Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI, FspEI or LpnPI

  1. Set up the following reaction in a sterile microcentrifuge tube (it is important to add the restriction enzyme last):

    DNA (0.5 to 1 μg)

    1–5 μl

    10X rCutSmart™ Buffer

    3 μl

    30X Enzyme Activator Solution

    1 μl

    Restriction Enzyme

    0.5–1 μl (2.5 to 5 units)

    Nuclease-free water

    to 30 μl

  2. Incubate at 37°C for 1-4 hours (extended reaction times may increase the yield of the 32-mer but will also increase star activity).

Note: These conditions are optimized to efficiently generate 32 bp fragments from fully modified CpG sites in genomic DNA. Under these conditions star activity may occur however, the star activity has minimal effect on generating the correct 32 bp fragments to be isolated and used in subsequent analysis