6.3. First PCR with Anchor Primers

This step utilizes universal Anchor PCR primers to amplify the target CDR3 DNA fragments flanked with the universal Anchor 1 and Anchor 2 sequences generated during the previous Forward FR3 Gene-Specific Primer Extension step.

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	10
   dNTP Mix	1
   Anchor Primer Mix, 10X	10
   Water	28.5
   DNA Polymerase	0.5
   Total	50

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 50 μl of Anchor PCR Master Mix to each reaction well:
    
   

   Component	Volume per sample, µl
   Forward GS Primer Extension DNA (after primer removal step)	54
   Anchor PCR Master Mix (prepared above)	50
   Total	104

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

*Note: Take the plate from the PCR-free room and perform all follow-up amplification, purification, and NGS steps in a location dedicated to PCR work. Do not bring amplified library products back to 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	1 min	1
   98°C	20 sec	22-25* Note below
   65°C	10 sec
   72°C	30 sec
   72°C	30 sec	1
   4°C	∞	1

Note: To avoid over-cycling biases, we recommend starting with 22 PCR cycles for TCR or 25 PCR cycles for BCR assay for samples that contained 5 μg of DNA isolated from lymphocyte-rich samples (e.g., whole blood, PBMC, lymphoid tissues, sorted cells, and positive control PBMC DNA). For similar samples with less DNA amount, you need to add extra cycles (e.g., if using 0.5 ug DNA, do three extra cycles). For small DNA samples isolated from sorted cells (or in DirectCell protocol), we recommend starting from 25 cycles for 250 ng of DNA (or 50,000 sorted cells) and using proportionally more cycles for using less starting DNA amount. For most samples, we recommend not exceeding 28 cycles. For DNA samples with low content of immune cells, use 5-10 ug DNA and 28 PCR cycles. The recommended number of cycles, in many cases, needs to be optimized and adjusted based on specific cell types, quality of DNA, type of tissue sample, the content of T/B cells, etc.

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