Taqman universal master mix
TaqMan Universal Master Mix is a pre-formulated, ready-to-use mixture designed for real-time PCR applications. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to perform quantitative gene expression analysis.
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11 protocols using taqman universal master mix
mRNA Quantification by RT-qPCR
Multiplex PCR for Mycobacterium paratuberculosis
Quantification of sncRNA715 in Mice Sciatic Nerve
Gene Expression Analysis by RT-PCR
Transcriptional Profiling of Oligodendrocytes
Quantitative PCR of Ca. Clavichlamydia salmonicola
The detection limit for Ca. Clavichlamydia salmonicola was 1000 copies due to increased variation between replicates in the standard curve for low DNA concentration (Ct SD >0.5) (Forootan et al., 2017 (link)). Prevalence at time point was determined by the percentage of positive samples per time point and infection intensity reflects the number of copies based on qPCR. Standard curve for quantification of Ca. Clavichlamydia salmonicola had a slope −2.974, ɣ‐intercept 39.7 and efficiency 116.8.
RNA Extraction and RT-PCR Analysis
Cardiac Tissue RNA Extraction and Gene Expression Analysis
The PCR was performed in a LightCycler 480-II Instrument (Roche, Basel, Switzerland) using TaqMan Universal Master Mix and specific Universal ProbeLibrary (UPL) probes (Roche) of each gene (MPC1, MPC2). PCR was performed in a 10 μl reaction mixture volume with 20 μM forward primer and 20 μM reverse primer for mRNA. The PCR cycling was as follows: a denaturing step of 10 min at 95°C, followed by 50 amplification cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s, followed by cooling to 40°C. Analysis of the results was carried out using Qbase + version 2.5 software and the relative expression levels of genes were estimated by the 2−ΔΔCT method (30 (link)) as the average value of each sample normalized to the geometric average of the housekeeping genes GAPDH, HPRT1, and ACTB, selected after doing a geNorm study. DNA primer sequences and UPL probes are described in
RT-PCR Analysis of Gene Expression
Leptospira DNA Detection via qPCR
The DNA templates obtained from the above protocol were analyzed in a qPCR system (Roche LightCycler 2.0) using a TaqMan probe targeting the LipL32 gene [22 (link)], which is specific only for pathogenic Leptospira spp. The primers lipL32-45F (5′-AAG CAT TAC CGC TTG TGG TG-3′) and lipL32-286R (5′-GAA CTC CCA TTT CAG CGA TT-3′) were used to amplify a fragment of 242 bp, which was detected by the above-mentioned probe, LipL32-189P (FAM-5′-AA AGC CAG GAC AAG CGCCG-3′-BHQ1). The amplification mixture for each sample contained 0.7 μM primers, 0.15 μM probe, 10 μL Master Mix TaqMan universal (Roche), and 5 μL DNA template in a total volume of 20 μL. Samples were amplified with the following program: (1) initial denaturation at 95 °C for 2 min, (2) 40 cycles of denaturation for 5 s at 95 °C, and (3) annealing/elongation for 30 s at 58 °C. The system contained a negative and positive control in order to survey the proficiency of the reaction in addition to negative and positive DNA controls.
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