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Methylated DNA Immunoprecipitation

Zymo Research: Magnetic Kit (D5101): ammounts for 400ng DNA

Dilute and denature input DNA samples as follows:

  1. Dilute 1-40uL of sample containing 400ng DNA in the DNA. Denaturing Buffer to a final volume of 50uL
  2. Denature the diluted DNA at 98°C for 5 minutes

 

While DNA is denaturing, set up in tubes.  In order, add the following to a 1.5mL tube

  1. 205uL MIP Buffer
  2. 15uL ZymoMag Protein A (gently flick and invert tube prior to use, pipet up and down to expel the beads from tip)
  3. 4uL Mouse Anti-5-Methylcytosine
  4. Invert the tubes 2-4 times to mix the antibody/protein A mixture

 

Add denatured DNA immediately to the antibody/Protein A mixture after step 1 is complete.

Incubate at 37°C for 1 hour on a rotator/rocker.  Also invert every 10 minutes during the incubation

Use magnet strips to allow the beads to cluster, then remove and discard supernatant.

Add 500uL MIP buffer to each tube and secure the cap.  Invert several times and vortex briefly to resuspend the beads.  Remove and discard the supernatant.

Repeat wash once more with MIP buffer.  Wash again with 500uL of DNA Elution Buffer

Add 20uL water to each tube and resuspend the beads by pipetting.  Transfer the suspension to a 0.2mL PCR tube

Incubate the tubes at 75°C for 5 minutes and spin for 2 minutes

Transfer the supernatant to a new 1.5mL tube without disturbing the beads.  If beads are disturbed, respin the tubes.

Quantify and store at -20 or -80 until proceeding with WGA2