PCR Amplification of Adaptor Ligated DNA - NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent (E6285)

Protocol

1. Mix the following components in a sterile microfuge tube:

For 10 ng – 100 ng
Adaptor Ligated DNA   1-40 μl
Primers   4 μl
Sterile H2O   variable
NEBNext High-Fidelity 2X PCR Master Mix   50 µl
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Total volume   100 μl

For 1 μg
Adaptor Ligated DNA   1-40 μl
Primers   10 μl
Sterile H2O   variable
NEBNext High-Fidelity 2X PCR Master Mix   50 µl
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Total volume 100 μl 

PCR Cycling Conditions

STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
4–12 Cycles 98°C 10 seconds
58°C 30 seconds
65°C 30 seconds
1 Cycle 65°C 5 minutes
Hold 4°C

Cycling Suggestions
DNA CYCLES
10 ng 10-12
100 ng 6–8
1 μg 4–6