Sybr green 2 pcr master mix

Manufactured by Takara Bio

Sourced in United States, China, Germany

SYBR Green II PCR Master Mix is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green II, a fluorescent dye that binds to double-stranded DNA, enabling the monitoring of amplification during the PCR process.

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SYBR Green Master Mix II SYBR Green II PCR Mix SYBR Green PCR Master SYBR Green II Master 2× SYBR green mix II SYBR Master Mix SYBR Green Mix PCR Master Mix SYBR Green II SYBR Green

13 protocols using sybr green 2 pcr master mix

Quantifying Mitochondrial Dynamics and Apoptosis

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Total RNA was extracted from the spleen tissue by TRIzol reagent (Invitrogen, China). The RNA was reverse-transcripted to first-strand complementary DNA (cDNA) according to the manufacturer’s protocol (Invitrogen, China) and then stored at −80°C for PCR.
Primer Premier Software 5.0 (PREMIER Biosoft International, USA) was used to design specific primers of mitochondrial dynamics-related and apoptosis-related genes were designed by Primer Premier Software 5.0 (PREMIER Biosoft International, USA) (Table 1). β-actin was used as an internal reference. Quantitative real-time PCR (qPCR) reactions were run in a 20μL reaction mixture using an ABI 7500 Detection System (Applied Biosystems, USA). Each RT reaction was comprised of 0.4 μl of 50× ROX reference Dye II, 10 μl of 2× SYBR Green II PCR Master Mix (TaKaRa, China), 2 μl of cDNA, 0.4 μl of each primer (10 μM), and 6.8μl of PCR-grade water. The PCR procedure consisted of 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 34 s. The mRNA relative abundance was determined via the method of Pfaffl [43 (link)].

Xu D., Li B., Cao N., Li W., Tian Y, & Huang Y. (2017). The protective effects of polysaccharide of Atractylodes macrocephala Koidz (PAMK) on the chicken spleen under heat stress via antagonizing apoptosis and restoring the immune function. Oncotarget, 8(41), 70394-70405.

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Quantitative Real-Time PCR Protocol

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Total RNAs of the spleen in each group were extracted by TRIzol reagent (Invitrogen, China). The content and purity of the RNA were measured spectrophotometrically at 260/ 280 nm. First-strand complementary DNA was synthesized according to the manufacturer's protocol (Invitrogen, China), and then was stored at −80°C for PCR.
Specific primers of the target genes were designed by uing Primer Premier Software 5.0 (PREMIER Biosoft International, USA) (Table 1). The β-actin was used as an internal reference. Quantitative real-time PCR reactions were run in a 20μL reaction mixture using an ABI 7500 Detection System (Applied Biosystems, USA). Each RT reaction was comprised of 10 μl of 2× SYBR Green II PCR Master Mix (TaKaRa, China), 0.4 μl of each primer (10 μM), 0.4 μl of 50× ROX reference Dye II, 2 μl of cDNA, and 6.8μl of PCR-grade water. The PCR procedure consisted of 95°C for 30 s followed by 40 cycles of 95°C for 5 s, 60°C for 34 s. The mRNA relative abundance was determined by using the method of Pfaffl [53 (link)].

Han Y., Li C., Su M., Wang Z., Jiang N, & Sun D. (2017). Antagonistic effects of selenium on lead-induced autophagy by influencing mitochondrial dynamics in the spleen of chickens. Oncotarget, 8(20), 33725-33735.

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Evaluating Immune Cell Markers in Yak Small Intestine

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Total RNA was isolated from the small intestine using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed to single-stranded cDNA using a reverse transcription kit (MBI Fermentas, Burlington, ON, Canada) according to the manufacturer's instructions. The RT-qPCR primers were designed according to the Bos grunniens CD3e, CD79a, IgA, IgG, CD68, SIRPa, and b-actin gene sequences (GenBank accession numbers: KY911279, KY911280, MG432919, MF099643, KY921959, MH347358, and DQ838049, respectively) using Primer 5 software and synthesised by the Beijing Genomics Institute BGI Company (China). The RT-qPCR primer sequences are presented in Table 1. RT-qPCR was conducted using a Light-Cycler480 thermocycler (Roche, Manheim, Germany) in 20-μL reaction volumes consisting of 1 μL cDNA, 1 μL forward primer, 1 μL reverse primer, 10 μL 2× SYBR Green II PCR Master Mix (TaKaRa, Shiga, Japan), 0.4 μL Rox, and 6.4 μL nuclease-free H 2 O. Four replicates were set for each sample to ensure the accuracy of the relative expression of the target genes in the sample. After amplification, according to the system-generated Ct value, the 2 -DDCt method was used with b-actin as an internal standard to obtain the relative expression of CD3e, CD79a, IgA, IgG, CD68 and SIRPa mRNA.

