---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-12-17 00:25:13+00:00
layout: post
slug: dna-isolation-oly-gdna-for-bs-seq
title: DNA Isolation - Oly gDNA for BS-seq
categories:
  - 2015
  - BS-seq Libraries for Sequencing at Genewiz
  - Olympia oyster reciprocal transplant
tags:
  - 1NF11
  - 1NF12
  - 1NF13
  - 1NF14
  - 1NF15
  - 1NF16
  - 1NF17
  - 1NF18
  - 2NF1
  - 2NF2
  - 2NF3
  - 2NF4
  - 2NF5
  - 2NF6
  - 2NF7
  - 2NF8
  - BS-seq
  - chloroform
  - DNA Isolation
  - DNA Quantification
  - DNazol
  - Fidalgo Bay
  - olympia oyster
  - Ostrea lurida
  - Oyster Bay
  - Qubit 3.0
  - Qubit dsDNA BR
  - RNAse A
---

Need DNA to prep our own libraries for bisulfite-treated high-throughput sequencing (BS-seq).

Isolated gDNA from the following tissue samples stored in RNAlater (tissue was not weighed) using DNAzol:

<table cellpadding="0" cellspacing="0" border="0" dir="ltr" > 
<tbody >
<tr >

<td data-sheets-value="[null,2,"2NF1"]" >2NF1
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF2"]" >2NF2
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF3"]" >2NF3
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF4"]" >2NF4
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF5"]" >2NF5
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF6"]" >2NF6
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF7"]" >2NF7
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF8"]" >2NF8
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF11"]" >1NF11
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF12"]" >1NF12
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF13"]" >1NF13
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF14"]" >1NF14
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF15"]" >1NF15
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF16"]" >1NF16
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF17"]" >1NF17
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF18"]" >1NF18
</td>
</tr>
</tbody>
</table>

The sample coding breaks down as follows ([see the project wiki for a full explanation](https://olyo.wikispaces.com/August+2013+outplanting)):

2NF#

2 = Oysters outplanted in Fidalgo Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number

1NF#

1 = Oysters outplanted in Oyster Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number



DNA was isolated in the following manner:




    
  * Homogenized tissues in 500μL of DNAzol (Molecular Research Center; MRC).

    
  * Added additional 500μL of DNAzol.

    
  * Added 10μL of RNase A (10mg/mL, ThermoFisher); incubated 10mins @ RT.

    
  * Added 300μL of chloroform and mixed moderately fast by hand.

    
  * Incubated 5mins @ RT.

    
  * Centrifuged 12,000g, 10mins, RT.

    
  * Transferred aqueous phase to clean tube.

    
  * Added 500μL of 100% EtOH and mixed by inversion.

    
  * Pelleted DNA 5,000g, 5mins @ RT.

    
  * Performed 3 washes w/70% EtOH.

    
  * Dried pellet 3mins.

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

    
  * Centrifuged 12,000g, 10mins, RT to pellet insoluble material.

    
  * Transferred supe to clean tube.



The samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit data (Google Sheet): [20151216_Oly_gDNA_qubit_quants](https://docs.google.com/spreadsheets/d/17GXiTjinn8QPMY7tKXsTtUbJimi27hb0NklLS3Zfn48/edit?usp=sharing)

<table cellpadding="0" cellspacing="0" border="0" dir="ltr" > 
<tbody >
<tr >

<td data-sheets-value="[null,2,"2NF1"]" >**SAMPLE**
</td>

<td data-sheets-value="[null,3,null,76.4]" >**CONCENTRATION (ng/μL)**
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF1"]" >2NF1
</td>

<td data-sheets-value="[null,3,null,76.4]" >76.4
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF2"]" >2NF2
</td>

<td data-sheets-value="[null,3,null,175]" >175
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF3"]" >2NF3
</td>

<td data-sheets-value="[null,3,null,690]" >690
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF4"]" >2NF4
</td>

<td data-sheets-value="[null,3,null,11.7]" >11.7
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF5"]" >2NF5
</td>

<td data-sheets-value="[null,3,null,142]" >142
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF6"]" >2NF6
</td>

<td data-sheets-value="[null,3,null,244]" >244
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF7"]" >2NF7
</td>

<td data-sheets-value="[null,3,null,25]" >25
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF8"]" >2NF8
</td>

<td data-sheets-value="[null,3,null,456]" >456
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF11"]" >1NF11
</td>

<td data-sheets-value="[null,3,null,182]" >182
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF12"]" >1NF12
</td>

<td data-sheets-value="[null,3,null,432]" >432
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF13"]" >1NF13
</td>

<td data-sheets-value="[null,3,null,155]" >155
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF14"]" >1NF14
</td>

<td data-sheets-value="[null,3,null,21]" >21
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF15"]" >1NF15
</td>

<td data-sheets-value="[null,3,null,244]" >244
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF16"]" >1NF16
</td>

<td data-sheets-value="[null,3,null,112]" >112
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF17"]" >1NF17
</td>

<td data-sheets-value="[null,3,null,25.2]" >25.2
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF18"]" >1NF18
</td>

<td data-sheets-value="[null,3,null,278]" >278
</td>
</tr>
</tbody>
</table>



Will run samples on gel tomorrow to evaluate gDNA integrity.