6.1. Reverse J Gene-Specific (GS) Primers Extension

In this step, the pool of Reverse J GS primers with adjoining VBC’s and AP2 sequences (see Outline of AIR Assay, Fig.1) is annealed to J regions (top, sense strand) of TCR or BCR genes, extended by DNA polymerase to generate Reverse GSP extended cDNA and purified from non-extended primers by nuclease treatment. The protocol assumes the reactions will be set off in a 96-well plate. For small numbers of samples, reactions can be done in tubes or strips also.

*Note: Reverse and Forward Primer Extension steps and assembly of 1st PCR reactions should be set up in a PCR-free room.

1. Prepare the Reverse J GS Primer Extension Master Mix as described below for all samples and controls (make 5% extra to account for pipetting error) and aliquot 20 µl in the wells of a 96-well plate:
    
   

   Reverse J GS Primer Extension Master Mix Component	Volume per sample, µl
   EXT Buffer, 4x	12.5
   dNTP Mix	1.25
   Reverse J Primer mix, 20x	2.5
   Water	3
   DNA polymerase	0.75
   Total	20

*Note: If you decide to run biological or amplification triplicates, increase the number of wells and amount of DNA allocated for each sample, respectively.

2. Adjust the volume of each DNA Sample to 30 µl with water, as shown in the table.
    
   

   Component	Volume per sample, µl
   Genomic DNA (~0.5-2 µg)	1 – 30*
   Water, to 30 µl final volume	0 – 29

*Note: For optimal input recommendations, please refer to Section 5.3 General Notes

3. Add 30 µl of DNA Sample to each well with pre-aliquoted 20 µl of Reverse J GS Primer Extension Master Mix.
    
   

   Component	Volume, µl
   DNA	30
   Reverse J GS Primer Extension Master Mix	20
   Total	50

4. Mix contents by pipetting 3 times. Seal the plate with adhesive film, and spin down to collect droplets. Load the plate in the thermal cycler and run the following program:
    
   

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

5. 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 three times, seal the plate and spin down to collect droplets.

6. 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 (Forward FR3 Gene-Specific Primer Extension)