---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-11-13 01:36:56+00:00
layout: post
slug: pcr-oly-rad-seq-test-scale-pcr-4
title: PCR – Oly RAD-seq Test-scale PCR
categories:
  - 2015
  - 2bRAD Library Tests for Sequencing at Genewiz
  - Olympia oyster reciprocal transplant
tags:
  - gel
  - ILL-BC1
  - ILL-HT1
  - ILL-LIB1
  - ILL-LIB2
  - O'geneRuler DNA Ladder Mix
  - olympia oyster
  - Ostrea lurida
  - PCR
  - PTC-200
  - RAD
  - RAD-seq
---

Continuing with the RAD-seq library prep. [Following the Meyer Lab 2bRAD protocol](https://github.com/sr320/LabDocs/blob/master/protocols/External_Protocols/2bRAD_11Aug2015.pdf).

Prior to generating full-blown libraries, we needed to run a “test-scale” PCR to identify the minimum number of cycles needed to produce the intended product size (166bp).

I ran PCR reactions on a subset (Sample #: 2, 3, 17, & 30) of the 10 samples that [I performed adaptor ligations on 20151029](https://robertslab.github.io/sams-notebook/posts/2015/2015-10-09-adaptor-ligation-oly-alfi-digested-gdna-for-rad-seq-2/).

PCR reactions were set up on ice in 0.5mL PCR tubes.

<table >
<tbody >
<tr >

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

<td >**SINGLE REACTION (μL)**
</td>

<td >**x4.4**
</td>
</tr>
<tr >

<td >Template
</td>

<td >8
</td>

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

<td >NanoPure H2O
</td>

<td >1
</td>

<td >4.4
</td>
</tr>
<tr >

<td >dNTPs (1mM)
</td>

<td >4
</td>

<td >17.6
</td>
</tr>
<tr >

<td >ILL-LIB1 (10μM)
</td>

<td >0.4
</td>

<td >1.76
</td>
</tr>
<tr >

<td >ILL-LIB2 (10μM)
</td>

<td >0.4
</td>

<td >1.76
</td>
</tr>
<tr >

<td >ILL-HT1 (1μM)
</td>

<td >1
</td>

<td >4.4
</td>
</tr>
<tr >

<td >ILL-BC1 (1μM)
</td>

<td >1
</td>

<td >4.4
</td>
</tr>
<tr >

<td >5x Q5 Reaction Buffer
</td>

<td >4
</td>

<td >17.6
</td>
</tr>
<tr >

<td >Q5 DNA Polymerase
</td>

<td >0.2
</td>

<td >0.88
</td>
</tr>
<tr >

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

<td >**20**
</td>

<td >**52.8**
</td>
</tr>
</tbody>
</table>



Combined 12μL of master mix with 8μL of the ligation reaction from earlier today.

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

<table >
<tbody >
<tr >

<td >**STEP**
</td>

<td >**TEMP (C)**
</td>

<td >**TIME (s)**
</td>
</tr>
<tr >

<td >Initial Denaturation
</td>

<td >



    
  * 98



</td>

<td >



    
  * 30



</td>
</tr>
<tr >

<td >27 cycles
</td>

<td >



    
  * 98

    
  * 60

    
  * 72



</td>

<td >



    
  * 5

    
  * 20

    
  * 10



</td>
</tr>
</tbody>
</table>

We’re following the “1/4 reduced representation” aspect of the protocol. As such, 5μL of each reaction was pulled immediately after the extension (72C – machine was paused) of cycles 12, 17, 22, & 27 in order to determine the ideal number of cycles to use. Also ran the ligation reactions (labeled “Ligations” on the gel below) of the samples as a pre-PCR comparison. Treated them the same as the PCR reactions: mixed 8μL of the ligation with 12μL of H2O, used 5μL of that mix to load on gel.

These samples were run on a 1x modified TAE 1.2% agarose gel (w/EtBr).



Results:

![](https://raw.githubusercontent.com/sr320/LabDocs/master/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg)(https://raw.githubusercontent.com/sr320/LabDocs/master/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg)

[caption id="" align="alignnone" width="701"]![](https://eagle.fish.washington.edu/Arabidopsis/20151112_gel_oly_RAD_test_scale_PCR.png)(http://eagle.fish.washington.edu/Arabidopsis/20151112_gel_oly_RAD_test_scale_PCR.png) Gel image denoting sample numbers within each cycle number. Green arrow indicates the expected migration of our target band size of 166bp.[/caption]

Looks like cycle 17 is the minimum cycle number with which we begin to see a consistent ~166bp band. Will continue on with the "prep-scale" PCR using 17 cycles.