5.5. First PCR with Anchor Primers

This step utilizes universal Anchor PCR primers to amplify the target DNA fragments flanked with the Anchor 1 and Anchor 2 sequences generated during the previous Forward GSP 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	13
   dNTP Mix	1.3
   Anchor Primer Mix, 10X	10
   Water	40
   DNA Polymerase	0.7
   Total	65

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)	40
   Anchor PCR Master Mix (prepared above)	60
   Total	100

3. Mix content by pipetting 3 times or by using an Eppendorf plate shaker. Seal the plate with new adhesive film and spin it 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	18
   65°C	10 sec
   72°C	30 sec
   72°C	30 sec	1
   4°C	∞	1

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