Sep 26, 2024
General methanogens: mcrA mlas-mod – mcrA-rev
- 1Soil and Water Research Infrastructure
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: eva.petrova@bc.cas.cz
Protocol Citation: Eva Petrova, Roey Angel 2024. General methanogens: mcrA mlas-mod – mcrA-rev. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyk1qvx9k/v1
Manuscript citation:
Angel R, Matthies D, Conrad R. Activation of methanogenesis in arid biological soil crusts despite the presence of oxygen. PLoS ONE. 2011;6:e20453.
Steinberg LM, Regan JM. Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge. Appl
Environ Microbiol. 2008;74:6663–71.
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: September 26, 2024
Protocol Integer ID: 12423
Keywords: PCR, Methanogens
Abstract
Amplification of the marker gene methyl coenzyme M reductase alpha subunit (mcrA) using the general primers mlas-mod – mcrA-rev
mlas-mod GGY GGT GTM GGD TTC ACM CAR TA Angel et al. (2011), Plos One
mcrA-rev CGT TCA TBG CGT AGT TVG GRT AGT Steinberg and Regan (2008), AEM.
Fragment size: 472bp.
Protocol materials
dNTP Set, PCR GradeBiotechrabbitCatalog #BR0600601
Step 1
DreamTaq Green DNA Polymerase (5 U/µL)Thermo Fisher ScientificCatalog #EP0711
Step 1
Bovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
Step 1
PCR mixture
PCR mixture
| A | B | C | D | E | F | |
| Reagent | Final. conc. | 1 tube (50μl) | 1 tube(25μl) | 96 tubes | 96 tubes | |
| PCR H2O | 25.1 | 12.55 | 1255 | 627.5 | ||
| 1x | 10 | 5 | 500 | 250 | ||
| dNTP mixture (2.0 mM each) | 0.2mM | 5 | 2.5 | 250 | 125 | |
| 2.5mM | 5 | 2.5 | 250 | 125 | ||
| BSA (20μg/μl) | 0.8 μg/μl | 2 | 1 | 100 | 50 | |
| mlas-mod | 0.4 μM | 0.8 | 0.4 | 40 | 20 | |
| mcrA-rev | 0.4 μM | 0.8 | 0.4 | 40 | 20 | |
| 1.5U/50μl | 0.3 | 0.15 | 15 | 7.5 | ||
| Template | 1 | 0.5 | 0.5 x 100 | 0.25 x 100 | ||
| Final volume | 50 | 25 | 2500 | 1250 |
dNTP Set, PCR GradeBiotechrabbitCatalog #BR0600601
DreamTaq Green DNA Polymerase (5 U/µL)Thermo Fisher ScientificCatalog #EP0711
Bovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
PCR program
PCR program
1. 94◦C – 5′
2. x 5 {
a. 60◦C – 1°C - 45''
b. 72◦C – 30''
c. 94◦C – 30'
}
3. x 29 {
a. 55◦C – 45''
b. 72◦C – 30''
c. 94◦C – 30''
}
4. 55◦C – 45''
5. 72°C - 10'