Oct 02, 2024
pMOA 189F-682R
- Roey Angel1,
- Eva Petrova1
- 1Institute of Soil Biology and Biogeochemistry, Biology Centre CAS
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: [email protected]
Protocol Citation: Roey Angel, Eva Petrova 2024. pMOA 189F-682R. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzz1rvx1w/v1
Manuscript citation:
Holmes AJ, Costello A, Lidstrom ME, Murrell JC. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett. 1995;132:203–8.
http://www.sciencedirect.com/science/article/pii/037810979500311R
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2018
Last Modified: October 02, 2024
Protocol Integer ID: 12426
Keywords: PCR, pmoA, PMMO, Methanotrophs, Methane oxidising bacteria, Particulate methane monooxygenase, particulate methane monooxygenase gene, alpha subunit of the particulate methane monooxygenase gene, amoa gene of ammonia, general assay for methane, pcr assay, oxidising bacteria, amoa gene, methane, ammonia, pmoa, pcr, general assay, bacteria, assay, using primer, alpha subunit, 682r, primer
Abstract
A PCR assay targeting the alpha subunit of the particulate methane monooxygenase gene (pmoA).
This is a general assay for methane oxidising bacteria using primers 189F–682R.
Note: these primers might occasionally also amplify the amoA gene of ammonia-oxidising bacteria.
189F GGN GAC TGG GAC TTC TGG (target seq. 189 - 207) Holmes et al. (1995),
682R GAA SGC NGA GAA GAA SGC (target seq. 664 - 682) Holmes et al. (1995).
Fragment size: 508bp.
Troubleshooting
PCR mixture
| Reagent | Final. conc. | 1 tube (50μl) | 1 tube(25μl) | 96 tubes (25μl x100) | 96 tubes (12.5μl x100) | |
| PCR H2O | 37.9 | 18.95 | 1255 | 627.5 | ||
| 10X Buffer (I for RT and II for DNA) | 1x | 5 | 2.5 | 500 (Promega) | 250 (Promega) | |
| dNTP mixture (2.0 mM each | 0.2mM | 5 | 2.5 | 250 | 125 | |
| MgCl2 (50 mM)* | 3mM | 1.5* | 0.75* | 250 | 125 | |
| BSA (20μg/μl) | 0.8 μg/μl | 2 | 1 | 100 | 50 | |
| 189f (25μM) | 0.4 μM | 0.8 | 0.4 | 40 | 20 | |
| 682r (25 μM) | 0.4 μM | 0.8 | 0.4 | 40 | 20 | |
| AccuPrimeTM Taq | ? | 1 | 0.5 | 15 | 7.5 | |
| Template | 1 | 0.5 | 0.5 x 100 | 0.25 x 100 | ||
| Final volume | 50 | 25 | 2500 | 1250 |
* Buffer contains 1.5 mM MgCl2 at final concentration
PCR program
1. 94◦C – 4′
2. x 10 {
a. 94◦C – 1′
b. 62◦C – 1°C – 1’
c. 68◦C – 1′
}
3. x 25 {
a. 94◦C – 1′
b. 52◦C – 1′
c. 68◦C – 1′
}
4. 68◦C – 10'