For all downstream techniques, including endpoint and real-time PCR assays, bisulfite conversion, cloning and sequencing, direct sequencing, library preparation for high-throughput sequencing, labeling for DNA microarray analysis or methylation-sensitive restriction enzyme-based assays, heating the sample at 65°C for 15 minutes is the recommended method of choice for elution of DNA. The appendix section also includes a protocol for NaCl multi-fraction elution series, followed by Qiagen spin column cleanup if necessary.
- Heat Method
Add 50–100 μl of DNase-free water to the sample pellet. Mix beads by gentle vortexing (1200 rpm) or by flicking the tube. Incubate the slurry in a heat block or thermomixer set at 65°C for 15 minutes, with frequent mixing.
- Briefly centrifuge the sample at 14,000 rpm in a microcentrifuge.
- Place the tube on the magnetic rack for 2–5 minutes to concentrate the beads on the inner wall of the tube.
- Carefully remove the supernatant to a fresh microcentrifuge tube. Eluent contains enriched methyl CpG containing DNA. Proceed directly to downstream analysis or store the eluted DNA sample at -20°C.