Sep 01, 2020

Public workspaceqPCR quantification of methanogens using general mcrA primers

DOI
dx.doi.org/10.17504/protocols.io.qcudsww
  • 1Soil and Water Research Infrastructure
  • 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.qcudsww
Protocol Citation: Roey Angel, Eva Petrova 2020. qPCR quantification of methanogens using general mcrA primers. protocols.io https://dx.doi.org/10.17504/protocols.io.qcudsww
Manuscript citation:
Angel, R., Matthies, D., and Conrad, R. (2011). Activation of methanogenesis in arid biological soil crusts despite the presence of oxygen. PLoS ONE 6, e20453. doi:10.1371/journal.pone.0020453.
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 01, 2020
Protocol Integer ID: 12404
Keywords: PCR, qPCR, mcrA, Methanogens, Archaea,
Abstract
General mcrA primers for quantifying methanogens using a SYBR Green-based assay.

NameDirectionSequenceCitation
mlas_modFwdGGY GGT GTM GGD TTC ACM CAR TAAngel et al., 2011
mcrA_revRevCGT TCA TBG CGT AGT TVG GRT AGTSteinberg and Regan, 2008


CITATION
Steinberg L M, Regen J M (2008). Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge. Applied and Environmental Microbiology.
LINK
10.1128/AEM.00553-08

CITATION
Angel R, Matthies D, Conrad R (2011). Activation of methanogenesis in arid biological soil crusts despite the presence of oxygen.. PloS one.
LINK
https://doi.org/10.1371/journal.pone.0020453

Attachments
Icon for Introduction_QPCR_Stratagene.pdf
Introduction_QPCR_St...
10.3MB
Icon for AB_rt-QPCRguide.pdf
AB_rt-QPCRguide.pdf
1,011KB
QPCR mixture
QPCR mixture
ReagentFinal conc.1 tube (25 μl)plate (25 μl x 100)
PCR H2O 2.85285
qPCR master mix1x12.51250
MgCl2 (25mM)*3.5 mM3.5350
BSA (50 μg μl-1)0.8 μg μl-10.440
mlas-mod (25 μM)0.25 μM0.2525
mrcA-rev (25 μM)0.25 μM0.2525
Template 55 x 100
* Different master mixes vary in concentration of MgCl2 and the amount should be adjusted accordingly
Pipetting
Programme
Programme
2h
2h
1.  94◦C – 5′
2.  x 40 {
       a.  95◦C – 30′′
       b.  57◦C – 45′′
       c.   72◦C – 30′′
       d.  84°C -  10'' take a snapshot
      }
3.  Melt curve:  75°C – 94.8°C –6’’. Increase by 0.2°C
PCR
Citations
Steinberg L M, Regen J M. Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge
10.1128/AEM.00553-08
Angel R, Matthies D, Conrad R. Activation of methanogenesis in arid biological soil crusts despite the presence of oxygen.
https://doi.org/10.1371/journal.pone.0020453