The following sources may contribute to DNA yields after Library PCR that are below the recommended specifications (e.g., 2 ng/µL):
|Low template concentration
|Verify that the DNA concentration of the 1st PCR is correctly quantified.
Repeat the Library PCR with increased cycles of up to 12.
|Wrong primer concentration
|Verify that each sample is amplified with 10 µL of Index Primer Pair.
|Wrong thermal cycling protocol
|Ensure that the correct thermal cycling protocol conditions are used, including temperature and cycle number.
These may occur alone or in combination.