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  • Perform USER Excision and PCR Library Enrichment (E7445)

    Protocol

    The Universal PCR primer and Index (X) Primer, and USER Enzyme are contained in the NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335 or NEB #E7500) Oligos for Illumina. If you are using alternate oligos which are not supplied by New England Biolabs, it will be necessary to purchase USER Enzyme separately (NEB #M5505).

    1. To the size selected cDNA (20 μl) add the following components:
      USER Enzyme 3 μl
      NEBNext High-Fidelity PCR Master Mix, 2X   25 μl
      Universal PCR Primer (25 μM)   1 μl
      Index (X) Primer (25 μM)*   1 μl
      ------------------------------------------------------------------------
      Total volume 50 μl
      * If you are using the NEBNext Multiplex Oligos for Illumina (E7335 or E7500) for each reaction, only one of the 12 PCR primer indices is used during the PCR step.

    2. PCR Cycling Conditions
      CYCLE STEP TEMP TIME CYCLES
      User Digestion 37°C 15 minutes 1
      Initial Denaturation 98°C 10 seconds 1
      Denaturation 98°C 10 seconds 12-15*
      Annealing 65°C 30 seconds
      Extension 72°C 30 seconds
      Final Extension 72°C 5 minutes 1
      Hold 4°C  
      * T he number of PCR cycles should be adjusted based on RNA input. If 100 ng total RNA or 10 ng purified mRNA or ribosomal depleted RNA are the starting input, it is recommended to perform 15 cycles of PCR.

    3. Purify the PCR reaction using AMPure XP Beads (strongly recommended).

    CYCLE STEP TEMP TIME CYCLES
    User Digestion 37°C 15 minutes 1
    Initial Denaturation 98°C 10 seconds 1
    Denaturation 98°C 10 seconds 12-15*
    Annealing 65°C 30 seconds
    Extension 72°C 30 seconds
    Final Extension 72°C 5 minutes 1
    Hold 4°C