In this step, the pool of Forward Gene-Specific Primers with adjoining Anchor 1 sequences (see Procedure Overview generates sense strands of the target amplicons using the cDNA generated in the previous step as a template.

  1. Prepare the Forward Gene-Specific Primer Extension Master Mix as follows for all samples plus 5% extra volume of all components as a safeguard against pipetting error:

    Forward GS Primer Extension Master Mix Component Volume per sample, µl
    RT-EXT Buffer 2
    PCR-Grade Water 5.8
    Forward GS Primer Pool* 2
    DNA Polymerase 0.2
    Total 10

Note: There are different Forward GS Primer and Reverse GS Primer pools for each DriverMap Assay. Please refer to the Product Insert for your specific kit to identify the appropriate component in the Kit.

  1. Gently vortex the Master Mix, and spin down briefly to collect droplets. Spin down the cDNA plate, remove the seal, then add 10 μl of the Forward GS Primer Extension Master Mix to each reaction well of the plate:

    Component Volume, µl
    cDNA 10
    Forward GS Primer Extension Master Mix 10
    Total 20
  1. Mix contents by pipetting 3 times, seal the plate with a new adhesive film, and spin down to collect droplets.

Note: A new adhesive film is required in order to avoid cross-contamination.

  1. Load the plate in the thermal cycler, and run the following program:

    Temperature Time
    98°C 1 min
    64°C 30 min
    4°C

  1. Prepare Primer Removal Master Mix as follows for all samples plus 5% extra volume of all components as a safeguard against pipetting error:

    Primer Removal Master Mix Component Volume per sample, µl
    RT-EXT Buffer 2.5
    PCR-Grade Water 9.5
    Primer Removal Enzyme 0.5
    Total 12.5

Note: Sufficient Primer Removal Master Mix is prepared above to use at this point in the procedure (5 μl), plus after two other later steps: the Reverse Gene-Specific Primer Extension (5 μl) and when you Quantify and Combine Samples for NGS (2 μl).

  1. Spin down, remove the seal from the plate, then add 5 μl of the Primer Removal Master Mix to each reaction well of the plate (keeping the remainder for follow-up steps listed above):

    Component Volume, µl
    Forward GSP Extension products (above) 20
    Primer Removal Master Mix (prepared above) 5
    Total 25
  1. Mix contents by pipetting 3 times, seal the plate, and spin down to collect droplets.
  1. Load the plate in the thermal cycler, and run the following program:

    Temperature Time
    37°C 30 min
    95°C 5 min
    4°C

Immediately proceed to the next step (Reverse Gene-Specific Primer Extension)


Last modified: Aug 08, 2019

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