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) 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

Beads:

• 80uL beads per sample

MBD-biotion protein:

• 56uL per sample

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

• C_iV_i = C_fV_f

• (58.4ng/uL)(136uL) = (25ng/uL)(V_f)

• V_f = 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.