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.