Taqman environmental master mix 2

Manufactured by Thermo Fisher Scientific

Sourced in United States

TaqMan Environmental Master Mix 2.0 is a ready-to-use solution designed for real-time PCR applications. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, optimized for reliable and sensitive detection of environmental samples.

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TaqMan Master Mix (448 citations) Environmental master mix (4 citations) Master Mix II (4 citations) TaqMan Environmental Master Mix (3 citations) TaqMan environmental master mix (version 2.0) PCR buffer (3 citations) ABI TaqMan Environmental Master Mix 2.0 (2 citations)

39 protocols using taqman environmental master mix 2

Inhibition Testing and Quantification of Crayfish DNA

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The samples were tested for inhibition by performing a qPCR with a solution containing 3 μl of the synthetic gene (10⁻⁴ ng/μl), 3 μl of extracted DNA from each tank respectively, 12.5 μl of Taqman Environmental Master Mix 2.0 (Life Technologies^®), 1 μl of each primer for the synthetic gene, and 1 μl of probe. Thermal conditions were 50°C for 5 min and 95°C for 10 min, followed by 55°C cycles of 95°C for 30 s and 60°C for 1 min. All samples were run in duplicate, if one of these showed a quantification cycle value different to the expected value, the sample was considered inhibited and so a twofold dilution of the sample DNA was required (modified from Biggs et al., 2015).
Once the samples had been tested for inhibition, the extracted DNA was diluted if needed and amplified by qPCR using the P. leniusculus primers and probe. The qPCR solution contained 3 μl of template DNA, 12.5 μl of Taqman Environmental Master Mix 2.0 (Life Technologies^®), 1 μl of each primer (10 μmol/L), and 1 μl of probe (2.5 μmol/L). Thermal conditions were 50°C for 5 min and 95°C for 10 min, followed by 55°C cycles of 95°C for 30 s and 62°C for 1 min. Samples were run in triplicate, and standards of known DNA concentrations, 10⁻¹ to 10⁻³ ng/μl, were also run in triplicate on each plate along with three nontemplate controls containing nuclease‐free water instead of DNA.

Dunn N., Priestley V., Herraiz A., Arnold R, & Savolainen V. (2017). Behavior and season affect crayfish detection and density inference using environmental DNA. Ecology and Evolution, 7(19), 7777-7785.

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Optimized eDNA Detection Protocol for Crayfish

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The optimized qPCR protocol used for crayfish eDNA detection and quantification for all assays is as follows: One qPCR reaction of 25 μL contained 10 μL TaqMan® Environmental Master Mix 2.0 (Life Technologies), 7 μL ddH₂O, 1 μL of each primer and probe (1.2 μM and 0.1 μM, respectively) and 5 μL of extracted DNA template. Thermal settings includes 1) 5 min initial denaturation at 50°C, then 10 min at 95°C, 2) 50 cycles at 95°C for 30 s, annealing at 60°C for 1 min, 3) final extension for 10 min at 72°C.
We prepared standard dilution series of target DNA with known copy numbers, and established limit of detection (LOD) and quantification (LOQ) for the crayfish eDNA analyses. This was done using two different, but comparable, approaches in the two involved laboratories in Denmark (University of Copenhagen) and Norway (Norwegian Veterinary Institute) (Table E in S1 File). Below, these two approaches are described separately.

Agersnap S., Larsen W.B., Knudsen S.W., Strand D., Thomsen P.F., Hesselsøe M., Mortensen P.B., Vrålstad T, & Møller P.R. (2017). Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples. PLoS ONE, 12(6), e0179261.

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Viral Concentration and Detection from Environmental Water Samples

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At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.5–2 L water samples as previously described [17 (link)] and using glass fibre prefilters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18 (link),19 (link)], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR.
At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, otherwise the protocol was as described in Maunula et al. [14 (link)]. As a modification, Taqman primer–probe sets were applied as published in ISO/TS 15216–2 [20 ] for norovirus GI and GII. Mengovirus was added as a process control.
MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 9308–2 and ISO 7899–2, respectively [21 ,22 ].

Kauppinen A., Al-Hello H., Zacheus O., Kilponen J., Maunula L., Huusko S., Lappalainen M., Miettinen I., Blomqvist S, & Rimhanen-Finne R. (2017). Increase in outbreaks of gastroenteritis linked to bathing water in Finland in summer 2014. Eurosurveillance, 22(8), 30470.

