( ...)Amplification of the amoA Gene of Nitrosococcus oceani Bacteria Using ,,, Eppendorf MasterTaq

Introduction

This application describes the use of the MasterTaq polymerase kit for improving PCR efficiency when studying functional gene presence. Using this polymerase, it was possible to amplify a 675 bp region of the amoA gene in lab-grown Nitrosococcus oceani, which has previously been met with many obstacles when utilizing other commercially available enzyme kits.

The Polymerase Chain Reaction (PCR) is a now-commonplace method used for making several copies of a specific gene for further manipulation, or for simply observing whether or not a specific gene is present.

Lab cultures of Nitrosococcus oceani and other ammonia-oxidizing bacteria (AOB) are notoriously difficult to study. They exhibit slow growth and therefore are easily contaminated by hetero t rophs. In addition to this hurdle, the gene used in this study is often found with three clustered subunits (amoA, amoB, and amoC) within some members of AOB (Alzerecca et. al, Sayavedra-Soto et. al). The gene lengths are remarkably similar; there f o re, the study of a single gene in
this area of the chromosome can be quite challenging.

Materials and Methods

Bacterial Preparation
Nitrosococcus oceani (ATCC 19707) was cultured in ATCC Medium 928 in a shaking incubator (100 rpm) at 27 C. Using 12.5% K2CO3, the pH level was maintained at ~8.3. Twenty-five milliliters were extracted and purified using a Qiagen blood Mini Prep Kit.


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