---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2011-07-23 03:16:23+00:00
layout: post
slug: 53-race-c-gigas-cox2pgs2-race-pcr
title: 5'/3' RACE - C.gigas COX2/PGS2 RACE PCR
categories:
  - 2011
  - Miscellaneous
tags:
  - COX
  - COX2
  - Crassostrea gigas
  - cyclooxygenase
  - gel
  - Pacific oyster
  - PGS
  - PGS2
  - prostaglandin synthase
  - RACE PCR
  - SMART RACE
---

Additional RACE using gene specific primers (SR IDs: 1347 & 1348) for C.gigas COX2/PGS2 according to Clontech's SMARTer cDNA RACE Kit protocol. 3'/5' RACE cDNA libraries are from 20080619. [Master mix calcs and set up is here](https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20110722-01.jpg). Cycling params followed "Program 2" of the Clontech protocol and are as follows:

25 cycles:




    
  * 94°C 30 sec

    
  * 68°C 30 sec

    
  * 72°C 3 min



Reactions were run with both primers on both libraries, just to ensure that in case there was any confusion in primer design. When finished, I will remove 2uL of the PCR reaction for use in a nested PCR reaction. Will run a gel with both sets of products, once the nested PCR is completed.

Results:

![](https://eagle.fish.washington.edu/Arabidopsis/20110725-01%20Gel.jpg)

Gel Layout:

Lane 1 - Hyperladder 1

Lanes 2-6 = 5' RACE Library

Lane 2 - GSP1 (5' RACE primer)

Lane 3 - GSP2 (3' RACE primer)

Lane 4 - Neg. Control (no RACE primers)

Lane 5 - Neg. Control (GSP1, no Universal primer)

Lane 6 - Neg. Control (GSP2, no Universal primer)

Lane 7 - Empty

Lanes 8-12 = 3' RACE Library

Lane 8 - GSP1 (5' RACE primer)

Lane 9 - GSP2 (3' RACE primer)

Lane 10 - Neg. Control (no RACE primers)

Lane 11 - Neg. Control (GSP1, no Universal primer)

Lane 12 - Neg. Control (GSP2, no Universal primer)

As has generally been the case, our primary RACE PCRs failed to produce any products. This is why I performed the nested PCR (described above) before viewing the results of this primary PCR.