The qPCR was carried out to measure the total copy number of archaeal 16S rDNA in 1 µL of isolated DNA. Standards were generated using serial dilutions of purified DNA from Methanobrevibacter ruminantium, and Methanosphaera stadtmanae purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The standard curve was created using a dilution series of the standards ranging from 10 1 to 10 6 copies of the 16S rDNA. The qPCR was performed using the Applied Biosystems StepOne system (Applied Biosystems, Foster City, USA). The archaeal-specific primers Arch 1174-1195 F (5-GAG GAA GGA GTG GAC GAC GGTA-3) and Arch 1406-1389 R (5-ACG GGC GGT GTG TGC AAG -3) (Rabee et al. 2022) (link) were used to amplify DNA samples and diluted standards. The 10-µL qPCR reaction contained 1µL genomic DNA, 1 μL of each primer, and 7 μL of SYBER Green qPCR-master mix (iNtRON Biotechnology, Inc.). The qPCR conditions were 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. The total copy number of archaeal 16S rDNA per 1 µL of DNA was determined relying on the linear relationship between the threshold amplification (Ct) and the logarithm of 16S rDNA copy numbers of the standards.
Syber green qpcr master mix
Manufactured by iNtRON Biotechnology
Sourced in Cameroon
SYBER Green qPCR-master mix is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence upon excitation, enabling the detection and quantification of target DNA sequences.
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5 protocols using syber green qpcr master mix
1
Quantifying Archaeal 16S rDNA Copy Number
2
Quantifying Rumen Bacterial Diversity via Real-Time PCR
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Real-time PCR was conducted to determine the total bacterial 16S rRNA copy number in the rumen fluid. Standards were generated using dilutions of purified genomic DNA from Prevotella sp, Ruminococcus albus, Butyrivibrio hungatei purchased from DSMZ (Braunschweig, Germany). Dilution series of the standards ranging from 101 to 106 copies of the 16S rRNA gene were used. The qPCR was performed using the Applied Biosystems StepOne system (Applied Biosystems, Foster City, CA, USA).
The bacterial specific primers F (5′-CGGCAACGAGCGCAACCC-3′) and R (5′-CCATTGTAGCACGTGTGTAGCC-3′) (Denman & McSweeney, 2006 (link)) were applied to amplify DNA samples and diluted standards. The 10-μL reaction consisted of 1 μL genomic DNA, 1 μL of each primer, and 7 μL SYBER Green qPCR- master mix (iNtRON Biotechnology, Inc., Seongnam-si, South Korea). The PCR conditions were as follows: 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. The linear relationship between the threshold amplification (Ct) and the logarithm of 16S rDNA copy numbers of the standards was used to calculate the copy numbers of rumen bacteria per μL of DNA.
The bacterial specific primers F (5′-CGGCAACGAGCGCAACCC-3′) and R (5′-CCATTGTAGCACGTGTGTAGCC-3′) (Denman & McSweeney, 2006 (link)) were applied to amplify DNA samples and diluted standards. The 10-μL reaction consisted of 1 μL genomic DNA, 1 μL of each primer, and 7 μL SYBER Green qPCR- master mix (iNtRON Biotechnology, Inc., Seongnam-si, South Korea). The PCR conditions were as follows: 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. The linear relationship between the threshold amplification (Ct) and the logarithm of 16S rDNA copy numbers of the standards was used to calculate the copy numbers of rumen bacteria per μL of DNA.
3
Quantitative PCR for Archaeal 16S rDNA
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Quantitative PCR (qPCR) was carried out to measure the total copy number of archaeal 16S rDNA in 1 µL of isolated DNA. Standards were generated using dilutions of purified DNA from Methanobrevibacter ruminantium, and Methanosphaera stadtmanae purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The standard curve was generated using dilution series of the standards ranging from 101 to 106 copies of the 16S rDNA. The qPCR was conducted using Applied Biosystems StepOne system (Applied Biosystems, Foster City, USA). The archaeal specific primers Arch 1174-1195 F (5-GAGGAAGGAGTGGACGACGGTA-3) and Arch 1406-1389 R (5-ACGGGCGGTGTGTGCAAG-3) [5] (link) were used to amplify DNA samples and diluted standards. The 10-µL qPCR reaction contained 1µL genomic DNA, 1 µL of each primer, and 7 µL of SYBER Green qPCR- master mix (iNtRON Biotechnology, Inc.). The qPCR conditions were 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. The total copy number of archaeal 16S rDNA per 1 µL of DNA was determined relying on the linear relationship between the threshold amplification (Ct) and the logarithm of 16S rDNA copy numbers of the standards.
4
Quantifying Rumen Bacterial 16S rRNA
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Quantitative real-time PCR (qPCR) was used to determine the total bacterial 16S rRNA copy number in the rumen samples. Standards were generated using serial dilutions of DNA purified from Prevotella spp., Ruminococcus albus, and Butyrivibrio hungatei that were purchased from DSMZ (Braunschweig, Germany). Serial dilutions of the standards ranging from 101 to 106 copies of the 16S rRNA gene were used. The qPCR was performed using the Applied Biosystems StepOne system (Applied Biosystems, Foster City, USA). Bacterial primers F (5′-CGGCAACGAGCGCAACCC-3′) and R (5′-CCATTGTAGCACGTGTGTAGCC-3′) [35 (link)] were applied. The 10 μL reaction consisted of 1 μL genomic DNA, 1 μL of each primer, and 7 μL SYBER Green qPCR Master Mix (iNtRON Biotechnology, Inc.). The PCR conditions followed 40 cycles of 95°C for 15 s and 60°C for 60 s. The linear relationship between the threshold amplification (cycle threshold) and the logarithm of 16S rDNA copy numbers of the standards was used to calculate the copy numbers of rumen bacteria per μL of DNA.
5
Quantitative Real-time PCR for Rumen Microbiome
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Quantitative real-time PCR (qPCR) was used to determine the total bacterial 16S rDNA copy number in the rumen samples. Standards were generated using serial dilutions of DNA isolated from Prevotella sp, Ruminococcus albus, Butyrivibrio hungatei purchased from DSMZ (Braunschweig, Germany). Serial dilutions of the standards ranging from 101 to 106 copies of the 16S rDNA gene were used. The qPCR was performed using the Applied Biosystems StepOne system (Applied Biosystems, Foster City, USA). Bacterial primers F (5′-CGGCAACGAGCGCAACCC-3′) and R (5′-CCATTGTAGCACGTGTGTAGCC-3′)40 (link) were applied. The 10-μL reaction consisted of 1 μL genomic DNA, 1 μL of each primer, and 7 μL SYBER Green qPCR- master mix (iNtRON Biotechnology, Inc.). The PCR conditions were as follows 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The linear relationship between the threshold amplification (Ct) and the logarithm of 16S rDNA copy numbers of the standards was used to calculate the copy numbers of rumen bacteria per μL of DNA.
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