Bacterial DNA in cecal digesta was extracted using the EZNATM Stool DNA kit (Omega BioTek, Doraville, CA, USA). The abundance of total bacteria and specific strains (Escherichia coli, Lactobacillus, Bifidobacterium, and Bacillus) was assessed by RT—qPCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology, Dalian, China) and PrimerScriptTM PCR kit (TaKaRa Biotechnology, Dalian, China), respectively. Standard curves were generated using standard plasmids based on Chen et al.’s work (2013) [23 (link)]. Specific primer sequences and probes for RT—qPCR are provided in Table 2 .
Primerscript pcr kit
Manufactured by Takara Bio
Sourced in China
The PrimerScript™ PCR kit is a reagent designed for performing polymerase chain reaction (PCR) experiments. The kit contains the essential components required for amplifying DNA sequences, including a thermostable DNA polymerase, reaction buffer, and nucleotides.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq reagent, by Takara Bio (2 mentions)
E z n a stool dna kit, by Omega Bio-Tek (1 mentions)
Sybr premix ex taq 2 reagent, by Takara Bio (1 mentions)
Quanstudio 6 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Stool dna kit, by Omega Bio-Tek (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
3 protocols using primerscript pcr kit
1
Quantifying Gut Bacterial Populations
2
Quantifying Bacterial Diversity in Colonic Digesta
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Bacterial DNA in colonic digesta was extracted by using the Stool DNA Kit (Omega Bio-Tek, Doraville, CA, USA). All primers and probes were listed in Table 4 and designed following the previous report [25 (link)]. Microbial real-time quantitative PCR was performed in a QuanStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster, CA, USA). Briefly, the total bacteria was detected using SYBR® Premix Ex Taq™ II reagent (TaKaRa, Dalian, China), and the Bacillus, Lactobacillus, E. coli and Bifidobacterium were detected using PrimerScriptTM PCR kit (TaKaRa, Dalian, China) following the previous methods [26 (link)]. Furthermore, for the quantification of bacteria, specific standard curves were generated by constructing standard plasmids as presented by Chen et al. (2013) [26 (link)].
3
Caecal Microbiome Quantification Protocol
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Microbial genomic DNA in the caecal digesta was extracted by using the Stool DNA Kit (Omega Bio-Tech) according to the manufacturer's instruction. The microbial real-time quantitative PCR was determined as described previously (20) . All primers and probes for total bacteria, Escherichia coli, Lactobacillus, Bifidobacterium and Bacillus (21) (Table 3) were commercially synthesised from TaKaRa Biotechnology (Dalian) Co. Ltd. Briefly, the number of total bacteria was analysed by real-time quantitative PCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology (Dalian) Co. Ltd) and CFX-96 real-time PCR detection system (BioRad Laboratories), and the numbers of Bacillus, Lactobacillus, E. coli and Bifidobacterium were analysed by real-time quantitative PCR using PrimerScript TM PCR kit (perfect real time; TaKaRa Biotechnology (Dalian) Co. Ltd) and CFX-96 real-time PCR detection system (Bio-Rad Laboratories) as previously described (20) . For the quantification of bacteria in the test samples, specific standard curves were generated by constructing standard plasmids as presented by Chen et al. (20) . In addition, bacterial copies were transformed (log 10 ) before statistical analysis.
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