---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-01-23 03:23:55+00:00
layout: post
slug: library-cleanup-lsu-c-virginica-mbd-bs-library
title: Library Cleanup - LSU C.virginica MBD BS Library
categories:
  - 2015
  - LSU C.virginica Oil Spill MBD BS Sequencing
tags:
  - bioanalyzer
  - Crassostrea virginica
  - DNA Quantification
  - Eastern oyster
  - electropherogram
  - library prep
---

I was contacted by the sequencing facility at the University of Oregon regarding a sample quality issue with our library.  As evidenced by the electropherogram below, there is a great deal of adaptor primer dimer (the peak at 128bp):

![](https://eagle.fish.washington.edu/Arabidopsis/20150120_LSUoilNGSlibraryBioanalyzer.png)(http://eagle.fish.washington.edu/Arabidopsis/20150120_LSUoilNGSlibraryBioanalyzer.png)



This is a problem because such a high quantity of adaptor sequence will result in the majority of reads coming off the Illumina being just adaptor sequences.

With the remainder of the [library sample prepared earlier](https://robertslab.github.io/sams-notebook/posts/2014/2014-12-23-bisulfite-ngs-library-lsu-c-virginica-oil-spill-mbd-bisulfite-dna-sequencing-submission/), I performed the recommended clean up procedure for removing adaptor sequences in the EpiNext Post-Bisulfite DNA Library Preparation Kit – Illumina (Epigentek).    Briefly:





  * Brought sample volume up to 20uL with NanoPure H2O (added 9.99uL)



  * Added equal volume of MQ Beads



  * Washed beads 3x w/80% EtOH



  * Eluted DNA w/12uL Buffer EB (Qiagen)






After clean up, quantified the sample via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of the sample and the standards.  All standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Results are here: [20150122 - LSU_virginicaMBDlibraryCleanup](https://docs.google.com/spreadsheets/d/1g2frcvTw4lYq3jXhUhDYTORjOdWNyRN9s3ksDKQHiRI/edit?usp=sharing)

Library concentration = 2.46ng/uL

Brought the entire sample up to 20uL with Buffer EB (Qiagen) and a final concentration of 0.1% Tween-20 (required by the sequencing facility).

Sent sample to the University of Oregon to replace our previous submission.
