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  • Protocol for a Routine PCR (E0555)

    Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.

    Protocol

    Reaction Setup:
    We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.
    1.   25 μl RXN 50 μl RXN FINAL CONC.
      Q5 High-Fidelity 2X Master Mix 12.5 μl 25 μl 1X
      10 μM Forward Primer 1.25 μl 2.5 μl 0.5 μM
      10 μM Reverse Primer 1.25 μl 2.5 μl 0.5 μM
      Template DNA variable variable < 1,000 ng
      Nuclease-Free Water to 25 μl 20 μl*** 20 μl
      Notes: Gently mix the reaction. Collect all liquid at the bottom of the tube with a quick spin, if necessary.

    2. Transfer PCR tubes to a thermocycler and begin the cycling program.

    3. Thermocycling Conditions for a Routine PCR:
      STEP TEMP TIME
      Initial Denaturation 98°C 30 seconds
      25–35 Cycles 98°C 5–10 seconds
      50–72°C* 10–30 seconds
      72°C 20–30 seconds/kb
      Final Extension 72°C 2 minutes
      Hold 4°C
      *Use of the NEB Tm Calculator is highly recommended.