MBD Enrichment - Crassostrea virginica Sheared DNA Day 1

As part of a project with Qiagen to have them try out some of our DNA with their newest DNA bisulfite conversion kit, I previously [isolated DNA from Crassotrea virginica (Eastern oyster)(2017/12/11/dna-isolation-quantification-crassotrea-virginica-mantle-gdna.html) and sheared to ~420bp.

Next, I needed to enrich the samples for methylated DNA. Did this using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). Followed the manufacturer’s protocol for input DNA amounts of 1 -10ug (I am using 8ug in each of two samples). Below are the exact volumes used for various steps:

Made 1x Bind/Wash Buffer

  • 2.88mL 5x Bind/Wash Buffer

  • 720uL molecular biology grade H2O


  • 80uL beads per sample

MBD-biotion protein:

  • 56uL per sample

Diluted the two sheared DNA samples to 25ng/uL:

  • CiVi = CfVf

  • (58.4ng/uL)(136uL) = (25ng/uL)(Vf)

  • Vf = 317.7

  • Add 181.7uL H2O to DNA to get 317.7ul (i.e. 25ng/uL)

Samples were incubated O/N in the 4C in the rotator.