---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2018-01-08 22:54:07+00:00
layout: post
slug: mbd-enrichment-crassostrea-virginica-sheared-dna-day-1
title: MBD Enrichment - Crassostrea virginica Sheared DNA Day 1
categories:
  - 2018
  - Miscellaneous
tags:
  - Crassostrea virginica
  - Eastern oyster
  - MBD enrichment
  - MethylMiner Methylated DNA Enrichment Kit
---

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](https://robertslab.github.io/sams-notebook/posts/2017/2017-12-11-dna-sonication-bioanalzyer-c-virginica-gdna-for-medip/).

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:



  * 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.