Difference between revisions of "Team:Paris Bettencourt/Protocols/PCR"

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     <td><b>PCR protocol (Phusion)</b><br />
 
     <td><b>PCR protocol (Phusion)</b><br />
 
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       <li>Thaw PCR mastermix on ice for 10min.
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       <li>Thaw PCR Master Mix on ice for 10min.
 
       <li>add 2µL of each primers (at 10 µM) in a PCR tube.
 
       <li>add 2µL of each primers (at 10 µM) in a PCR tube.
 
       <li>add 1µL of the DNA (at 1 ng/µL) you want to amplify in the same tube.
 
       <li>add 1µL of the DNA (at 1 ng/µL) you want to amplify in the same tube.
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Revision as of 14:52, 29 August 2015

PCR protocol (Phusion)
  • Thaw PCR Master Mix on ice for 10min.
  • add 2µL of each primers (at 10 µM) in a PCR tube.
  • add 1µL of the DNA (at 1 ng/µL) you want to amplify in the same tube.
  • add 25µL of Life Technologies Phusion High-Fidelity PCR Master Mix with HF Buffer.
  • add 20µL of DNase/RNase-free water to the tube.
  • Thermocycling conditions
    • initial denaturation 98°C for 30 seconds
    • 35 cycles
      • 98°C for 30 seconds
      • 57°C for 30 seconds
      • 72°C 30 seconds/kb
    • final extension of 72°C for 10 minutes
    • infinite hold 12°C