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  • PCR Optimization of the Control Template using Phusion® High-Fidelity PCR Kit

    Protocol

    1. Set up the appropriate reactions on ice:
      Component 20 µl Reaction 50 µl Reaction Final Concentration
      Nuclease-free water 13.6 µl 34 µl  
      5X Phusion HF Buffer 4 µl 10 µl 1X
      10 mM dNTPs 0.4 µl 1 µl 200 µM
      Primers* 1 µl 2.5 µl 0.2 µM
      Control Template DNA 0.8 µl 2 µl 0.02 ng/µl
      Phusion DNA Polymerase 0.2 µl** 0.5 µl 0.02 U/µl
      *Reaction can be set up using either the 1.3 or 10 kb primer set
      ** Dilute polymerase with 1X reaction buffer to avoid pipetting errors

    2. Recommended cycling conditions for the 1.3 kb fragment using a 2-step protocol:
      Cycle step Temp Time
      Initial denaturation 98°C 1 Minute
      25 Cycles 98°C
      72°C
      5 Seconds
      20 Seconds
      Final extension 72°C 10 Minutes
      Hold 4°C

      Recommended cycling conditions for the 10 kb fragment using a 3-step protocol. Alternatively, this program can be used to amplify both fragments simultaneously.
      Cycle step Temp Time
      Initial denaturation 98°C 1 Minute
      25 Cycles 98°C
      60°C
      72°C
      5 Seconds
      15 Seconds
      2 Minutes 30 Seconds
      Final extension 72°C 10 Minutes
      Hold 4°C

      Note: Controls have been shown to work in a variety of conditions.