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

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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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                     <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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<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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<b>Methods:</b><br>
 
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For a 25 µL reaction<br>
 
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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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<p><br>For a 50 µL reaction<br></p>
 
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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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<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 preheated to 98°C to begin thermocycling</li>
 
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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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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.

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