Navigation Links
Amplification of the amoA Gene of Nitrosococcus oceani Bacteria Using ,,, Eppendorf MasterTaq

Kathryn L. Dudeck and Wendy A. Dustman
University of Georgia,
Department of Marine Sciences

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.

PCR Amplification
Two bacterial primers, A189, GGN GAC TGG GAC TCC TGG (Holmes et. al) and AmoA2-R, CCC CTC KGS AAA GCC TTC TTC (Rotthauwe et. al), were utilized to amplify a 675 bp region of the amoA gene of the cultures. PCR was performed as follows: 2 l of each primer (10 M), 1 l of N. oceani culture, 2.5 l of 10X buffer, 2 l of 2.5 mM dNTP, 2.5 U of Taq polymerase, and 0-5 l of 5X TaqMaster. This mixture was adjusted to a final volume of 25 l using
sterile water. A negative control (no DNA) was also amplified. Amplification of the target gene was carried out via the following program: 94 C for 2 minutes, 35 cycles of 94 C for 1 minute, 56 C for 1 minute, and 72 C for 1 minute, and a final extension step of 56 C for 5 minutes.

Visualization
PCR products were run in a 2.0% agarose gel and stained with ethidium bromide for visualization.

Results and Discussion

The 675 bp region of the amoA gene was successfully amplified using the Eppendorf MasterTaq polymerase kit. Varying the amount of TaqMaster thermostabilizer included in the PCR reaction had a minimal effect on the band obtained from N. oceani. The addition of 2.5 l of TaqMaster gave the most favorable results (Lane 4), though bands were visible at all concentrations of TaqMaster (Lanes 3 and 5). The additional bands seen in the N. oceani lanes are most likely the amo subunits. Internal probing after PCR verifies that the band seen at approximately 675 bp is indeed amoA (data not shown). Eppendorfs MasterTaq polymerase kit is a beneficial enzyme system to use when attempting to study known laboratory cultures of ammonia-oxidizing bacteria.

Figure 1: The use of MasterTaq for amplification of the amoA gene in two ammonia-oxidizing bacterial cultures
Lane 1 - Molecular weight marker VIII
Lane 2 - Negative control (no DNA)
Lane 3 - Nitrosococcus oceani with no Ta q M a s t e r
Lane 4 - Nitrosococcus oceani with 2.5 l Ta q M a s t e r
Lane 5 - Nitrosococcus oceani with 5 l Ta q M a s t e r



References

Alzerecca, J.J., J.M. Norton, and M.G. Klotz. 1999. The amo operon in marine, ammonia-oxidizing gamma-proteobacteria. FEMS Microbiology Letters 180:21-29.

Holmes, A. J., A. Costello, M. E. Lidstrom, and J. C. Murrell. 1995. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiology Letters. 132:203-208.

Rotthauwe, J. H., K. P. Witzel, and W. Liesack. 1997. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology. 63:4704-4712.

Sayavedra-Soto, L.A., N.G. Hommes, J.J. Alzerreca, D.J. Arp, J.M. Norton and M.G. Klotz. 1998. Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp. NpAV. FEMS Microbiology Letters 167:81-88.




'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Greater Amplification Specificity with New Hot Start PCR Enzyme
4. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
5. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
6. Improve Amplification Specificity with Hot Start PCR Enzyme
7. Amplification of Mouse cDNAs for Microarrays Using the Eppendorf MasterTaq Kit
8. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
9. Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler
10. Amplification of Epstein-Barr Virus Exon C with Difficult Primers Using Eppendorf Mastercycler gradient
11. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:3/27/2021)... ... March 24, 2021 , ... ... Life Sciences and Healthcare firms of all sizes, adds depth to its team ... specialist. Pardillo, who earned his doctorate in computational chemistry from Florida International University ...
(Date:3/27/2021)... ... March 24, 2021 , ... ABI Wellness, ... and reporting approach designed under CEO Mark Watson, today announced a webinar dedicated ... featuring guest speakers Dr. Cameron Clark, Neuropsychologist and Founder of Sharp Thinking, and ...
(Date:3/27/2021)... ... ... The Xtalks editorial team is pleased to announce the launch of the ... joined by editorial team members Ayesha Rashid, Sydney Perelmutter and Mira Nabulsi to discuss ... including insights from industry experts. , The Xtalks Life Science podcast will feature ...
(Date:3/23/2021)... Conn. (PRWEB) , ... March 23, 2021 , ... ... develops solutions for characterizing microbiome populations down to the strain level, recently unveiled ... applications. , Not all microbes are created equal: some are easy to ...
Breaking Biology Technology:
(Date:3/27/2021)... ... March 25, 2021 , ... Phlexglobal announced ... Planet Group, has selected Phlexglobal and its innovative regulatory SaaS software, PhlexSubmission, as ... a comprehensive review of five regulatory software companies, with the review team including ...
(Date:3/27/2021)... ... 25, 2021 , ... The 2021 Virtual Conference on Clinical Trial Supply-Europe ... More and more, clinical trial supply conferences are featuring speakers and forums that ... Asymmetrex’s founder and CEO, James L. Sherley, M.D., Ph.D., presented a talk ...
(Date:3/23/2021)... ... March 23, 2021 , ... G-CON Manufacturing (G-CON), the ... by Matica Biotechnology (Matica Bio), a contract development and manufacturing organization (CDMO) specializing ... the cleanroom build out for its new GMP production facility in College Station, ...
Breaking Biology News(10 mins):