Sep 26, 2024

Public workspace16S Arc 109F-934R

DOI
dx.doi.org/10.17504/protocols.io.n92ldkq9v5br/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
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.n92ldkq9v5br/v1
Protocol Citation: Eva Petrova, Roey Angel 2024. 16S Arc 109F-934R. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldkq9v5br/v1
Manuscript citation:
1. Grosskopf R, Janssen PH, Liesack W. Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Appl Environ Microbiol. 1998;64:960–9. http://aem.asm.org/cgi/content/abstract/64/3/960

2. Miyashita A, Mochimaru H, Kazama H, Ohashi A, Yamaguchi T, Nunoura T, et al. Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs). FEMS Microbiol Lett. 2009;297:31–7.
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2009.01648.x/full
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: 12411
Keywords: PCR, Archaea, 16S rRNA gene
Abstract
Amplification of the marker gene 16S rRNA for general Archaea using primers 109F-934R.
109F  or           ACK GCT CAG TAA CAC GT   (target seq. 109 – 125) Grosskopf  et al. (1998),
109F-mod      AHD GCT CAG TAA CAC RT    (target seq. 109 – 125) Miyashita et al. (2009)
934R                GTG CTC CCC CGC CAA TTC CT    (target seq. 915 – 934) Grosskopf  et al. (1998)
Expected fragment size: 791bp
Materials
MATERIALS
ReagentGoTaq® Flexi DNA PolymerasePromegaCatalog #M830
PCR mixture
PCR mixture
ReagentFinal. conc.1 tube (50μl)1 tube(25μl) 96 tubes (25μl x100)
PCR H2O  27.713.851385
GoTaq®Flexi 5x Green Buffer1x105500
dNTP mixture (2.0 mM each0.2mM52.5250
MgCl2 (25 mM) 1.5mM31.5150
BSA (20 μg/μl)0.8 μg/μl21100
109f/109f-mod (25 μM)0.25 μM0.50.2525
934r (25 μM)0.25 μM0.50.2525
Go Taq Flexi polymerase1.5U/50μl0.30.1515
Template 10.50.5 x 100
Final volume 50252500
PCR program
PCR program
1.  94◦C – 4′
2.  x 28 - 32 {
       a.  52◦C – 30′′
       b.  72◦C – 45′′
       c.  94◦C – 30′′
      }
3.  52◦C – 30′′
4.  72◦C – 10'