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  • PCR Amplification (E7370)

    1. Mix the following components in sterile strip tubes:

      Adaptor Ligated DNA Fragments   23 μl
      NEBNext High Fidelity 2X PCR Master Mix   25 μl
      Index Primer*   1 μl
      Universal PCR Primer*   1 μl
      ------------------------------------------------------------------
      Total volume   50 μl

      *The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500) Oligos for Illumina.

    2. PCR cycling conditions:

      CYCLE STEP TEMP TIME CYCLES
      Initial Denaturation 98°C 30 seconds 1
      Denaturation 98°C 10 seconds 6-15*
      Annealing 65°C 30 seconds
      Extension 72°C 30 seconds
      Final Extension 72°C 5 minutes 1
      Hold 4°C  
      *We suggest 6 PCR cycles for 1 μg DNA input 10 cycles for 50 ng, and 13–15 for 5 ng DNA input. Further optimization of PCR cycle number may be required.