---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-09-17 23:23:26+00:00
layout: post
slug: ethanol-precipitation-geoduck-olympia-oyster-gdna
title: Ethanol Precipitation - Geoduck & Olympia oyster gDNA
categories:
  - 2015
  - Geoduck Genome Sequencing
  - Olympia Oyster Genome Sequencing
tags:
  - DNA Quantification
  - EtOH precipitation
  - geoduck
  - NanoDrop1000
  - olympia oyster
  - Ostrea lurida
  - Panopea generosa
---

Pooled all of the geoduck gDNA from all the previous geoduck isolations for the [Geoduck Genome Sequencing Project](category/geoduck-genome-sequencing.html) and pooled all of the _Ostrea lurida_ gDNA from previous Ostrea lurida isolations for the [Olympia Oyster Genome Sequencing Project](category/olympia-oyster-genome-sequencing.html).

Both sets of gDNA were ethanol (EtOH) precipitated. This was done for two reasons - to concentrate the samples to the minimum necessary concentration required by BGI (>119ng/μL) and to try to improve the poor 260/230 ratios (which are likely due to high salt carryover).

Precipitation was performed by consolidating each species of DNA in 15mL conicals, adding 0.1 volumes of 3M sodium acetate (pH= 5.2) and then adding 2.5 volumes of ice cold (-20C) 100% EtOH. Volumes used are below.

<table >
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<td >
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<td >
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<td >
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<td >**Sample**
</td>

<td >**DNA Vol (μL)**
</td>

<td >**3M Sodium Acetate Vol. (μL)**
</td>

<td >**100% EtOH Vol (μL)**
</td>

<td >**Total Vol (μL)**
</td>
</tr>
<tr >

<td >Geoduck
</td>

<td style="text-align: center;" >1860
</td>

<td style="text-align: center;" >186
</td>

<td style="text-align: center;" >5115
</td>

<td style="text-align: center;" >7161
</td>
</tr>
<tr >

<td >Oly
</td>

<td style="text-align: center;" >780
</td>

<td style="text-align: center;" >78
</td>

<td style="text-align: center;" >2145
</td>

<td style="text-align: center;" >3003
</td>
</tr>
</tbody>
</table>

Samples were mixed by inversion and incubated @ -80C for 3hrs.

DNA was pelleted by spinning tubes at 12,000g for 30mins @ 4C in a SL-50T (Sorval) rotor in a T21 (Sorval) table top centrifuge.

Supernatant was decanted and pellets were transferred to 1.5mL snap cap tubes.

Pellets were washed three times with 70% EtOH.

After the last wash, the supe was removed and pellets were air dried for 5mins @ RT.

In order to exceed the target concentration (>110ng/μL) needed by BGI, the pellets were resuspended in 500μL (150ng/μL) of EB Buffer (Qiagen). This is assuming each sample has at least 75μg of DNA.

Samples were spec'd on the Roberts Lab NanoDrop1000.



Results:

![](https://eagle.fish.washington.edu/Arabidopsis/20150917_gDNA_geoduck_oly_ODs.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150917_gDNA_geoduck_oly_ODs.JPG)

![](https://eagle.fish.washington.edu/Arabidopsis/20150917_gDNA_geoduck_oly_plots.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150917_gDNA_geoduck_oly_plots.JPG)



Concentrations are on point, the 260/280 ratios are still good and the 260/230 ratios are greatly improved. Samples are ready to send off to BGI for sequencing!

Total yields:

Geoduck: 84μg

Oly: 86μg

Will run these samples out on a gel to verify that the gDNA is still intact and didn't get degraded during the precipitation.