Taqman universal polymerase chain reaction master mix

Manufactured by Thermo Fisher Scientific

Sourced in United States

TaqMan universal polymerase chain reaction (PCR) master mix is a pre-mixed reagent designed for real-time PCR applications. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and buffer, to perform PCR amplification.

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7 protocols using taqman universal polymerase chain reaction master mix

Genotyping of COMT and DRD2/ANKK1 Variants

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Allelic discrimination for the COMT Val¹⁵⁸Met (rs4680) and DRD2/ANKK1 TaqIA (rs1800497) polymorphisms were determined through real-time polymerase chain reaction (PCR) technique, using a TaqMan SNP genotyping assay with fluorogenic probes (Applied Biosystems, Foster City, CA). Briefly, 15 ng of DNA was amplified in a total volume of 8 μL containing 0.2 μL of a minor groove binder (MGB) probe solution (Applied Biosystems) and 4 μL of TaqMan universal polymerase chain reaction master mix (Applied Biosystems). PCR conditions were provided by the manufacturer: 40 cycles of 95 °C denaturation (15 sec), 60 °C anneal/extension (1 min).
Thermal cycling and fluorescence signal genotyping were performed through the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). A positive control for each possible genotype and a negative control were included in each 96-well plate.

da Costa Azevedo J.N., Carvalho C., Serrão M.P., Coelho R., Figueiredo-Braga M, & Vieira-Coelho M.A. (2022). Catechol-O-methyltransferase activity in individuals with substance use disorders: a case control study. BMC Psychiatry, 22, 412.

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Genotyping Protocol for Association Analysis

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All subjects were individually genotyped according to the manufacturer’s instructions using the TaqMan 7900HT sequence Detection System (Applied Biosystems, Foster City, Calif.). Each assay was carried out using 10 ng DNA in a 5-µL reaction consisting of TaqMan universal polymerase chain reaction master mix (Applied Biosystems, Foster City, Calif.), forward and reverse primers, and 6-carboxyfluorescein (FAM) and 4,7,2’-trichloro-7’-phenyl-6-carboxyfluorescein (VIC)-labeled probes designed by Applied Biosystems (ABI Assays-on-demand). Allelic discrimination was measured automatically using Sequence Detection Systems 2.1 software (automatic confidence level 95%). Approximately 8% of all genotypes were repeated in independent polymerase chain reactions to check for consistency and to ensure intraplate and interplate genotyping quality control. No genotyping discrepancies were detected between the repeated samples (Figure 1).
Figure 1

The workflow of association analysis.

Yao J., Mao X., Sun Q., Wu B., Yu W., Huang Y., Luo S., Zeng J, & Lin J. (2023). TBX5 Variants are Associated with Susceptibility to and the Incidence of Liver Cirrhosis and Hepatocellular Carcinoma in the Chinese Population: A Multicenter and Follow-Up Study. Infection and Drug Resistance, 16, 2653-2665.

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Osteogenic Differentiation of hMSCs

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Total messenger RNA (mRNA) was isolated from culture hMSCs on scaffolds and treated with OS for 5 days, and complementary deoxyribonucleic acid (cDNA) was transcribed with reverse transcriptase (Invitrogen) and oligodeoxythymidine primers. The cDNA was amplified with TaqMan universal polymerase chain reaction (PCR) master mix (Applied Biosystems, USA) and primers and TaqMan probe sets for alkaline phosphatase (ALP; Hs01029144_m1), osteocalcin (OC; Hs01587814_g1), and runt-related transcription factor 2 (Runx2; Hs00231692_m1) were purchased from Applied Biosciences. All TaqMan PCR was performed using StepOne Plus real-time PCR system (Applied Biosystems). Amount of cDNAs were normalized to that of 18S ribosomal RNA.

Kim B.S., Park K.E., Kim M.H., You H.K., Lee J, & Park W.H. (2015). Effect of nanofiber content on bone regeneration of silk fibroin/poly(ε-caprolactone) nano/microfibrous composite scaffolds. International Journal of Nanomedicine, 10, 485-502.

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Quantification of Pancreatic Triglycerides and Gene Expression

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Pancreatic triglycerides were assayed according to Castell et al. [22 (link)]. Total RNA was extracted using Trizol (Ambion, Austin, TX, USA) and trichloromethane-ethanol (Panreac, Barcelona, Spain) and purified using an RNA extraction kit (Qiagen, Hilden, Germany). Complementary DNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain), and the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) amplification was performed using TaqMan Universal polymerase chain reaction (PCR) Master Mix and the respective specific TaqMan probes (Applied Biosystems, Madrid, Spain). The relative expression of each mRNA was calculated against the control group using the 2^−ΔΔCt method, with cyclophilin A as reference.

Ginés I., Gil-Cardoso K., Serrano J., Casanova-Marti À., Lobato M., Terra X., Blay M.T., Ardévol A, & Pinent M. (2019). Proanthocyanidins Limit Adipose Accrual Induced by a Cafeteria Diet, Several Weeks after the End of the Treatment. Genes, 10(8), 598.

