediately (precipitate forms)
  • centrifuge 10min at 13.000 rpm
  • transfer supernatant onto column
  • centrifuge for 1min
  • add 500µl Washing Buffer PE (HBC Buffer) per tube
  • centrifugation for 1min throw filrate away
  • add 700µl DNA Wash Buffer per tube
  • centrifugation for 1min throw filrate away
  • add 30µl milipore per tube – let column sit for 2min
  • centrifuge for 2min
Gel Extraction (QIAquick Gel Extraction Kit)
  • band is cut out of the gel under UV light and transferred into a 1,5ml tube
  • weigh the gel with the tube
  • add QG (100mg gel → 100µl)
  • incubate for approx. 10min at 50-60°C, vortex to dissolve the gel
  • put spin colum into 2mlcollection tube
  • add dissolved gel onto column → centrifuge for 1min
  • discard flow-through
  • wash with 750µl Buffer PE → centrifuge for 1min
  • discard flow-through
  • place column in 1,5ml tube
  • elute with 50µl milipore water: incubate 5min at 37°C → centrifuge for 1min
Purification of PCR Products (QIAquick Gel Extraction Kit) short protocol

short protocol without gel extraction

  • add 3 volumes or QG Buffer to tubes and 1 volume isopropanol
  • place QIAquick spin column in 2ml collection tube
  • centrifuge for 1min →discard flow-through
  • add 750µl Buffer PE
  • centrifuge for 1min →discard flow-through
  • place column in 1,5 ml tube
  • eluate with 50µl milipore water
  • incubate for 5min at 37°C, centrifuge for 1min
PCR1
Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min
5 Go To 2 30x
6 72 4min
7 8 infinite
PCR2
Step Temperature °C Time
1 95 2 min
2 95 20 sec
3 58 20 sec
4 68 2,5 min
5 Go To 2 40x
6 68 4min
7 8 Infinite
PCR3
Step Temperature °C Time
1 95 3 min
2 95 30 sec
3 58 20 sec
4 72 1 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCR4
Step Temperature °C Time
1 95 5 min
2 95 30 sec
3 55-65 30 sec
4 72 2,5 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCR5
Step Temperature °C Time
1 95 5 min
2 95 20 sec
3 55 20 sec
4 72 20 sec
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCRQ5
Step Temperature °C Time
1 95 3 min
2 95 30 sec
3 55 30 sec
4 72 1 min
5 Go To 2 30x
6 72 5min
7 4 Infinite
PCRQ5-2
Step Temperature °C Time
1 95 5 min
2 95 30 sec
3 58 20 sec
4 72 50 sec
5 Go To 2 35x
6 72 5min
7 4 Infinite
PCR Q5 elongation for 2kb fragments
Step Temperature °C Time
1 95 3 min
2 95 30 sec
3 55 30 sec
4 72 2,5 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
Taq PCR
Step Temperature °C Time
1 95 5 min
2 95 30 sec
3 58 20 sec
4 72 2,5 min
5 Go To 2 35x
6 72 5min
7 4 Infinite
Annealing with PNK Inactivation
Step Temperature °C Time
1 37 1h
2 65 20 min
3 95 5 min
4 95-4 1 min/1°C
5 4 Infinite
Medium
LB-medium Concentration Final
Ampicilin Ampicilin (1000x) 100µl:1000ml
Final: 50µg/ml
Canamycin Canamycin (1000x) 100µl:100ml Final: 50µg/ml
Chloramphenicol Chloramphenicol (1000x) 100µl:100ml
Buffer
Buffer Additions
-2.0 Buffer 2.0 (10x) + BSA (10mg/ml -10) → BSA end concentration 100µg/ml (0,1µg/µl)

Retrieved from "http://2014.igem.org/Team:Marburg:Project:Notebook:Methods"