4.3. Reverse Gene-Specific Primer Extension

In this step, the pool of Reverse Gene-Specific Primers uses the template generated in the Forward Gene-Specific Primer Extension step to generate antisense target amplicon strands flanked by both Anchor 1 and Anchor 2 sequences.

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

   Reverse GS Primer Extension Master Mix Component	Volume, µl
   RT-EXT Buffer	1
   dNTP Mix	0.25
   PCR-Grade Water	0.65
   Reverse GS Primer Pool*	3
   DNA Polymerase	0.1
   Total	5

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.

2. Gently vortex the Master Mix, and spin down briefly to collect droplets. Spin down the Primer Extension plate, remove the seal, then add 5 μl of the Reverse GS Primer Extension Master Mix to each reaction well of the plate:
   

   Component	Volume, µl
   Forward GS Extension product (from previous step)	25
   Reverse GS Extension Master Mix (prepared above)	5
   Total	30

3. 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.

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

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

5. Spin down, remove the seal from the plate, and add 5 μl of the Primer Removal Master Mix (prepared in Forward Gene-Specific Primer Extension step) to each reaction well of the plate (keeping the remainder for the Quantify and Combine Samples for NGS step):
   

   Component	Volume, µl
   Reverse GSP Extension products (above)	30
   Primer Removal Master Mix (prepared in Forward Gene-Specific Primer Extension	5
   Total	35

6. Mix contents by pipetting 3 times, seal the plate, and spin down to collect droplets.

7. 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 (PCR with Anchor Primers)