---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2011-04-14 21:13:44+00:00
layout: post
slug: colony-pcr-5-race-colonies
title: Colony PCR - 5' RACE Colonies
categories:
  - 2011
  - Miscellaneous
tags:
  - colony screen
  - COX
  - COX2
  - Crassostrea gigas
  - cyclooxygenase
  - gel
  - Pacific oyster
  - PCR
  - PGS
  - PGS2
  - prostaglandin synthase
---

Two light blue colonies (there were no white colonies) were picked, restreaked on a new Kan50 plate (no X-gal) and PCR'd.

Master Mix:

2x Apex Red Master Mix - 37.5uL

10uM M13 Forward - 3uL

10uM M13 Reverse - 3uL

H2O - 46.5

Added 25uL to each PCR tube.

Cycling Params:




    
  * 95C - 10mins



40 cycles of:


    
  * 95C - 30s

    
  * 55C - 30s

    
  * 72C - 2mins



1 cycle:


    
  * 72C - 10mins



Results:

![](https://eagle.fish.washington.edu/Arabidopsis/20110415-01.jpg)

The two pale blue colonies do NOT contain our desired insert. Despite the presence of a larger, faint band (~950bp), that is far smaller than our 5'RACE insert size (~1500bp). And, clearly, the primary amplicon produced is ~250bp, which is the expected size for empty vector... Ladder is Hyperladder I (Bioline).

Will need to re-do ligation reaction and will do so at the recommended volumes in the TOP TA (Invitrogen) protocol.
