6.4. First PCR with Anchor Primers

This step utilizes universal Anchor PCR primers to amplify the target cDNA fragments flanked with the Anchor 1 and Anchor 2 sequences generated during the previous Forward Gene-Specific Primer Extension step (Outline).

1. Prepare the Anchor PCR Master Mix as shown below for all samples and controls plus 5% extra volume of all components:
    
   

   Anchor PCR Master Mix Component	Volume per sample, µl
   PCR Buffer, 5X	12
   dNTP Mix	1.2
   Anchor Primer Mix, 10X	10
   Water	36.2
   DNA Polymerase	0.6
   Total	60

2. Gently vortex Master Mix and spin down briefly to collect droplets. Spin down the Forward GS Primer Extension plate, remove the seal, then add 60 μl of Anchor PCR Master Mix to each reaction well:
    
   

   Component	Volume per sample, µl
   Forward GS Primer Extension DNA (after primer removal step)	42
   Anchor PCR Master Mix (prepared above)	60
   Total	102

3. Mix content by pipetting 3 times. Seal the plate with new adhesive film and spin down to collect droplets.

*Note: Take the plate from the PCR-free room and perform all follow-up Amplification, Purification, and NGS steps in the location dedicated to PCR work. Never bring amplified library products back to the PCR-free room.

4. Load the plate in the thermal cycler in a location dedicated to PCR work. Run the following program using the recommended number* of PCR cycles:
    
   

   Temperature	Time	Cycles
   98°C	30 sec	1
   98°C	20 sec	16-24* Note below
   65°C	10 sec
   72°C	30 sec
   72°C	30 sec	1
   4°C	∞	1

*Note: To avoid bias in gene expression levels by over-cycling samples, we recommend starting with 18 PCR cycles for samples that contained 25 ng or more RNA (e.g., 50 ng of PBMC or 100 ng of whole blood or lymphocyte-rich samples) or at least 25,000 immune cells. For samples with less RNA or cells (or low lymphocyte RNA content), you may have to add extra cycles, but in general, we recommend not exceeding 20 cycles. For very small RNA samples (1-5 ng) or experiments with less than ~1000 immune cells, 22-26 cycles may be required. The recommended number of cycles needs to be optimized and adjusted based on specific cell types, sample types, RNA quality, and the level of TCR/BCR mRNAs, etc. In order to optimize cycle number we recommend analyzing 5 ul of amplified products in a 3% agarose gel or fragment analyzer. For optimal cycle number, you will detect weak cDNA amplification products in the range of 300-500 bp. If you can’t see any products, add 3 more cycles and analyze the yield of PCR products again.

!Stopping Point in procedure: PCR products may be stored at 4°C overnight.