Post Library Construction Quantification

1. Thaw KAPA Library Quantification Kit for Illumina Platforms.

2. Dilute 2 μl sample with deionized water to appropriate dilutions that fall within the linear detection range of the KAPA Library Quantification Kit for Illumina Platforms. (For more accurate quantification, make the dilution(s) in duplicate).

3. Make enough Quantification Master Mix for the DNA dilutions per sample and the DNA Standards (plus 10% excess) using the guidance for 1 reaction volume below.

   Quantification Master Mix	1X (µl)
   SYBR Fast Master Mix + Primer	12
   Water	4
   Total	16

4. Dispense 16 μl Quantification Master Mix for sample dilutions and DNA Standards into a 96 well PCR plate.

5. Add 4 μl sample dilutions and 4 μl DNA Standards to appropriate wells. Centrifuge briefly.

6. Incubate in a thermal cycler with the following protocol.

   Step	Temperature	Run Time
   1	95°C	00:03:00
   2	95°C	00:00:05
   3	67°C Read Signal	00:00:30
   4	Go to Step 2, 29X (Total 30 cycles)

7. Follow the manufacturer’s recommendations for qPCR-based quantification. For library quantification for sequencer clustering, determine the concentration based on average insert size derived from the Bioanalyzer/TapeStation trace.