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Real-Time PCR of Prostaglandin Pathway

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Real-Time PCR was performed by using TaqMan® Environmental Master Mix 2.0 (Life Tech, Grand Island, NY). A mixture of cDNA from the sample pool was used to construct a standard curve for each gene. All samples and standard curves were run in duplicate in the real-time PCR reactions. The primers and probes used for real time PCR were purchased from Applied Biosystems (Foster City, CA, USA). The primers used were as follows: COX-1 (PTGS1), Hs00377726_ml), COX-2 (PTGS2, Hs00153133_m1), mPGES1 (PTGES1, Hs01115610_ml), cPGES (PTGES3, Hs00832847_gH), 15 HPGD (HPGD, Hs00168359_ml), EP2 (PTGER2, Hs04183523_m1), and EP4 (PTGER4, Hs00168761_m1). Cytokeratin 20 Krt20, a marker of colonic epithelial cell mass, was used as an internal control for normalization (Hs00300643_m1) (19 (link)). The real-time PCR thermal conditions were: 50°C 2 min, 95°C 10 min followed by 40 cycles of 95°C 15 sec and 60°C 1 min.
The mean efficiency for the PCR standard curve for each primer was: PTGS1 (95%), PTGS2 (90.4%), PTGES (105%), PTGES3 (97.2%), HPGD (99%), PTGER2 (93%), PTGER4 (94%), and Krt20 (94%). For quantification, the standard curve method was used, and the average amount of each target mRNA expression and Krt20 mRNA expression was established from the standard curve. Each target gene expression was then normalized by Krt20 expression.

Sidahmed E., Sen A., Ren J., Patel A., Turgeon D.K., Ruffin M.T., Brenner D, & Djuric Z. (2016). Colonic Saturated Fatty Acid Concentrations and Expression of COX-1, but not Diet, Predict Prostaglandin E2 in Normal Human Colon Tissue. Nutrition and cancer, 68(7), 1192-1201.

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Quantification of Lso in Psyllid and Potato

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Absolute quantification method was used to quantify Lso copy numbers in both the potato psyllid vector and potato tuber tissue. The qPCR was performed with 15 μl of reactions containing TaqMan® Environmental Master Mix 2.0 (Life Technologies, Carlsbad, CA), 400 nM primer LsoF (5ʹ-GTC GAG CGC TTA TTT TTA ATA GGA-3ʹ; [24 (link)]), 400 nM primer HLBr (5 ʹ-GCG TTA TCC CGT AGA AAA AGG TAG-3ʹ; [25 (link)]), 200 nM probe HLBp (6FAM 5 ʹ - AGA CGG GTG AGT AAC GCG -3', TAMRA, Life Technologies, Carlsbad, CA; [25 (link)]), and 1 μl of DNA template. Both forward and reverse primers were synthetized by the Integrated DNA Technologies, Inc., Skokie, IL. The real time PCR program consisted of 1 cycle at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Reactions were performed in a CFX Connect Real Time System (Bio Rad, Hercules, CA, USA). A positive control (DNA of Lso positive), a negative control (DNA of healthy tuber), and water control (no template control, NTC) were included in all qPCR.

Rashidi M., Novy R.G., Wallis C.M, & Rashed A. (2017). Characterization of host plant resistance to zebra chip disease from species-derived potato genotypes and the identification of new sources of zebra chip resistance. PLoS ONE, 12(8), e0183283.

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Quantitative PCR for stx and 16S rRNA

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qPCR assays for stx and 16S rRNA genes were performed as previously described^{18 (link),52 (link)} under standard conditions in a Step One RT PCR System (Applied Biosystems). Genes were amplified in a 20 μl reaction mixture with the TaqMan Environmental Master Mix 2.0 (Thermo Fisher Scientific. Waltham, MA). The reaction contained 1 μl of the DNA sample or quantified plasmid DNA^{18 (link),52 (link)}. All samples were run in triplicate, as well as the standards and negative controls. Gene copy number (GC) was defined as the mean of the triplicate data obtained.

Blanco-Picazo P., Fernández-Orth D., Brown-Jaque M., Miró E., Espinal P., Rodríguez-Rubio L., Muniesa M, & Navarro F. (2020). Unravelling the consequences of the bacteriophages in human samples. Scientific Reports, 10, 6737.

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Standardized Multiplex qPCR Protocol

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All participants received standardized protocols, including detailed instructions for completing the study. Laboratories were supplied with sterile PCR-grade OmniPur water (VWR, Radnor, PA), 1.7 ml Axygen^™ MaxyClear microcentrifuge tubes (Thermo Fisher Scientific, Grand Island, NY), SRM 2917 (NIST, Gaithersburg, MD), internal amplification control (IAC) for multiplex HF183/BacR287, HumM2, and CowM2 qPCR assays (10² copies/2 μL), a 1.5 mL aliquot of 2 mg/ml bovine serum albumin fraction V stock solution (Thermo Fisher Scientific), primer/hydrolysis probe stock solutions for each qPCR assay (Table 1), MicroAmp^™ optical 96-well reaction plates (Thermo Fisher Scientific), MicroAmp^™ optical adhesive film (Thermo Fisher Scientific), and TaqMan^™ Environmental Master Mix 2.0 (Thermo Fisher Scientific). Participants were required to use a StepOnePlus^™, 7500, 7500 Fast Dx, 7500 Fast^™, QuantStudio^™ 3, QuantStudio^™ 5, or QuantStudio^™ 7 Pro Dx real-time PCR system (Thermo Fisher Scientific). Using the required supplies, participants were instructed to (i) generate six calibration curves for each qPCR assay on separate instrument runs and (ii) submit data to the EPA (Cincinnati, OH). Participants were instructed to perform all experiments within 30 days.

