Realmaster mix sybr green fp202

Manufactured by Tiangen Biotech

Sourced in China

RealMaster Mix (SYBR Green; FP202) is a ready-to-use solution for real-time quantitative PCR (qPCR) experiments. It contains SYBR Green I dye, DNA polymerase, and necessary buffer components for efficient amplification and detection of target DNA sequences.

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Lab products found in correlation

Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Icycler iqtm optical module, by Beckman Coulter (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Revertra ace qpcr kit, by Toyobo (1 mentions) M1701, by Promega (1 mentions) Fsk 201, by Toyobo (1 mentions) M mlv reverse transcriptase, by Merck Group (1 mentions) Pikoreal96, by Thermo Fisher Scientific (1 mentions)

3 protocols using realmaster mix sybr green fp202

Quantitative Real-Time PCR Analysis of Gene Expression

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After resuscitation, the H1299, OE, and NC groups were cultured according to ATCC protocol for several days. Total RNA were then isolated using an RNeasy mini kit (Qiagen, Dusseldorf, Germany), from each group on the first, third, fifth, and seventh days, separately. A ReverTra Ace qPCR kit (FSQ −101; Toyobo, Kagoshima, Japan) was used to synthesize cDNA. The conditions for reverse transcription were 65°C for five minutes, 37°C for 15 minutes, then 98°C for five minutes.
All of the PCR primers used were designed according to data provided by GenBank (Table 1). RealMaster Mix (SYBR Green; FP202; Tiangen, Beijing, China) was used to perform qPCR. All procedures were fulfilled in an iCycler iQTM Optical Module (Beckman Coulter, Fullerton, CA, USA) under the following conditions: initial denaturation at 95°C for 30 seconds, followed by 40 cycles at 95°C for 30 seconds, then 58°C for 30 seconds and 72°C for 30 seconds, followed by a melt curve from 55 to 95°C in 0.5°C increments and 10 second intervals. All tests were repeated three times.

Li L., Zhang L., Liu D., Cheng Y., Jing Y.T., Yu H., Zhou P., Song J, & Li W.M. (2015). Overexpression of eukaryotic translation initiation factor 4E-binding protein 1 induces the alteration of immune status in H1299 lung cancer cells. Thoracic Cancer, 6(4), 427-432.

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Validating DEG Expression by qRT-PCR

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To validate the differential expression pattern of DEGs obtained by Illumina sequencing, qRT-PCR of all of 16 differentially expressed TFs and 21 randomly chosen DEGs was conducted using the total RNA extracted from PRs of six 4-day-old plants of each accession. Gene-specific primers were designed using Primer 3 (http://fokker.wi.mit.edu/primer3/input.htm), and the primer sequences are presented in Table S2. Approximately 3.0 μg of total RNA for each sample was reversed following the instructions of a reverse transcription kit (Cat. M1701, Promega), and first-strand cDNA was amplified according to the instructions for RealMasterMix (SYBR Green [FP202]; TIANGEN). The B. napus ACTIN2 gene-specific primer (Table S2) was used as a control to normalize the expression data. The results were analyzed using CFX Manager Software using the 2^−ΔΔCT method.

Dun X., Tao Z., Wang J., Wang X., Liu G, & Wang H. (2016). Comparative Transcriptome Analysis of Primary Roots of Brassica napus Seedlings with Extremely Different Primary Root Lengths Using RNA Sequencing. Frontiers in Plant Science, 7, 1238.

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Analysis of Stem Cell Gene Expression

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Total RNA was extracted and isolated from MSCs in each stimulation condition with Trizol Reagent (New Industry) following the standard protocol and quantified with Nanodrop spectrophotometer (ND-1000, Thermo). Then, the reverse transcription reaction was performed on 500 ng of RNA with M-MLV reverse transcriptase (C28025, Sigma) and oligo (dT) (FSK-201, TOYOBO) in a PCR thermal. Quantitative real-time polymerase chain reactions (qRT-PCR) were performed in the PCR system (Pikoreal 96, Thermo) with RealMasterMix SYBR Green (FP202, Tiangen) following the manufacturer’s procedures. The expression levels of stem cell pluripotency genes and trilineage differentiation-related genes were analyzed. The primers were listed in Supplementary Table 1. The target genes of each sample were normalized to the values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Relative expression of each gene was expressed as fold changes by the 2^−ΔΔCt method. The experiment was repeated three times, with five technological repeats for each assay.

Li K., Ning T., Wang H., Jiang Y., Zhang J, & Ge Z. (2020). Nanosecond pulsed electric fields enhance mesenchymal stem cells differentiation via DNMT1-regulated OCT4/NANOG gene expression. Stem Cell Research & Therapy, 11, 308.

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