5.4. Forward Gene-Specific Primer Extension

In this step, the pool of Forward hGW19K Primers (Fwd GSPs) with adjoining Anchor 1 sequences generates sense strands of the target amplicons flanked from both sides by Anchor 1 and Anchor 2 sequences using the cDNA generated in the previous step as a template.

1. Prepare the Forward GSP Extension Master Mix as follows for all samples plus 5% extra volume of all components:
    
   

   Forward GS Primer Extension Master Mix Component	Volume per sample, µl
   RT-EXT Buffer, 5X	4.4
   Water	8.5
   Forward hGW19K Primer Mix, 5x	8.4
   DNA Polymerase	0.7
   Total	22

2. Gently vortex Master Mix and spin down briefly to collect droplets. Spin down the cDNA plate, remove the seal, then add 20 μl of the Forward GS Primer Extension Master Mix to each reaction well of the plate:
    
   

   Component	Volume, µl
   cDNA (step above)	20
   Forward GS Primer Extension Master Mix	20
   Total	40

3. Mix contents by pipetting three 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.

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

   Temperature	Time
   98°C	1 min
   68°C	30 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 using an Eppendorf plate shaker or pipetting 3 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	30 min
   95°C	5 min
   4°C	∞

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