Sivaganesan M., Willis J.R., Karim M., Babatola A., Catoe D., Boehm A.B., Wilder M., Green H., Lobos A., Harwood V.J., Hertel S., Klepikow R., Howard M.F., Laksanalamai P., Roundtree A., Mattioli M., Eytcheson S., Molina M., Lane M., Rediske R., Ronan A., D’Souza N., Rose J.B., Shrestha A., Hoar C., Silverman A.I., Faulkner W., Wickman K., Kralj J.G., Servetas S.L., Hunter M.E., Jackson S.A, & Shanks O.C. (2022). Interlaboratory performance and quantitative PCR data acceptance metrics for NIST SRM® 2917. Water research, 225, 119162.

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Quantifying Theileria orientalis Infection

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Real-time PCR targeting the T.orientalis MPSP gene was completed on DNA extracted from blood of the four steers and from salivary glands of ticks fed on the three transmission-fed steers. PCR reaction components consisted of TaqMan™ Environmental Master Mix 2.0 (Thermo Fisher Scientific), 300 µM of Forward Primer [34 (link)] (5′-GCA AAC AAG GAT TTG CAC GC-3′), 300 µM of Reverse Primer (5′-TGT GAG ACT CAA TGC GCC TAG A-3′), 100 µM of Probe (5′-NED–TCG ACA AGT TCT CAC CAC-MGB-NFQ-3′), and 2 µL of DNA in a 20 µL reaction. Serial ten-fold dilutions of plasmid DNA pASK-IBA2 (IBA Life Sciences, Goettingen, Germany) ligated with a partial T.orientalis Ikeda MPSP gene were included in the assay as a standard curve. qPCR was run on a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) using standard mode and the following cycling conditions: 10 min at 95C followed by 45 cycles of 15 s at 95C and 1 min at 60C (Additional file 2: table S1).

Dinkel K.D., Herndon D.R., Noh S.M., Lahmers K.K., Todd S.M., Ueti M.W., Scoles G.A., Mason K.L, & Fry L.M. (2021). A U.S. isolate of Theileria orientalis, Ikeda genotype, is transmitted to cattle by the invasive Asian longhorned tick, Haemaphysalis longicornis. Parasites & Vectors, 14, 157.

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Real-Time PCR Amplification of Environmental Samples

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The real-time PCR was carried out using the Applied Biosystems™ QuantStudio™ 6 Pro Real-Time PCR System (Thermo Fisher Scientific Waltham, MA, USA) in an optical 96-well reaction plate (0.2 mL) sealed with optical adhesive film (Thermo Fisher Scientific, Waltham, MA, USA). The PCR was performed in a total volume of 25 μL, with the reaction mixture consisting of 12.5 μL of TaqMan™ Environmental Master Mix 2.0 (Thermo Fisher Scientific, Waltham, MA, USA), 4.75 μL of nuclease-free water, 2.5 μL of 10x primer/probe mix (Table 2), 0.25 μL of Uracil-DNA glycosylase (UNG) (New England Biolabs, Ipswich, MA, USA), and 5 μL of isolated DNA. The amplification program included a 2 min hold at 50 °C, an initial denaturation step at 95 °C for 10 min, followed by 45 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min.

Wang Y., Teo E., Lin K.J., Wu Y., Chan J.S, & Tan L.K. (2023). Quantification of Pork, Chicken, Beef, and Sheep Contents in Meat Products Using Duplex Real-Time PCR. Foods, 12(15), 2971.

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eDNA Extraction and qPCR for Bonamia

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DNA from whole filters was purified with the DNeasy PowerWater Kit (cat.no. 14900-100-NFm, QIAGEN, Denmark) according to the manufacturer (eluted in 50 µL), validated on a nanodrop, and subsequently stored at − 20 °C. Like for PCR, purification was conducted in a dedicated room and qPCR was set up in a different room. Filter tips were used at all time points and decontamination procedures were carefully and regularly conducted. The qPCR method was chosen due to high sensitivity and low concentrations of eDNA (30–280 ng/µL) from the filters. Our qPCR assays included already published Bonamia sp. specific primer sets and probes for ITS1 and 18S (Table 1) and we furthermore designed primers for ELF 1α from O. edulis as previously described²⁴. A TaqMan Environmental Master Mix 2.0 was used for the qPCR. All qPCR reactions except for ELF 1α were run twice and the Ct cut off value was set at 37. qPCR reactions were as follows: 5 µL sample, 10 µM primers and 5 µM probes, TaqMan Environmental Master Mix 2.0 (cat.no. 4396838, Thermo Fisher Scientific, Denmark). The qPCR conditions consisted of 10 min of pre-denaturation at 95 °C, 45 cycles of 15 s denaturation at 95 °C and 1 min elongation at 60 °C. A positive and a negative control (as described above for PCR) for B. ostreae and three no template controls were included in all runs.

Jørgensen L.V., Nielsen J.W., Villadsen M.K., Vismann B., Dalvin S., Mathiessen H., Madsen L., Kania P.W, & Buchmann K. (2020). A non-lethal method for detection of Bonamia ostreae in flat oyster (Ostrea edulis) using environmental DNA. Scientific Reports, 10, 16143.

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