---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2009-07-22 16:18:09+00:00
layout: post
slug: qpcr-gigas-dna-for-opticon-testing
title: qPCR - Gigas DNA for Opticon testing
categories:
  - 2009
  - Miscellaneous
tags:
  - Opticon2
  - qPCR
  - troubleshooting
---

Due to some weird anomolies seen during my previous qPCRs with the H.crach RNA/cDNA samples (positive controls produced good fluorescence when tested in Column 1 of the Opticon, but consistently failed to produce virtually any fluorescence when in Column 6 of the Opticon), I've decided to check the Opticon's fluorescence detection.

[qPCR setup/plate layout is here](https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20090722-01.jpg). I've made a master mix using Cg_HSP70_F/R primers designed by Mac. gDNA is BB #11 (0.49ug/uL) from [20090519](/Sam%27s+Working+Notebook+Jan-May+2009#sjw20090519). The gDNA will be added to the master mix. All wells will be tested.

Results: Unfortunately, this does not look as tight as it should. The Cts range from 23.9 - 26.6 (Cts from Opticon 2 with default threshold settings). This is nearly a 3 cycle difference which represents a nearly 10-fold difference in "expression."