Continued MeDIP process from yesterday. Added 20uL of Protein A/G Plus Agarose (Santa Cruz Biotech) beads to each sample and continued incubation with rotation @ 4C for 2hrs. Pelleted the Protein A/G beads 3300g, 2mins, 4C.
Removed and saved supe (to retain unmethylated DNA). Washed beads with 1mL 1x MeDIP Buffer. Repeated two more times. Saved supe after each wash.
Resuspended beads in 250uL MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS). Added 75ug of Proteinase K. Incubated 20hrs @ RT with end-over-end rotation.
Note: The protocol we have says to incubate the Proteinase K digest @ 55C. However, we don’t have a means to do so, since we need a rocker/rotator to keep the agarose beads in suspension. According to various sources, Proteinase K retains >80% of it’s enzymatic activity between 20C-50C. So, I’ve allowed the digest to run longer (24hrs) than recommended (O/N).