---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-09-15 23:46:52+00:00
layout: post
slug: genomic-dna-isolation-olympia-oyster-adductor-musle-mantle-3
title: Genomic DNA Isolation – Olympia oyster adductor musle & mantle
categories:
  - 2015
  - Olympia Oyster Genome Sequencing
tags:
  - DNA Isolation
  - DNA Quantification
  - DNazol
  - gDNA
  - NanoDrop1000
  - olympia oyster
  - Ostrea lurida
---

Isolated gDNA from _Ostrea lurida_ (Olympia oyster) [adductor muscle & mantle samples collected by Brent & Steven on 20150812](https://onsnetwork.org/halfshell/2015/08/12/another-day-another-species/) using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:




    
  * 73.5mg of adductor muscle

    
  * 146mg of mantle

    
  * Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes

    
  * After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.

    
  * Performed optional centrifugation step (10,000g, 10mins @ RT)

    
  * Initial pellet wash was performed using a 70%/30% DNAzol/EtOH

    
  * Pellets were resuspended in 200μL of Buffer EB (Qiagen)

    
  * Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes



Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing.



Results:

![](https://eagle.fish.washington.edu/Arabidopsis/20150915_gDNA_oly_ODs.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150915_gDNA_oly_ODs.JPG)



![](https://eagle.fish.washington.edu/Arabidopsis/20150915_gDNA_oly_plots.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150915_gDNA_oly_plots.JPG)



There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will evaluate gDNA integrity on agarose gel.



Yields from this isolation:

Adductor muscle: 18.75μg

Mantle: 15.9μg



Total Olympia oyster gDNA from this isolation: 34.65μg



Total Olympia oyster gDNA accumulated for this project: 88.75μg



Great! Have sufficient gDNA to send to BGI (minimum of 73μg needed). Assuming gDNA integrity looks good on a gel, I will pool samples tomorrow, quantify the pooled gDNA and prepare for submission.
