Manufacturer’s instructions

Library Amplification

5.1 Assemble each library amplification reaction as follows per pool:

Component Volume
KAPA HiFi HotStart ReadyMix (2X) 12.5 μl
PCR1_F Primer (Universal) 1.25 μl
PCR2_R Primer (Indexed) 1.25 μl
Adapter-ligated library pool 10.0 μl
Total Volume 25 μl

5.2 Mix thoroughly and centrifuge briefly.

5.3 Amplify using the following cycling protocol:

Step Temp Duration Cycles
Initial denaturation 98 °C 45 sec 1
Denaturation 98 °C 15 sec X
Annealing- 60 °C 30 sec X
Extension 72 °C 30 sec X
Final Extension 72 °C 1 min 1
Hold 4 °C ∞ 1
Amount of adapter-ligated DNA in amplification reaction Number of cycles required to generate 1 μg of library DNA
500 ng 1-2
100 ng 3-4
50 ng 5-6
25 ng 7-8
10 ng 8-9
5 ng 11-12
1 ng 12-13

For Puritz and Lotterhos (2017), 6 PCR cycles were used.

5.4 Proceed immediately to the next step

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