---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-11-25 01:54:41+00:00
layout: post
slug: dna-isolation-geoduck-adductor-muscle-gdna
title: DNA Isolation - Geoduck Adductor Muscle gDNA
categories:
  - 2015
  - Geoduck Genome Sequencing
tags:
  - DNA Isolation
  - DNA Quantification
  - gDNA
  - geoduck
  - NanoDrop1000
  - Panopea generosa
  - Quant-iT DNA BR Kit
  - Victor 1420 plate reader
---

Since we still don't have sufficient gDNA for the full scope of the genome sequencing, I isolated more gDNA.

Isolated gDNA from 257mg [adductor muscle tissue collected by Steven & Brent on 20150811](https://onsnetwork.org/halfshell/2015/08/11/big-day-big-clam/).

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:




    
  * Doubled solution volumes for steps before sample was loaded on columns

    
  * Sample was split equally in two tubes prior to addition of 100% EtOH

    
  * All mixing was done by shaking - no vortexing! Done this way to, hopefully, maintain gDNA integrity

    
  * Elution volume = 50μL

    
  * Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.

    
  * The two preps were pooled - final volume = 79μL



DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer's protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab's Victor 1420 plate reader (Perkin Elmer).

Results:

<table >
<tbody >
<tr >

<td >METHOD
</td>

<td >CONCENTRATION (ng/μL)
</td>

<td >VOLUME (μL)
</td>

<td >YIELD (ng)
</td>
</tr>
<tr >

<td >NanoDrop1000
</td>

<td >54.93
</td>

<td >79
</td>

<td >4,339
</td>
</tr>
<tr >

<td >Quant-IT
</td>

<td >34.52
</td>

<td >79
</td>

<td >2,727
</td>
</tr>
</tbody>
</table>



The NanoDrop1000 overestimates the concentration of the sample by 1.6x!

Regardless, the yield isn't all that great, which has generally been the case for all of the geoduck gDNA isolations I've performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve...

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): [20151124_geoduck_oly_gDNA_quants](https://docs.google.com/spreadsheets/d/167If9r5fDNJb6xenUo-bKy52794_O1LgN1twKAOmElw/edit?usp=sharing)



NanoDrop1000 Measurements and Plots

![](https://eagle.fish.washington.edu/Arabidopsis/20151124_gDNA_geoduck_oly_ODs.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20151124_gDNA_geoduck_oly_ODs.JPG)

![](https://eagle.fish.washington.edu/Arabidopsis/20151124_gDNA_geoduck_oly_plots.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20151124_gDNA_geoduck_oly_plots.JPG)