Oct 01, 2024

Public workspacepMOA 189F-682R

DOI
dx.doi.org/10.17504/protocols.io.rm7vzz1rvx1w/v1
  • 1Institute of Soil Biology and Biogeochemistry, Biology Centre CAS
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: eva.petrova@bc.cas.cz
Eva Petrova
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzz1rvx1w/v1
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 01, 2024
Protocol Integer ID: 12426
Keywords: PCR, pmoA, PMMO, Methanotrophs, Methane oxidising bacteria, Particulate methane monooxygenase
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.
PCR mixture
PCR mixture
ReagentFinal. conc.1 tube (50μl)1 tube(25μl) 96 tubes (25μl x100) 96 tubes (12.5μl x100)
PCR H2O  37.918.951255627.5
10X Buffer (I for RT and II for DNA)1x52.5 500 (Promega) 250 (Promega)
dNTP mixture (2.0 mM each0.2mM52.5 250125
MgCl2 (50 mM)* 3mM1.5*0.75*250125
BSA (20μg/μl)0.8 μg/μl2110050
189f (25μM) 0.4 μM0.80.4 4020
682r (25 μM)0.4 μM0.80.44020
AccuPrimeTM Taq ?10.5 157.5
Template 10.50.5 x 1000.25 x 100
Final volume 502525001250
* Buffer contains 1.5 mM MgCl2 at final concentration
PCR program
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'