---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2009-06-10 01:49:28+00:00
layout: post
slug: pcr-c-pugetti-gdna-from-20090526
title: PCR - C.pugetti gDNA from 20090526
categories:
  - 2009
  - Miscellaneous
tags:
  - 1492R
  - 16s
  - 27F
  - Cycloclasticus pugetii
  - gDNA
  - gel
  - gel extraction
  - PCR
  - Ultrafree-DA
---

This is a repeat of yesterday's PCR due to the presence of bands in the water-only samples. Will use reagents and universal 16s bacterial primers (27F & 1492R) provide by the Horner-Devine lab in hopes of: 1) getting this two work and, 2) figuring out the source of the contamination.

All rxns were prepared sterily and all instruments, racks, tubes, tips and water were UV-sterilized for ~45mins in the biological hood. Rxns were prepared in the biological hood. [PCR setups are here.](https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20090609-01.jpg) Anneal 60C. Cycling params same as yesterday.

![](https://eagle.fish.washington.edu/Arabidopsis/20090610.JPG)

Lane 1 - 100bp ladder

Lane 2 - DNA (HD Rxn 1)

Lane 2 - H2O (HD Rxn 1)

Lane 3 - H2O (HD Rxn 1)

Lane 4 - DNA (HD Rxn 2)

Lane 5 - H2O (HD Rxn 2)

Lane 6 - H2O (HD Rxn 2)

Lane 7 - DNA (SR Rxn)

Lane 8 - H2O (SR Rxn)

Lane 9 - H2O (SR Rxn)

Lane 10 - 100bp ladder

Results: Well, we got our band and NO contamination in any H2O lanes. The super-bright, 1500bp band will be excised and purified using Millipore spin columns and submitted for sequencing. However, this gel is interesting because the primers provided by Mike (used in HD Rxn 1 and SR Rxn) did not amplify anything...