---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-10-09 22:30:03+00:00
layout: post
slug: restriction-digest-oly-gdna-for-rad-seq-walfi-2
title: Restriction Digest – Oly gDNA for RAD-seq w/AlfI
categories:
  - 2015
  - 2bRAD Library Tests for Sequencing at Genewiz
  - Olympia oyster reciprocal transplant
tags:
  - AlfI
  - gDNA
  - olympia oyster
  - Ostrea lurida
  - RAD-seq
  - restriction digestion
---


Used a subset (10 samples) from [the _Ostrea lurida_ gDNA isolated 20150916](https://robertslab.github.io/sams-notebook/posts/2015/2015-09-17-dna-isolation-olympia-oyster-whole-body/) to prepare RAD libraries.

Followed the [2bRAD protocol (PDF) developed by Eli Meyer’s lab](https://github.com/sr320/LabDocs/blob/master/protocols/External_Protocols/2bRAD_11Aug2015.pdf).

Prepared 9.0μg of each of the following samples in a volume of 10μL:

Google Sheet: [20151009_RADseq_DNA_calcs](https://docs.google.com/spreadsheets/d/1jAA9lAhKaG1ZMYczH2rGc1mK4ovltWgbTGzrMN8ZNKQ/edit?usp=sharing)





Prepared a 150μM working stock of the SAM buffer needed for the restriction digestion by diluting 30μL of the supplied stock (500μM) in 70μL NanoPure H2O (total volume = 100μL). This working stock was stored @ -20C in FTR 209 in the “RAD-seq Reagents” box.

Prepared master mix for restriction enzyme reaction:

<table >
<tbody >
<tr >

<td >**REAGENT**
</td>

<td >**SINGLE REACTION (μL)**
</td>

<td >**x11**
</td>
</tr>
<tr >

<td >DNA
</td>

<td >8
</td>

<td >NA
</td>
</tr>
<tr >

<td >10x Buffer R
</td>

<td >1.2μL
</td>

<td >13.2μL
</td>
</tr>
<tr >

<td >150μM SAM
</td>

<td >0.8μL
</td>

<td >8.8μL
</td>
</tr>
<tr >

<td >AlfI
</td>

<td >0.5μL
</td>

<td >5.5μL
</td>
</tr>
<tr >

<td >H2O
</td>

<td >1.5μL
</td>

<td >16.5μL
</td>
</tr>
</tbody>
</table>



Combined 4μL of the master mix with 8μL of each sample in 0.5mL snap cap tubes. Incubated @ 37C 2hrs. in thermal cycler (PTC-200; no heated lid). Heat inactivated the digest @ 65C for 10mins.