The quantifications of fungi and bacteria in moromi fermentation samples were determined by qPCR. Genomic DNA extracted from moromi samples were used as the templates. The V3–V4 region of the 16S rRNA bacterial gene was amplified by using the primers Eub338 (5′-ACTCCTACGGGA GGCAGCAG-3′) and Eub518 (5′-ATTACCGCGGCTGCTGG-3′) [28 (link)]. For fungi, the ITS2 region was amplified by using the primers of ITS1 f (5′-TCCGTAGGTGAACCTGCGG-3′) and 5.8 S (5′-CGCTGCGTTCTTCATCG-3′) [28 (link)]. The qPCR was performed by using the QuantiFast SYBR green PCR kit commercial kit (Vazyme Biotech Co. Ltd., Nanjing, China) according to our previous protocol [29 (link)]. Standard curves of bacterial and fungal biomasses in moromi were created through plotting the CT values of different concentrations of Bacillus pumilus’s 16S rRNA gene and Candida zeylanoides’s ITS gene. The StepOnePlus instrument (Applied Biosystems, Foster, CA, USA) was used for the qPCR experiment [24 (link),30 (link)].
Quantifast sybr green pcr kit
Manufactured by Vazyme
Sourced in China
The QuantiFast SYBR green PCR kit is a real-time PCR reagent designed for fast and sensitive quantification of DNA targets. It contains a fast-cycling SYBR Green I master mix formulation that enables rapid thermal cycling and accurate quantification of samples.
Automatically generated - may contain errors
2 protocols using quantifast sybr green pcr kit
1
Quantification of Fungi and Bacteria in Moromi Fermentation
2
Quantification of AGE Formation and Gene Expression in Chondrocytes
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After treatment, the supernatant of culture medium was collected. The AGE formation level in the supernatants was quanti ed by speci c AGEs kits according to the manufacturer's instruction (Cloud-Clone, Wuhan, China). The absorbance was read immediately at 450 nm.
Total RNA Extraction and Real-time Fluorescent Quantitative PCR Total RNA was extracted from chondrocytes using TRIzol (Invitrogen, USA) according to the protocol of the kit. 1 µg total RNA was reverse transcribed to synthesize cDNA according to the reverse transcription kit (VAZYME, Nanjing, China). cDNA was ampli ed with real-time PCR on ABI7900 real-time PCR detection system using QuantiFast SYBR Green PCR Kit (VAZYME, Nanjing, China) accroding to the manufacturer's instructions. Real-time PCR was performed using the speci c primers: β-actin forward 5'-AGCGAGCATCCCCCAAAGTT-3' and reverse 5'-GGGCACGAAGGCTCATCATT-3', TNF-α forward 5'-TCAGAGGGCCTGTACCTCAT - 3' and reverse 5'-GGAAGACCCCTCCCAGATAG - 3', IL-1β forward 5'-TCCAGCTACGAATCTCCGAC - 3' and reverse 5'-TGATCGTACAGGTGCATCGT - 3', MMP13 forward 5'-CCCAACCCTAAACATCCAA - 3' reverse 5'-AAACAGCTCCGCATCAACC - 3', PPARγ forward 5'-GGAGGTCCGCATCTTTCACT - 3' reverse 5'-GCTACCAGCATCCCGTCTTT - 3'. The relative mRNA expression abundance was normalized to the housekeeping gene β-actin using the formula Y = 2 -△△Ct .
Total RNA Extraction and Real-time Fluorescent Quantitative PCR Total RNA was extracted from chondrocytes using TRIzol (Invitrogen, USA) according to the protocol of the kit. 1 µg total RNA was reverse transcribed to synthesize cDNA according to the reverse transcription kit (VAZYME, Nanjing, China). cDNA was ampli ed with real-time PCR on ABI7900 real-time PCR detection system using QuantiFast SYBR Green PCR Kit (VAZYME, Nanjing, China) accroding to the manufacturer's instructions. Real-time PCR was performed using the speci c primers: β-actin forward 5'-AGCGAGCATCCCCCAAAGTT-3' and reverse 5'-GGGCACGAAGGCTCATCATT-3', TNF-α forward 5'-TCAGAGGGCCTGTACCTCAT - 3' and reverse 5'-GGAAGACCCCTCCCAGATAG - 3', IL-1β forward 5'-TCCAGCTACGAATCTCCGAC - 3' and reverse 5'-TGATCGTACAGGTGCATCGT - 3', MMP13 forward 5'-CCCAACCCTAAACATCCAA - 3' reverse 5'-AAACAGCTCCGCATCAACC - 3', PPARγ forward 5'-GGAGGTCCGCATCTTTCACT - 3' reverse 5'-GCTACCAGCATCCCGTCTTT - 3'. The relative mRNA expression abundance was normalized to the housekeeping gene β-actin using the formula Y = 2 -△△Ct .
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