For quantitative real-time-PCR (qRT-PCR), cultures of P. polymyxa WLY78, ∆nifS-like, ∆yutI, ∆sufCDB, ∆sufC, and ∆sufD were grown under N2-fixing conditions (2 mM glutamate and without O2) and harvested after 8 h of incubation. To detect the expression of the nifU gene, total RNA was extracted from ∆sufC, ∆sufD, ∆sufB, ∆sufCDB, ∆sufC/nifU-K.o, ∆sufD/nifU-K.o, ∆sufB/nifU-K.o, and ∆sufCDB/nifU-K.o. Total RNA was isolated using TRIzol (Takara Bio, Tokyo, Japan). The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I (Takara Bio, Tokyo, Japan). The integrity and size distribution of the RNA were verified by agarose gel electrophoresis, and the concentrations were determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specifications (Takara Bio, Tokyo, Japan). cDNA (0.4 µg) was used for qRT-PCR. The relative transcript levels of sufD, sufS, sufU, and sufB were determined with 16S rDNA as a control by the SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara Bio, Tokyo, Japan). Primers for sufC, sufD, sufS, sufU, sufB, nifH, nifD, nifU, and 16S rDNA used for RT-PCR or qRT-PCR are listed in Table S3 .
Rt prime mix
Manufactured by Takara Bio
Sourced in Japan
RT Prime Mix is a ready-to-use solution for reverse transcription and subsequent PCR amplification of RNA targets. It contains all the necessary components, including reverse transcriptase, RNase inhibitor, and optimized buffer, for efficient conversion of RNA to cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Rnase free dnase 1, by Takara Bio (3 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Sybr premix ex taq tli rnaseh plus kit, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
7500 real time system, by Thermo Fisher Scientific (1 mentions)
Pcr mastermix, by Transgene (1 mentions)
Trizol reagent, by Sangon (1 mentions)
5 protocols using rt prime mix
1
Quantitative Analysis of Nitrogen Fixation Genes
2
Determining Operons in Arsenic Clusters
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In order to determine the operons in ars clusters, an RT-PCR experiment with primers designed to span across intergenic regions (Table S3 ) was carried out. A culture of Pantoea sp. IMH was grown in LB medium with 1 mM As(V). After 8 h, the IMH strains were harvested by centrifugation at 4 °C, and the total RNA was isolated using the PrimeScript® RT reagent Kit with gDNA Eraser (Takara Bio) according to the manufacturer’s instructions. The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I (Takara Bio). The integrity and size distribution of the RNA were verified by agarose gel electrophoresis, and the concentration was determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specification (Takara Bio). 1.0 μg of cDNA was used for the template of RT-PCR.
3
RT-PCR and qRT-PCR analysis of nitrogen-fixing genes in Paenibacillus sabinae
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For RT-PCR, P. sabinae T27 was grown in N2-fixing conditions (without NH4Cl and O2). For qRT-PCR, P. sabinae T27 was grown in N2-fixing conditions (without NH4Cl and O2) and non- N2-fixing conditions (100 mM ammonium and 21% O2). The culture was harvested by centrifugation at 4 °C, and total RNA was isolated using the PrimeScript® RT reagent Kit with gDNA Eraser (Takara Bio) according to the manufacturer’s instructions. The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I (Takara Bio). The integrity and size distribution of the RNA was verified by agarose gel electrophoresis, and the concentration was determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specifications (Takara Bio). 0.8 μg of cDNA was used for RT-PCR. The nif and nif-like gene transcripts were detected by using an RT-PCR Kit with 16S rDNA as a control. Primers for nif, nif-like genes and 16S rDNA used for PCR are listed in (Additional file 11 : Table S2).
4
Quantifying Nitrogen Fixation in Fungus
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For RT-PCR, P. graminis RSA19 was grown in N2-fixing conditions (without NH4Cl and O2). For qRT-PCR, P. graminis RSA19 and Δorf1 mutant strain were grown in N2-fixing conditions (without NH4Cl and O2) and non-N2-fixing conditions (100 mM ammonium and 21% O2). The integrity and size distribution of the RNA was verified by agarose gel electrophoresis, and the concentration was determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specifications (Takara Bio, Tokyo, Japan). The nif gene transcripts were detected by using an RT-PCR Kit with 16S rDNA as a control. Primers for nif genes and 16S rDNA used for PCR are listed in Table 2 . qRT-PCR was performed on Applied Biosystems 7500 Real-Time System and detected by the SYBR Green detection system with the following program: 95 °C for 15 min, 1 cycle; 95 °C for 10 s and 65 °C for 30 s, 40 cycles. The relative expression level was calculated using the 2−ΔΔCT method [45 (link)], where ΔΔCT = (CT gene of nif − CT gene of 16S rRNA) N2-fixing condition − (CT gene of nif − CT gene of 16S rRNA) non-N2-fixing condition The CT value is the cycle threshold at which the detected fluorescence crosses an arbitrarily placed threshold. Each experiment was performed in triplicate.
5
Cloning armc10 Coding Sequence from Zebrafish
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Total RNA was extracted from 80 embryos at 24 h post-fertilization (hpf) using TRIzol reagent (Sangon Biotech Co., Ltd., Shanghai, China). Following extraction, 1 µg of RNA was reverse transcribed into cDNA using RT Prime mix (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. The primers were designed based on the sequences of armc10 (ENSDARG00000062960) provided by the Ensembl database (http://asia.ensembl.org/index.html ) to clone the coding sequence of armc10. The primers used were as follows: armc10 F1, 5′-TGGGAGATGGCAGATGAT-3′ and R1, 5′-AGGAGCCGTCCAGTAAAA-3′; armc10 F2, 5′-CTCTGCTGGGGATTGTGG-3′ and R2, 5′-GAGAGTCCGGTCTCCTCCTC-3′. The RT product was used as a template for nested-PCR with 10 µl 2X PCR Mastermix (Beijing TransGen Biotech Co., Ltd., Beijing, China), 1 µl cDNA, 2 µl F/R primers and 7 µl H2O. The conditions for the nested-PCR were as follows: 95°C for 3 min, and 35 cycles of 95°C for 30 sec, 56°C for 30 sec and 72°C for 1 min, followed by incubation for 10 min at 72°C.
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