Zhang Q., Cui Y., Yu S.J., Huang Y.F., Pan Y.Y, & Bai Z.C. (2022). Immune cells in the small intestinal mucosa of newborn yaks. Folia morphologica, 81(1).

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Transcriptomic Data Validation via qRT-PCR

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Thirty-eight DEGs were selected to confirm the transcriptomic data through qRT-PCR analysis. Gene-specific primers were designed using Primer 3 software (Additional file 1). The three independent biological replicates for three flowering development times were mixed as T1, T2, and T3. First-strand cDNA was generated from purified total RNA using a PrimeScript™ RT reagent kit (Takara, Japan). Longan actin (Dlo_028674), which was used in our previous study, was selected as the reference gene [35 (link)]. qRT-PCR was conducted using the LightCycler® 480 Real-Time PCR System (Roche, Germany) and SYBR Green II PCR Master Mix (Takara, Japan). The amplification program was as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Each reaction was performed in triplicate. The relative expression levels of the candidate genes were calculated using the 2^-∆∆Ct method [35 (link)]. Pearson’s correlation values between the RNA-Seq and qRT-PCR data of selected genes were calculated using “cor.test” in R version 3.3.

Jue D., Sang X., Liu L., Shu B., Wang Y., Liu C., Wang Y., Xie J, & Shi S. (2019). Comprehensive analysis of the longan transcriptome reveals distinct regulatory programs during the floral transition. BMC Genomics, 20, 126.

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Gene Expression Determination Protocol

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For gene expression determination, the samples were first stored at −80 °C and were prepared for RNA isolation and gene expression analysis. Total RNA was isolated from about 0.1 g of crushed leaves by a plant RNA kit (Huayueyang, Beijing, China) according to the manufacturer’s protocol. The first strand of cDNA was synthesized from 1 μg of total RNA using PrimeScript RT reagent Kit (Takara, Shiga, Japan). The primers for the corresponding genes were designed on primer 5, and actin2 was used as an internal control (Table 4). The qPCR was performed in Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with SYBR Green II PCR Master Mix (Takara, Shiga, Japan). The qPCR was carried out in a final volume of 20 μL, which contained 4 μL of cDNA, and the conditions were the following: initial denaturation at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 5 s, and annealing and extension at 60 °C for 34 s. A melting curve was obtained at 95 °C for 15 s and at 60 °C for 1 min followed by continuous heating. The analysis of the qPCR results was performed with the REST 2009 software.

Wang T., Li L., Cheng G., Shu X., Wang N., Zhang F., Zhuang W, & Wang Z. (2021). Physiological and Molecular Analysis Reveals the Differences of Photosynthesis between Colored and Green Leaf Poplars. International Journal of Molecular Sciences, 22(16), 8982.

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Transcriptome Analysis and Validation in Brassica

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Gene ontology (GO) enrichment analysis was conducted on the DEGs using GOseq R software [25 (link)]. KOBAS software was used to perform DEG enrichment statistics in the KEGG pathway [26 (link)]. Brassica Database v3.0 was used as reference lists [19 (link)]. Twelve DEGs were randomly selected to confirm the transcriptome data by qRT-PCR analysis. Gene-specific primers were designed using Primer v5.0 (Table S2). The normalized eEF1Bα2 gene(BraA10g020830.3C.gene) was used as an internal control of gene expression [27 (link)]. The quality-assured RNA was used as template, with the synthesis of cDNA processed by a Takara PrimeScript RT reagent Kit (Takara, Nojihigashi, Kusatsu, Japan). The Bio-Rad CFX96 RT-PCR Detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green II PCR Master mix (Takara) were used for the qRT-PCR reactions. The gene expression data were analyzed using the 2^−ΔΔCt method [28 (link)]. SPSS v19.0 (SPSS, Chicago, IL, USA) was used to conduct the one-way analysis of variance (ANOVA) with Duncan’s multiple range test using a significance threshold of p < 0.05. The results were visualized using SigmaPlot v10.0 (Systat Software Inc., San Jose, CA, USA).

Dai Y., Zhang S., Sun X., Li G., Yuan L., Li F., Zhang H., Zhang S., Chen G., Wang C, & Sun R. (2020). Comparative Transcriptome Analysis of Gene Expression and Regulatory Characteristics Associated with Different Vernalization Periods in Brassica rapa. Genes, 11(4), 392.

