---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-11-14 00:27:24+00:00
layout: post
slug: pcr-oly-rad-seq-prep-scale-pcr-2
title: PCR – Oly RAD-seq Prep Scale PCR
categories:
  - 2015
  - 2bRAD Library Tests for Sequencing at Genewiz
  - Olympia oyster reciprocal transplant
tags:
  - barcodes
  - gel
  - ILL-BC1
  - ILL-BC10
  - ILL-BC2
  - ILL-BC3
  - ILL-BC4
  - ILL-BC5
  - ILL-BC6
  - ILL-BC7
  - ILL-BC8
  - ILL-BC9
  - ILL-HT1
  - olympia oyster
  - Ostrea lurida
  - PCR
  - PTC-200
  - Q5 High-Fidelity DNA Polymerase
  - 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).
[After determining the minimum number of PCR cycles to run to generate a visible, 166bp band on a gel yesterday](https://robertslab.github.io/sams-notebook/posts/2015/2015-11-13-pcr-oly-rad-seq-test-scale-pcr-4/), ran a full library “prep scale” PCR.



<table >
<tbody >
<tr >

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

<td >**SINGLE REACTION (μL)**
</td>

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

<td >Template
</td>

<td >40
</td>

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

<td >ILL-HT1 (1μM)
</td>

<td >5
</td>

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

<td >ILL-BC# (1μM)
</td>

<td >5
</td>

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

<td >NanoPure H2O
</td>

<td >5
</td>

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

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

<td >20
</td>

<td >220
</td>
</tr>
<tr >

<td >ILL-LIB1 (10μM)
</td>

<td >2
</td>

<td >22
</td>
</tr>
<tr >

<td >ILL-LIB2 (10μM)
</td>

<td >2
</td>

<td >22
</td>
</tr>
<tr >

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

<td >20
</td>

<td >220
</td>
</tr>
<tr >

<td >Q5 DNA Polymerase
</td>

<td >1
</td>

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

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

<td >**100**
</td>

<td >**550**
</td>
</tr>
</tbody>
</table>



Combined the following for PCR reactions:




    
  * 55μL PCR master mix

    
  * 40μL ligation mix

    
  * 5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.





<table >
<tbody >
<tr >

<td >**SAMPLE**
</td>

<td >**BARCODE**
</td>

<td >**SEQUENCE**
</td>
</tr>
<tr >

<td >Oly RAD 02
</td>

<td > 1
</td>

<td > CGTGAT
</td>
</tr>
<tr >

<td >Oly RAD 03
</td>

<td > 2
</td>

<td > ACATCG
</td>
</tr>
<tr >

<td >Oly RAD 04
</td>

<td > 3
</td>

<td > GCCTAA
</td>
</tr>
<tr >

<td >Oly RAD 06
</td>

<td > 4
</td>

<td > TGGTCA
</td>
</tr>
<tr >

<td >Oly RAD 07
</td>

<td > 5
</td>

<td > CACTGT
</td>
</tr>
<tr >

<td >Oly RAD 08
</td>

<td > 6
</td>

<td > ATTGGC
</td>
</tr>
<tr >

<td >Oly RAD 14
</td>

<td > 7
</td>

<td > GATCTG
</td>
</tr>
<tr >

<td >Oly RAD 17
</td>

<td > 8
</td>

<td > TCAAGT
</td>
</tr>
<tr >

<td >Oly RAD 23
</td>

<td > 9
</td>

<td > CTGATC
</td>
</tr>
<tr >

<td >Oly RAD 30
</td>

<td >10
</td>

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



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 >17 cycles
</td>

<td >



    
  * 98

    
  * 60

    
  * 72



</td>

<td >



    
  * 5

    
  * 20

    
  * 10



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



After cycling, added 16μL of 6x loading dye to each sample.

Loaded 10μL of ladder on each of the two gels.

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)

![](https://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_01.png)(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_01.png)

![](https://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_02.png)(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_02.png)

![](https://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_03.png)(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_03.png)

![](https://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_04.png)(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_04.png)



Things looked fine. Excised the bands from each sample indicated by the green arrow. Before and after gel images show regions excised. Will purify the bands and quantify library yields.