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  • PCR Amplification of Adaptor Ligated DNA (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 
    --------------------------------------------------------------
    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
    --------------------------------------------------------------
    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