6.3. Forward GSP Extension

In this step, the pool of Forward Gene-Specific Primers with adjoining Anchor 1 sequences (Fig.1) is annealed to cDNA template generated in previous step, extended by DNA polymerase and generates sense strands of the target amplicons flanked from both sides by Anchor 1 and Anchor 2 sequences. The protocol below specifies volumes of reagents and Master Mixes for one pooled cDNA sample.

1. Prepare the Forward GS Primer Extension Master Mix as follows:
    
   

   Forward GS Primer Extension Master Mix Component	Volume per sample, µl
   RT-EXT Buffer, 5X	6
   Water	20.4
   Forward GSP TCR-Mark36 or Forward GSP BCR-Mark30 primers, 20X	3
   DNA Polymerase, 100X	0.6
   Total	30

2. Gently vortex Master Mix and spin down briefly to collect droplets. Spin down the sample test tube (from step 6.2), and add 25 μl of the Forward GS Primer Extension Master Mix:
    
   

   Component	Volume, µl
   cDNA (from the step above)	26
   Forward GS Primer Extension Master Mix	25
   Total	51

3. Mix contents by pipetting three times, briefly spin down the tube, load the tube in the thermal cycler, and run the following program:
    
   

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

4. Add 2 µl of the Primer Removal Enzyme to the sample, mix contents by pipetting 3 times, spin down to collect droplets, load the sample test tube 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)