Difference between revisions of "Team:ETH Zurich/Experiments"

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<h2>Polymerase Chain Reaction</h2>
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<h2>Polymerase Chain Reaction - for Construction</h2>
 
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</div>
 
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<h3>Phusion &#174; High-Fidelity DNA Polymerase</h3>
 
<h3>Phusion &#174; High-Fidelity DNA Polymerase</h3>
 
   <h4>Composition:</h4>
 
   <h4>Composition:</h4>
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         <table>
 
         <tbody>
 
         <tbody>
 
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             <tr>
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<h3>Kap Hifi Hotstart Ready Mix</h3>
 
<h3>Kap Hifi Hotstart Ready Mix</h3>
 
    
 
    
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<div class="sec white" id="PCR">
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<div>
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<h2>Polymerase Chain Reaction - for Analysis</h2>
 +
</div>
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</div>
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 +
<div class="sec white">
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<div>
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<h3>Solis BioDyne FirePol<sup>&#174;</sup> DNA Polymerase</h3>
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  <h4>Composition:</h4>
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>8 x 20 &micro;l Reaction (+ 10 &micro;l backup) </strong></td>
 +
                </tr>
 +
            <tr>
 +
                <td>FIREPol DNA Polymerase</td>
 +
               
 +
                <td>0.85 &micro;l</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>MgCl<sup>2</sup> (25 mM)</td>
 +
               
 +
                <td>10.2 &micro;l</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>Reaction Buffer B (10x)</td>
 +
               
 +
                <td>17 &micro;l</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>dNTPs (10 mM)</td>
 +
               
 +
                <td>3.4 &micro;l</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>Forward Primer (10 &micro;M)</td>
 +
               
 +
                <td>3.4 &micro;l</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>Reverse Primer (10 &micro;M)</td>
 +
             
 +
                <td>3.4 &micro;l</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>Template DNA</td>
 +
             
 +
                <td>1 &micro;l</td>
 +
             
 +
            </tr>
 +
           
 +
            <tr>
 +
                <td>Nuclease-free water</td>
 +
               
 +
                <td>125 &micro;l</td>
 +
               
 +
            </tr>
 +
           
 +
        </tbody>
 +
    </table>
 +
 +
<h4>Thermocycling Conditions:</h4>
 +
<table>
 +
        <tbody>
 +
            <tr>
 +
               
 +
                <td align="center"><strong>Temperatur <br />
 +
                </strong></td>
 +
                <td align="center"><strong>Time<br />
 +
                </strong></td>
 +
            </tr>
 +
            <tr>
 +
             
 +
                <td align="center">98&deg;C </td>
 +
                <td>5 minutes</td>
 +
            </tr>
 +
            <tr>
 +
             
 +
                <td align="center">95&deg;C<br />
 +
                45-72&deg;C<br />
 +
                72&deg;C </td>
 +
                <td>30 seconds<br />
 +
                45 seconds<br />
 +
                1 minute per kb </td>
 +
            </tr>
 +
            <tr>
 +
             
 +
                <td align="center">72&deg;C </td>
 +
                <td>5-10 minutes </td>
 +
            </tr>
 +
       
 +
        </tbody>
 +
    </table>
 +
 +
</div>
 +
</div>
  
  

Revision as of 00:56, 9 October 2016

PAVLOV'S COLI

Experimental: Protocols, Methods and Material

Polymerase Chain Reaction - for Construction

Phusion ® High-Fidelity DNA Polymerase

Composition:

Component 50 µl Reaction
Phusion HF Buffer (5x) 10 µl
dNTPs (10 mM) 1 µl
Forward Primer (10 µM) 2.5 µl
Reverse Primer (10 µM) 2.5 µl
Template DNA variable
DMSO (optional) up to 3%
Nuclease-free water up to 50 µl
Phusion Polymerase 0.5 µl

Thermocycling Conditions:

Temperatur
Time
98°C 30 seconds
98°C
45-72°C
72°C 		5-10 seconds
10-30 seconds
15-30 seconds per kb
72°C 5-10 minutes

Kap Hifi Hotstart Ready Mix

Component 50 µl Reaction
Master Mix (2x) 25 µl
Forward Primer (10 µM) 2.5 µl
Reverse Primer (10 µM) 2.5 µl
Template DNA variable
Nuclease-free water up to 50 µl

Thermocycling Conditions:

Temperatur
Time
95°C 3 minutes
98°C
55-75°C
72°C 		20 seconds
15 seconds
15-60 seconds per kb
72°C 1 minute per kb

Polymerase Chain Reaction - for Analysis

Solis BioDyne FirePol® DNA Polymerase

Composition:

Component 8 x 20 µl Reaction (+ 10 µl backup)
FIREPol DNA Polymerase 0.85 µl
MgCl2 (25 mM) 10.2 µl
Reaction Buffer B (10x) 17 µl
dNTPs (10 mM) 3.4 µl
Forward Primer (10 µM) 3.4 µl
Reverse Primer (10 µM) 3.4 µl
Template DNA 1 µl
Nuclease-free water 125 µl

Thermocycling Conditions:

Temperatur
Time
98°C 5 minutes
95°C
45-72°C
72°C 		30 seconds
45 seconds
1 minute per kb
72°C 5-10 minutes
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