Team:Goettingen/protocol PCR

From 2014.igem.org

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<div class="proRP">
<div class="proRP">
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            <h3 id="Fusi_PCR">Fusion PCR</h3>
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             <h3 id="Pfus_PCR"><i>PfuS</i> PCR</h3>
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            <p>Reaction protocol 50 µl:<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 μl Primer forward (5 pmol)<br />
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-
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 μl Primer reverse (5 pmol)<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;100 ng template-DNA of each fragment<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10 μl 5x HF-Buffer <br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 μl PfuS-Polymerase<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 μl dNTPs (12.5 mM)<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ad. 50 μl sterile water<br /><br />
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        PCR cycles:</p>
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      <table class="table1"  align="center">
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      <tr><th>Amount of cycles</th><th>Reaction </th><th> Temp.</th><th> Time</th></tr>
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      <tr><td>1</td><td>Pre-running</td><td>98°C</td><td>1 min</td></tr>
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      <tr><td>10</td><td>Denaturation</td><td>98°C</td><td>15 sec</td></tr>
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      <tr><td>10</td><td>Annealing</td><td>52°C</td><td>30 sec</td></tr>
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      <tr><td>10</td><td>Primer Extension </td><td>72°C</td><td>2 min</td></tr>
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      <tr><td>1</td><td>Pause</td><td>8°C</td><td>add primer</td></tr>
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      <tr><td>21</td><td>Denaturation</td><td>98°C</td><td>15 sec</td></tr>
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      <tr><td>21</td><td> Annealing</td><td>52°C</td><td>30 sec</td></tr>
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      <tr><td>21</td><td>Primer Extension</td><td>72°C</td><td>2 min + 5 sec/cycle</td></tr>
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      <tr><td>1</td><td>Final elongation</td><td>72°C</td><td>10 min</td></tr>
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      <tr><td>1</td><td> Hold </td><td>4°C</td><td> ∞ </td></tr>
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      </table>
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            <br /><br />
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             <h3 id="Pfus_PCR"><i>Pfus</i> PCR</h3>
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             <p>Watch the following tutorial to learn about the whole process:<br /><br /></p>
             <p>Watch the following tutorial to learn about the whole process:<br /><br /></p>
             <center><iframe width="640" height="390" src="//www.youtube.com/embed/gT2yVxLlfCQ" frameborder="0" allowfullscreen></iframe> </center>
             <center><iframe width="640" height="390" src="//www.youtube.com/embed/gT2yVxLlfCQ" frameborder="0" allowfullscreen></iframe> </center>
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             </table>
             </table>
       <br /><br />           
       <br /><br />           
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             <h3 id="Asse_PCR">Assembly PCR</h3>
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             <h3 id="Fusi_PCR">Fusion PCR</h3>
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             <p>Reaction protocol 100 µl:<br />
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             <p>Reaction protocol 50 µl:<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;20 μl 5 x PCR buffer<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 μl Primer forward (5 pmol)<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 μl dNTP-Mix(12.5 mM each)<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 μl Primer reverse (5 pmol)<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;8 μl Forward oligo<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;100 ng template-DNA of each fragment<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;8 μl Reverse oligo<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10 μl 5x HF-Buffer <br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;100 ng Region I<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 μl PfuS-Polymerase<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;100 ng Region II<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 μl dNTPs (12.5 mM)<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;100 ng Region III<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ad. 50 μl sterile water<br /><br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ad. 100 μl sterile water<br /><br />
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         PCR cycles:</p>
         PCR cycles:</p>
       <table class="table1"  align="center">
       <table class="table1"  align="center">
       <tr><th>Amount of cycles</th><th>Reaction </th><th> Temp.</th><th> Time</th></tr>
       <tr><th>Amount of cycles</th><th>Reaction </th><th> Temp.</th><th> Time</th></tr>
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       <tr><td>1</td><td>Initial Denaturation</td><td>98°C</td><td>10 min</td></tr>
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       <tr><td>1</td><td>Pre-running</td><td>98°C</td><td>1 min</td></tr>
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       <tr><td>10</td><td>Denaturation</td><td>98°C</td><td>10 sec</td></tr>
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       <tr><td>10</td><td>Denaturation</td><td>98°C</td><td>15 sec</td></tr>
       <tr><td>10</td><td>Annealing</td><td>52°C</td><td>30 sec</td></tr>
       <tr><td>10</td><td>Annealing</td><td>52°C</td><td>30 sec</td></tr>
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       <tr><td>10</td><td>Elongation</td><td>72°C</td><td>2 min</td></tr>
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       <tr><td>10</td><td>Primer Extension </td><td>72°C</td><td>2 min</td></tr>
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       <tr><td>1</td><td>Hold</td><td>12°C</td><td>add oligos</td></tr>
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       <tr><td>1</td><td>Pause</td><td>8°C</td><td>add primer</td></tr>
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       <tr><td>21</td><td>Denaturation</td><td>98°C</td><td>10 sec</td></tr>
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       <tr><td>21</td><td>Denaturation</td><td>98°C</td><td>15 sec</td></tr>
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       <tr><td>21</td><td>Annealing</td><td>47°C</td><td>1 min</td></tr>
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       <tr><td>21</td><td> Annealing</td><td>52°C</td><td>30 sec</td></tr>
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       <tr><td>21</td><td>Elongation</td><td>72°C</td><td>2 min</td></tr>
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       <tr><td>21</td><td>Primer Extension</td><td>72°C</td><td>2 min + 5 sec/cycle</td></tr>
       <tr><td>1</td><td>Final elongation</td><td>72°C</td><td>10 min</td></tr>
       <tr><td>1</td><td>Final elongation</td><td>72°C</td><td>10 min</td></tr>
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       <tr><td>1</td><td> Hold </td><td>12°C</td><td> ∞ </td></tr>
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       <tr><td>1</td><td> Hold </td><td>4°C</td><td> ∞ </td></tr>
       </table>
       </table>
             <br /><br />
             <br /><br />
 +
 +
         </div>
         </div>
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Revision as of 11:50, 10 October 2014

PfuS PCR

Watch the following tutorial to learn about the whole process:



Reaction protocol for 50 µl
      1 μl Primer fwd (5 pmol)
      1 μl Primer rev (5 pmol)
      1 μl Matrizen-DNA (ca. 100 ng)
      10 μl 5x HF-Polymerase Puffer
      0.5 μl PfuS-Polymerase (50 ng)
      1 μl dNTPs (12.5 μmol ml-1)
      35.5 μl dest. Water

PCR cycles:

CyclesReactionTemp.Time
1Pre-running98 °C10 min
30Denature9810 s
30Annealing54-60 °C1 min
30Primer Extension72 °C15 s - 2.5 min
1Final elongation72 °C10 min
1Hold4 °C ∞


Fusion PCR

Reaction protocol 50 µl:
      4 μl Primer forward (5 pmol)
      4 μl Primer reverse (5 pmol)
      100 ng template-DNA of each fragment
      10 μl 5x HF-Buffer
      1 μl PfuS-Polymerase
      2 μl dNTPs (12.5 mM)
      ad. 50 μl sterile water

PCR cycles:

Amount of cyclesReaction Temp. Time
1Pre-running98°C1 min
10Denaturation98°C15 sec
10Annealing52°C30 sec
10Primer Extension 72°C2 min
1Pause8°Cadd primer
21Denaturation98°C15 sec
21 Annealing52°C30 sec
21Primer Extension72°C2 min + 5 sec/cycle
1Final elongation72°C10 min
1 Hold 4°C ∞


Retrieved from "http://2014.igem.org/Team:Goettingen/protocol_PCR"