PCR Amplification of Adaptor Ligated DNA - NEBNext Fast DNA Library Prep Set for Ion Torrent (E6270)

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 Q5 Hot Start HiFi 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 Q5 Hot Start HiFi 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