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Quantitative Real-Time PCR Analysis

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Quantitative real-time PCR was performed by using LightCycler® 480 Real-Time PCR System (Roche, Germany) and SYBR Green II PCR Master Mix (Takara, Dalian, China). The amplification programme was performed as described in our previous study [40 (link)]. Three biological replicates were carried out for each sample. Transcript levels were calculated using the 2^–∆∆Ct method and normalized using the longan Actin gene (Dlo_028674) [5 (link)] or Arabidopsis Actin2 as an internal control. Genes that were up- or downregulated by at least twofold were considered differentially expressed.

Jue D., Li Z., Zhang W., Tang J., Xie T., Sang X, & Guo Q. (2024). Identification and functional analysis of the LEAFY gene in longan flower induction. BMC Genomics, 25, 308.

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Poplar Cultivars RNA Isolation and qRT-PCR

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The samples of different poplar cultivars were first prepared for RNA isolation. Total RNA was isolated from 0.1 g of crushed leaves with a plant RNA kit (Huayueyang, Beijing, China) according to the manufacturer’s protocol. Then, the first strand of cDNA was synthesized from 1 μg of total RNA using a PrimeScript RT reagent Kit (Takara, Dalian, China). The qPCR was performed in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with SYBR Green II PCR Master Mix (Takara, Dalian, China). The reaction was carried out in a final volume of 20 μL (containing 4 μL of cDNA) with the following conditions: initial denaturation at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 5 s, and annealing and extension at 60 °C for 34 s. A melting curve was obtained at 95 °C for 1 s and at 60 °C for 1 min followed by continuous heating. Finally, the qRT-PCR results were analyzed with the REST 2009 software (version 2.0.13). In this study, the primers for the corresponding genes were designed on primer 5, and actin2 was used as an internal control (Table 3).

Wang T., Xu D., Zhang F., Yan T., Li Y., Wang Z., Xie Y, & Zhuang W. (2024). Changes in Photosynthetic Characteristics between Green-Leaf Poplar Linn. “2025” and Its Bud-Sporting Colored-Leaf Cultivars. International Journal of Molecular Sciences, 25(2), 1225.

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Quantitative Analysis of Hub Gene Expression

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Five hub genes were selected to evaluate their expression levels by qRT-PCR analysis. Gene-specific primers were designed using Primer v5.0. Actin was used as an internal control for gene expression (Table S7). The Bio-Rad CFX96 RT-PCR Detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green II PCR Master mix (Takara, Nojihigashi, Kusatsu, Japan) were used for the qRT-PCR reactions. The gene expression data were analyzed using the 2^-ΔΔCt method [65 (link)]. SPSS v19.0 (SPSS, Chicago, IL, USA) was used to conduct a one-way analysis of variance (ANOVA) with Duncan’s multiple range post-hoc test and a significance threshold of p < 0.05. Results were visualized using Sigmaplot v10.0 (Systat Software Inc., San Jose, CA, USA).

Dai Y., Sun X., Wang C., Li F., Zhang S., Zhang H., Li G., Yuan L., Chen G., Sun R, & Zhang S. (2021). Gene co-expression network analysis reveals key pathways and hub genes in Chinese cabbage (Brassica rapa L.) during vernalization. BMC Genomics, 22, 236.

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Quantifying Auditory Cortex Gene Expression

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The auditory cortex tissues from 6 rats in each subgroup were separated and prepared for RNA extraction using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). Following this, an aliquot of the extracted RNA was immediately reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Bio, Inc., Otsu, Japan). qPCR was performed with a LightCycler 480 RT-PCR system (Roche Diagnostics) using SYBR-Green II PCR Master Mix (Takara Bio, Inc.). The primers were designed by Takara Bio, Inc., and are listed in Table II. β-actin was included as an endogenous control. The amplification conditions were as follows: 95°C for 5 min; 45 cycles of 95°C for 10 sec, 60°C for 20 sec, and 72°C for 20 sec; followed by 95°C for 5 sec and 65°C for 60 sec. The relative mRNA expression levels in the control and mimetic aging groups were calculated using the 2^−ΔΔCq method.

Yuan J., Zhao X., Hu Y., Sun H., Gong G., Huang X., Chen X., Xia M., Sun C., Huang Q., Sun Y., Kong W, & Kong W. (2018). Autophagy regulates the degeneration of the auditory cortex through the AMPK-mTOR-ULK1 signaling pathway. International Journal of Molecular Medicine, 41(4), 2086-2098.

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