Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using FspEI

Protocol

  1. Set up the following reaction in a sterile microcentrifuge tube (it is important to add FspEl last):
    DNA (0.5 to 1 μg) 1–5 μl
    10X CutSmart Buffer
    3 μl
    30X Enzyme Activator Solution 1 μl
    FspEl 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.

Note: Selling concentration is 5,000 U/ml this used to be 4,000 U/ml.