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

Line 13: Line 13:
 
           <li>35 cycles  
 
           <li>35 cycles  
 
               <ul>
 
               <ul>
−
               <li>94°C for 30 seconds
+
               <li>98°C for 30 seconds
 
               <li>57°C for 30 seconds
 
               <li>57°C for 30 seconds
 
               <li> 72°C 30 seconds/kb
 
               <li> 72°C 30 seconds/kb

Revision as of 10:59, 29 August 2015

PCR protocol (Phusion)
  • Thaw PCR mastermix 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