Summary
As the most widely used method in microbial community analysis, qPCR can deal with quantification of the number of target genes in a community sample. In this post, we are going to discuss some simple methods utilized in this field.
Materials
Bacterial culture
1.Strains and cultivation conditions
19 bacterial strains containing the nifH gene
2.DNA extraction and quantification
3.Washing bacterial colonies three times in phosphate-buffered saline. According to the manufacturers’ instructions, DNA is extracted and eluted with 100 ul diethyl pyrocarbonate-treated water. Checking DNA purity on a nanodrop spectrophotometer and qiantified with SYBR green I.
4.Environmental samples
DNA, extracted from natural habitats, soil, plant and lake, should be used in this study to represent a broad range of samples typically studied in environmental microbiology. The DNA extracts are obtained in three previously conducted studies and stored at −80°C until analysis.
5.Real-time PCR assays
6.Determination of CT and Efi
Methods
1.SC method
SC method is very popular in environmental microbiology and capable of the employment of a defined target concentration, which is calculated from the DNA concentration, the length of the DNA fragments, the number of targets per DNA fragment, the Avogadro constant and the average weight of a double-standed base pair.
2.OPC method
This method is an alternative approach for absolute quantification from qPCR data. OPC method is processed by defining one standard, which contains a known number of template copies.
Efi of bacterial strains and environmental samples
Through qPCR assays and the fluorescence increase, the amplification efficiencies of the bacterial strains and environmental samples can be detected.
Analysis of template mixtures from two species
Preparing serial dilutions of G. sulfurreducens and N. commune DNA templates. And preparing template mixtures of G. sulfurreducens and N. Commune from the different dilution steps. Running the template mixtures and dilution series as triplecates in one qPCR assay by using nifHF/nifHR primers.
Analysis of environmental samples
Using qPCR to amplify four replicates of each environmental sample and triplicates of a B. japonicum dilution series with the presence of nifHF/nifHR primer pair. This method is same as that of the measurement of Efi of the environmental samples.
These are some simple approaches used for the microbial community samples’ quantitative PCR efficiency variations.
As the most widely used method in microbial community analysis, qPCR can deal with quantification of the number of target genes in a community sample. In this post, we are going to discuss some simple methods utilized in this field.
Materials
Bacterial culture
1.Strains and cultivation conditions
19 bacterial strains containing the nifH gene
2.DNA extraction and quantification
3.Washing bacterial colonies three times in phosphate-buffered saline. According to the manufacturers’ instructions, DNA is extracted and eluted with 100 ul diethyl pyrocarbonate-treated water. Checking DNA purity on a nanodrop spectrophotometer and qiantified with SYBR green I.
4.Environmental samples
DNA, extracted from natural habitats, soil, plant and lake, should be used in this study to represent a broad range of samples typically studied in environmental microbiology. The DNA extracts are obtained in three previously conducted studies and stored at −80°C until analysis.
5.Real-time PCR assays
6.Determination of CT and Efi
Methods
1.SC method
SC method is very popular in environmental microbiology and capable of the employment of a defined target concentration, which is calculated from the DNA concentration, the length of the DNA fragments, the number of targets per DNA fragment, the Avogadro constant and the average weight of a double-standed base pair.
2.OPC method
This method is an alternative approach for absolute quantification from qPCR data. OPC method is processed by defining one standard, which contains a known number of template copies.
Efi of bacterial strains and environmental samples
Through qPCR assays and the fluorescence increase, the amplification efficiencies of the bacterial strains and environmental samples can be detected.
Analysis of template mixtures from two species
Preparing serial dilutions of G. sulfurreducens and N. commune DNA templates. And preparing template mixtures of G. sulfurreducens and N. Commune from the different dilution steps. Running the template mixtures and dilution series as triplecates in one qPCR assay by using nifHF/nifHR primers.
Analysis of environmental samples
Using qPCR to amplify four replicates of each environmental sample and triplicates of a B. japonicum dilution series with the presence of nifHF/nifHR primer pair. This method is same as that of the measurement of Efi of the environmental samples.
These are some simple approaches used for the microbial community samples’ quantitative PCR efficiency variations.