6.2. Forward FR3 Gene-Specific (GS) Primer Extension

In this step, the pool of Forward FR3 GS Primers with adjoining Anchor 1 sequences (see Outline of AIR Assay, Fig.1) is annealed to Reverse GSP extended cDNA template generated in the previous step, extended by DNA polymerase, and generates the target CDR3 DNA region (top, sense strand) flanked from both sides by Anchor 1 and Anchor 2 sequences.

1. Spin down the plate from the previous step, remove the seal from the plate, then add 2.5 μl of the Forward FR3 GS Primer Mix to each reaction well of the plate:
    
   

   Component	Volume, µl
   Reverse J GS extension reaction	52
   Forward FR3 GS Primer Mix	2.5
   Total	54.5

2. 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 to avoid cross-contamination.

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

   Temperature	Time
   98°C	1 min
   68°C	10 min
   4°C	∞

4. Spin down the plate, remove the seal from the plate, then add 2 μl of the Primer Removal Enzyme to each reaction well of the plate. Mix contents by pipetting 3 times, seal the plate, and spin down to collect droplets

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

   Temperature	Time
   37°C	20 min
   95°C	5 min
   4°C	∞

!Immediately proceed to the next step (PCR with Anchor Primers)