Difference between revisions of "Team:Gaston Day School/Notebook"

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                     <div class="col-md-11">09/01/2017 PCR Primer</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
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                     <div class="col-md-11">PCR From Plasmid DNA Template</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
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<p>
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<b>Materials:</b><br>
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2x Phusion Mastermix<br>
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10 µM forward primer<br>
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10 µM forward primer<br>
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PCR tube<br>
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Sterile water<br>
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Plasmid DNA<br>
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<br>
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<b>Methods:</b><br>
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For a 25 µL reaction<br>
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<ol style="font-size:16px;">
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<li>In a PCR tube on ice, combine  1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
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<br><i>Note: It is important to add Phusion Master Mix last in order to prevent primer degradation
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caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
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<li>Gently mix the reaction</li>
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<li>If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly</li>
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<li>Transfer the PCR tube from ice to a PCR machine to begin thermocycling</li>
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</ol>
  
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<p><br>For a 50 µL reaction<br></p>
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    <thead>
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<ol style="font-size:16px;">
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      <tr>
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<li>In a PCR tube on ice, combine  1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
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        <th>14.3</th>
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<br><i>Note: It is important to add Phusion Master Mix last in order to prevent primer degradation
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        <th>H2O</th>
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caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
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        <th>114.4</th>
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<li>Gently mix the reaction</li>
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      </tr>
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<li>If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly</li>
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<li>Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling</li>
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</ol>
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<p><br><b>Thermocycling</b><br>
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The PCR machine should be set to run the following steps: </p>
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<thead>
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        <tr>
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            <th>Step</th>
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            <th>Temperature (°C)</th>
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            <th>Time</th>
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        </tr>
 
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     </thead>
 
     <tbody>
 
     <tbody>
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      <tr>
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        <tr>
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         <td>2.5</td>
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            <td>Initial denaturation</td>
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         <td>buffer</td>
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            <td>98</td>
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         <td>20</td>
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            <td>30 seconds</td>
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      </tr>
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         </tr>
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        <tr>
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            <td>25-35 cycles</td>
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            <td>98 (denaturation)<br>
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                45-72 (annealing) <a href="#Note1">see Note 1</a><br>
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                72 (extension)</td>
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            <td>5-10 seconds <br>
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                10-30 seconds<br>
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                15-30 seconds per kb</td>
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         </tr>
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        <tr>
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            <td>Final extension</td>
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            <td>72</td>
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            <td>2-5 minutes</td>
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         </tr>
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        <tr>
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            <td>Hold</td>
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            <td>4</td>
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            <td>Indefinitely</td>
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        </tr>
  
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</table>
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<p id="Note1">Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: <a target="_blank" href="http://tmcalculator.neb.com/#!/">http://tmcalculator.neb.com/#!/</a></p>
  
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Revision as of 04:20, 24 October 2017

Notebook

On this page, Gaston Day School iGEM team would like to share with you our notes during the experiments from 09/01/2017 to 10/23/2017. The team leader Heena Saqib records the data patiently.

Materials:
2x Phusion Mastermix
10 µM forward primer
10 µM forward primer
PCR tube
Sterile water
Plasmid DNA

Methods:
For a 25 µL reaction

  1. In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
    Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
  2. Gently mix the reaction
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
  4. Transfer the PCR tube from ice to a PCR machine to begin thermocycling


For a 50 µL reaction

  1. In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
    Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
  2. Gently mix the reaction
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
  4. Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling


Thermocycling
The PCR machine should be set to run the following steps:

Step Temperature (°C) Time
Initial denaturation 98 30 seconds
25-35 cycles 98 (denaturation)
45-72 (annealing) see Note 1
72 (extension)		 5-10 seconds
10-30 seconds
15-30 seconds per kb
Final extension 72 2-5 minutes
Hold 4 Indefinitely

Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: http://tmcalculator.neb.com/#!/

 

Email: iGEM@gastonday.org

Gaston Day School

2001 Gaston Day School Rd

Gastonia 28056