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Real-Time PCR for Coccidioides Detection

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A single-tube nested real-time PCR assay^{38 (link)} which is based on the CocciEnv real-time PCR target^{39 (link)} was run in duplicate to analyze DNA samples for presence of Coccidioides DNA. At lab A, each 10 µL reaction mixture contained 2 µL DNA template, 5 µL 2× SSOAdvanced Universal Probes Supermix (Bio-rad), 240 nM each of primers and probe, and 2.5 µL nuclease-free water. Thermocycling conditions consisted of an initial denaturation for 10 min at 95 °C, followed by 11 outer amplification cycles at 95 °C 10 s, 65 °C 30 s, 72 °C 15 s, and 45 inner amplification cycles at 95 °C 10 s, 52 °C 30 s, 72 °C 15 s on a Biorad CFX Connect. At lab B, the assay was run in 20 µL reactions containing TaqMan Universal Polymerase Chain Reaction (PCR) Master Mix (Applied Biosystems, Grand Island, NY, USA), 240 nM each of primers and probe, BSA (2 ng/μl; BSA) and 2 μl DNA template. Thermocycling conditions consisted of an initial denaturation for 10 min at 95 °C, followed by 25 outer amplification cycles at 95 °C 10 s, 65 °C 30 s, 72 °C 15 s, and 45 inner amplification cycles at 95 °C 10 s, 52 °C 30 s, 72 °C 15 s on a Rotor-Gene 6000 thermocycler (Qiagen; Valencia, CA, USA). Samples were considered positive if they displayed a Ct less than 45 and displayed logarithmic amplification plots on at least one of the duplicate real-time PCR reactions.

Porter W.T., Gade L., Montfort P., Mihaljevic J.R., Bowers J.R., Willman A., Klimowski B.A., LaFleur B.J., Sunenshine R.H., Collins J., Adame G., Brady S., Komatsu K.K., Williams S., Toda M., Chiller T., Litvintseva A.P, & Engelthaler D.M. (2024). Understanding the exposure risk of aerosolized Coccidioides in a Valley fever endemic metropolis. Scientific Reports, 14, 1311.

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Quantifying Gene Expression in Swine Tissues

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Total RNA was isolated from the normal liver and LNs of a nontransplanted swine and from an ectopic and native liver of P147‐13 using the RNAeasy kit (Qiagen, Hilden, Germany). RNA was quantified with the Nanodrop 2000c Spectrophotometer (Thermo Scientific). Reverse transcription was performed using TaqMan Universal polymerase chain reaction (PCR) Master Mix (Life Technologies, Carlsbad, CA) followed by real‐time PCR on an StepOne Plus real‐time PCR thermal cycler (Applied Biosystems, Foster City, CA). TaqMan probes and primers were purchased from Life Technologies (Table 1). Expression was normalized to β‐actin (ACTB). Values represent mean ± standard deviation (SD; n = 3 in each group).

Fontes P., Komori J., Lopez R., Marsh W, & Lagasse E. (2020). Development of Ectopic Livers by Hepatocyte Transplantation Into Swine Lymph Nodes. Liver Transplantation, 26(12), 1629-1643.

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Quantification of VDAC Genes in Endometrial Cancer

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The TaqMan Gene Expression Assay (Applied Biosystems) was used to analyze the expression of the VDAC genes in 33 normal and 93 endometrial cancer tissues. The fluorogenic, FAM-labeled probes, the sequencespecific primers for VDAC1, VDAC2, and VDAC3, and the internal control hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) were purchased as inventoried assays (Hs04978484, Hs01075603, Hs00366592, and Hs02800695, respectively). Each PCR reaction contained 1 mL of complementary DNA (cDNA), 5 mL of 2 3 TaqMan Universal polymerase chain reaction (PCR) MasterMix (Life Technology, USA), 3.5 mL of water, and 0.5 mL of TaqMan Gene Expression Assays, which consisted of a pair of unlabeled PCR primer and a TaqMan probe with FAMä dye label on the 5# end and minor groove binder (MGB) nonfluorescent quencher on the 3# end. The quantitative real-time PCR (RT-qPCR) reactions were performed in triplicate using the Mastercycler ep realplex (Eppendorf, Germany) according to the following PCR program: 95°C for 10 min, 40 cycles at 95°C for 15 s, 1 min annealing, and extension at 60°C. The 2 DCt (Ct gene-Ct HPRT1) method was employed to quantify the expression levels of the studied genes. The obtained 2 2DCt values were recalculated into relative copy number values (number of studied gene mRNA copies per 1.000 copies of HPRT1 mRNA).

Jóźwiak P., Ciesielski P., Forma E., Kozal K., Wójcik-Krowiranda K., Cwonda Ł., Bieńkiewicz A., Bryś M, & Krześlak A. (2020). Expression of voltage-dependent anion channels in endometrial cancer and its potential prognostic significance. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 42(8).

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