---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-11-25 02:03:04+00:00
layout: post
slug: phenol-chloroform-dna-cleanup-geoduck-gdna
title: Phenol-Chloroform DNA Cleanup - Geoduck gDNA
categories:
  - 2015
  - Geoduck Genome Sequencing
tags:
  - DNA Quantification
  - gDNA
  - geoduck
  - NanoDrop1000
  - Panopea generosa
  - phenol-chloroform cleanup
  - Quant-iT DNA BR Kit
  - Victor 1420 plate reader
---

The [gDNA I extracted on 20151104](https://robertslab.github.io/sams-notebook/posts/2015/2015-11-05-dna-quantification-quality-assessment-geoduck-oly-gdna/) didn't look great on the NanoDrop so I decided to perform a phenol-chloroform cleanup to see if I could improve the NanoDrop1000 absorbance spectrum and, in turn, the quality of the gDNA.




    
  * Added an equal volume (500μL) of phenol:chloroform:isoamyl alcohol (25:24:1) to the DNA

    
  * Mixed by hand - moderate shaking

    
  * Centrifuged 2mins, 10,000g, RT

    
  * Transferred aqueous phase to clean tube and discarded interphase & organic phase

    
  * Added an equal volume (280μL) of chlforoform:isoamyl alcohol (24:1)

    
  * Mixed by hand - moderate shaking

    
  * Centrifuged 2mins, 10,000g, RT

    
  * Transferred aqueous phase (210μL) to clean tube

    
  * Added 0.1vols (21μL) of 3M sodium acetate (pH = 5.2)

    
  * Added 2vols (420μL) of 100% EtOH

    
  * Mixed by inversion

    
  * Incubated @ -20C, 1hr (probably not necessary since gDNA clearly precipitated out as soon as I mixed the sample)

    
  * Pelleted DNA by centrifuging 15mins, 12,000g, RT

    
  * Discarded supe

    
  * Washed pellet with 1000μL cold (-20C) 70% EtOH

    
  * Centrifuged 5mins, 12,000g, RT

    
  * Discarded supe

    
  * Repeated was steps three more times

    
  * Resuspended pellet in 100μL of Buffer EB (Qiagen)



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 >371.83
</td>

<td >100
</td>

<td >37,183
</td>
</tr>
<tr >

<td >Quant-IT
</td>

<td >100.83
</td>

<td >100
</td>

<td >10,082
</td>
</tr>
</tbody>
</table>



The NanoDrop1000 overestimates the concentration of the sample by 3.7x!

Regardless, the yield isn't all that great (using yield from Quant-IT), 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

The clean up procedure didn't really seem to help with the geoduck sample, as we're still seeing a significant amount of absorbance from 230 - 250nm.

![](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)