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  • Perform PCR Library Enrichment (E7530)

    1. To the size selected cDNA (23 μl) add the following components:

      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.

      Note: 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.

    2. PCR Cycling Conditions
      CYCLE STEP TEMP TIME CYCLES
      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  
      * The 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.
      ** It is important to limit the number of PCR cycles to avoid overamplification. If overamplification
      occurs, larger molecular weight products (> 500 bp) will appear on the bioanalyzer trace.
    Purify the PCR Reaction using AMPure XP Beads

    Note: X refers to the original sample volume from the above step.

    1. Vortex AMPure XP beads to resuspend.
    2. Add 50 μl (1.0X) of resuspended AMPure XP beads to the PCR reaction (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
    3. Incubate for 5 minutes at room temperature.
    4. Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
    5. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
    6. Repeat Step 5 once for a total of 2 washing steps.
    7. Air dry the beads for 5 minutes while the tube is on the magnetic rack with the lid open.
    8. Elute the DNA target from the beads into 23 μl nuclease free water. Mix well on a vortex mixer or by pipetting up and down, quickly spin the tube in a microcentrifuge and place it in the magnetic rack until the solution is clear.
    9. Transfer 20 μl of the supernatant to a clean PCR tube, and store at -20°C.