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
- 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.