---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2016-12-15 00:24:41+00:00
layout: post
slug: dna-isolation-ostrea-lurida-dna-for-pacbio-sequencing
title: DNA Isolation - Ostrea lurida DNA for PacBio Sequencing
categories:
  - 2016
  - Olympia Oyster Genome Sequencing
tags:
  - DNA Isolation
  - DNA Quantification
  - E.Z.N.A. Mollusc Kit
  - gDNA
  - gel
  - olympia oyster
  - Ostrea lurida
  - Qubit 3.0
  - Qubit dsDNA BR
---

In an attempt to improve upon the partial genome assembly we received from BGI, we will be sending DNA to the [UW PacBio core facility](https://pacbio.gs.washington.edu/) for additional sequencing.

Isolated DNA from mantle tissue from the same Ostrea lurida individual used for the BGI sequencing efforts. [Tissue was collected by Brent & Steven on 20150812](https://onsnetwork.org/halfshell/2015/08/12/another-day-another-species/).

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of mantle tissue according to the manufacturer's protocol, with the following changes:




    
  * Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer

    
  * Incubated homogenate at 60C for 1.5hrs

    
  * No optional steps were used

    
  * Performed three rounds of 24:1 chloroform:IAA treatment

    
  * Eluted each in 50μL of Elution Buffer and pooled into a single sample



Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 326ng/μL (Quant data is here [Google Sheet]: [20161214_gDNA_Olurida_qubit_quant](https://docs.google.com/spreadsheets/d/1KklkRZYSbHOx6CCIG9thUC5p1e4a8w1NUXKlKzwf1NI/edit?usp=sharing)

Yield is good and we have more than enough (~5μg is required for sequencing) to proceed with sequencing.

Evaluated gDNA quality (i.e. integrity) by running ~500ng (1.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O'GeneRuler DNA Ladder Mix (ThermoFisher).

Results:

![](https://github.com/sr320/LabDocs/blob/9073dc7caf2dcf40e7739fc7ce9d922b28468dc3/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg?raw=true)(https://github.com/sr320/LabDocs/blob/9073dc7caf2dcf40e7739fc7ce9d922b28468dc3/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg?raw=true)



![](https://eagle.fish.washington.edu/Arabidopsis/20161214_gel_Oly_gDNA.jpg)(http://eagle.fish.washington.edu/Arabidopsis/20161214_gel_Oly_gDNA.jpg)



Overall, the gel looks OK. A fair amount of smearing, but a strong, high molecular weight band is present. The intensity of the smearing is likely due to the fact that the gel is overloaded for this particular well size. If I had used a broader comb and/or loaded less DNA, the band would be more defined and the smearing would be less prominent.

Will submit sample to [the UW PacBio facility](https://pacbio.gs.washington.edu/) tomorrow!