---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-10-30 00:04:26+00:00
layout: post
slug: adaptor-ligation-oly-alfi-digested-gdna-for-rad-seq-3
title: Adaptor Ligation – Oly AlfI-Digested gDNA for RAD-seq
categories:
  - 2015
  - 2bRAD Library Tests for Sequencing at Genewiz
  - Olympia oyster reciprocal transplant
tags:
  - 3ILL-NR
  - 5ILL-NR
  - anti-ILL
  - gDNA
  - ligation
  - olympia oyster
  - Ostrea lurida
  - PTC-200
  - RAD-seq
  - T4 DNA Ligase
---

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

[Digested DNA from yesterday](https://robertslab.github.io/sams-notebook/posts/2015/2015-10-28-restriction-digest-oly-gdna-for-rad-seq-walfi-3/) was heat inactivated for 10mins @ 65C and was _not_ run out on a gel due to the fact that the input gDNA was degraded and a shift in the high molecular weight band (indicating the digestion was successful) would not exist because [a high molecular weight band is absent in these samples](https://robertslab.github.io/sams-notebook/2015/09/17/agarose-gel-olympia-oyster-whole-body-gdna-integrity-check/).





### Anneal Adaptors



After preparing the two adaptors below, they were incubated for 10mins @ RT:




    
  * Adaptor 1 (2μM final concentration of each oligo): 1.5μL of 5ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total

    
  * Adaptor 2 (2μM final concentration of each oligo): 1.5μL of 3ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total



After annealing, the adaptors were stored on ice.





### Adaptor Ligation



All components were stored on ice. Ligation reactions were prepared on ice and performed in 0.5mL snap cap tubes.

<table >
<tbody >
<tr >

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

<td >**SINGLE REACTION (μL)**
</td>

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

<td >Digested DNA
</td>

<td >10
</td>

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

<td >ATP (10mM)
</td>

<td >1
</td>

<td >11
</td>
</tr>
<tr >

<td >10x T4 Ligase Buffer
</td>

<td >4
</td>

<td >44
</td>
</tr>
<tr >

<td >Adaptor 1 (2μM)
</td>

<td >5
</td>

<td >55
</td>
</tr>
<tr >

<td >Adaptor 2 (2μM)
</td>

<td >5
</td>

<td >55
</td>
</tr>
<tr >

<td >T4 DNA Ligase
</td>

<td >1
</td>

<td >11
</td>
</tr>
<tr >

<td >NanoPure H2O
</td>

<td >24
</td>

<td >264
</td>
</tr>
<tr >

<td >**TOTAL**
</td>

<td >**50**
</td>

<td >**440**
</td>
</tr>
</tbody>
</table>

Combined 40μL of the master mix with 10μL of AlfI-digested DNA in a 0.5mL snap cap tube.

Incubated ligation reaction @ 16C O/N in PTC-200 thermal cycler (MJ Research) – no heated lid.

Ligations will be stored @ -20C until I can continue working with them on Tuesday.