---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-10-29 00:11:05+00:00
layout: post
slug: restriction-digest-oly-gdna-for-rad-seq-walfi-3
title: Restriction Digest – Oly gDNA for RAD-seq w/AlfI
categories:
  - 2015
  - 2bRAD Library Tests for Sequencing at Genewiz
  - Olympia oyster reciprocal transplant
tags:
  - 2bRAD
  - AlfI
  - gDNA
  - olympia oyster
  - Ostrea lurida
  - RAD-seq
  - restriction digestion
---

T[he previous attempt at making these RAD libraries failed during the prep-scale PCR](https://robertslab.github.io/sams-notebook/posts/2015/2015-10-13-pcr-oly-rad-seq-prep-scale-pcr/), likely due to [a discrepancy in the version of the Meyer Lab protocol I was following](https://robertslab.github.io/sams-notebook/2015/10/23/troubleshooting-oly-rad-seq/), so I have to start at the beginning to try to make these libraries again.

Since [the input DNA is so degraded](https://robertslab.github.io/sams-notebook/posts/2015/2015-09-17-agarose-gel-olympia-oyster-whole-body-gdna-integrity-check/), I’ve repeated this using 9μg of input DNA (instead of the recommended 1.2μg). This should increase the number of available cleavage sites for AlfI, thus improving the number of available ligation sites for the adaptors.

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 9.5μL:

Google Sheet: [20151028_RADseq_DNA_calcs](https://docs.google.com/spreadsheets/d/15gSMJX-n6jIkR0kqaupCvhcKWmJI7KgfqJqb9rRT1wU/edit?usp=sharing)







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 >9.5
</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>
</tbody>
</table>



Combined 2.5μL of the master mix with 9.5μL of each DNA sample in 0.5mL snap cap tubes. Incubated @ 37C O/N in thermal cycler (PTC-200; no heated lid).