---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-09-16 19:03:07+00:00
layout: post
slug: genomic-dna-isolation-geoduck-adductor-muscle-foot-4
title: Genomic DNA Isolation – Geoduck Adductor Muscle & Foot
categories:
  - 2015
  - Geoduck Genome Sequencing
tags:
  - DNA Isolation
  - DNA Quantification
  - DNazol
  - gDNA
  - geoduck
  - NanoDrop1000
  - Panopea generosa
---

Just need a tad bit more gDNA for the geoduck genome sequencing project with BGI. Currently have ~69 and need a minimum of 73μg.

Isolated gDNA from _Panopea generosa_ (geoduck) [adductor muscle & foot samples collected by Brent & Steven on 20150811](https://onsnetwork.org/halfshell/2015/08/11/big-day-big-clam/) using the DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:




    
  * 124.4mg of adductor muscle 1

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

    
  * After homogenization, topped off tube 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

    
  * Pellet was resuspended in 400μL of Buffer EB (Qiagen)

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



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



Results:



![](https://eagle.fish.washington.edu/Arabidopsis/20150916_gDNA_geoduck_ODs.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150916_gDNA_geoduck_ODs.JPG)

![](https://eagle.fish.washington.edu/Arabidopsis/20150916_gDNA_geoduck_plots.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150916_gDNA_geoduck_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 ratio looks pretty good, but the 260/230 ratio is 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 assess gDNA integrity on a gel.



Yield of geoduck gDNA from this isolation: ~48μg

Total accumulated geoduck gDNA for this project: ~117μg

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.