Sybr green supermix

Manufactured by Quantabio

Sourced in United States

SYBR Green SuperMix is a ready-to-use reaction mix for real-time quantitative PCR (qPCR) that contains SYBR Green I dye, a DNA-binding fluorescent dye, and all necessary components for qPCR.

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Lab products found in correlation

9 protocols using sybr green supermix

Decaffeinated Green Tea Extract Analysis

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Decaffeinated green tea extract (GTE) was a gift from Nature’s Sunshine Products, Inc. (Spanish Fork, UT, USA) and the composition is shown in Table S1. Experimental diets were purchased from Research Diets, Inc (New Brunswick, NJ, USA) and the formulations have been previously published [24 (link)]. Tri reagent was purchased from Sigma-Aldrich (St. Louis, MO). RT² HT First Strand Kit was purchased from Qiagen (Germany). Custom primers were purchased from Integrated DNA Technologies (Coralville, IA) and SYBR® Green SuperMix was purchased from Quantabio (Beverly, MA). All other chemicals were of the highest grade commercially available.

Khoo W.Y., Chrisfield B.J., Sae-tan S, & Lambert J.D. (2019). Mitigation of non-alcoholic fatty liver disease in high fat-fed mice by the combination of decaffeinated green tea extract and voluntary exercise. The Journal of nutritional biochemistry, 76, 108262.

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Quantifying Gene Expression by qRT-PCR

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Gene expression was quantified by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) as previously described [34 (link)]. In brief, liver RNA was extracted using TRI reagent (Sigma-Aldrich, St. Louis, MO) and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). cDNA was synthesized with the RT² HT First Strand Kit (Qiagen, Germany). After which, qRT-PCR was performed by using cDNA, custom primers (Integrated DNA Technologies, Coralville, Iowa) and SYBR® Green SuperMix (Quantabio, Beverly, MA) on a QuantStudio 12K Flex Real-Time PCR System according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA). Relative quantification of PCR products was accomplished using Thermo Fisher Connect™ (Thermo Fisher Scientific, Waltham, MA). Hepatic expression of genes involved in lipolysis, cholesterol synthesis and uptake and mitochondrial biogenesis were quantified relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) using a comparative method (2^−ΔΔCT). Primer sequences are available in Table S2.

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PCR and RT-PCR Amplification Protocols

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PCR was performed using red load Taq master according to the instructions of the manufacturer (Larova, Gena, Germany), with 400 nM forward and reverse oligonucleotide primers (Supplementary Table 1) and 10 ng cDNA or 20 ng gDNA template per reaction (in a total volume of 25 μl). RT-PCR was performed using the perfeCTa SYBR Green SuperMix according to the manufacturer’s instructions (Quanta bio, Beverly, MA, USA) with 150 nM forward and reverse oligonucleotide primers (Supplementary Table 1) and 5 ng cDNA or gDNA template per reaction (total volume—20 μl). All gene expression levels were normalized to glucuronidase beta (GUSB), and gDNA levels were normalized to folate receptor α (gFR-α). RT-PCR reactions were conducted using the 7300 Real-Time PCR System (Applied Biosystems, CA, USA) and results were analyzed with the 7300-system sequence detection software version 1.4 (Applied Biosystems). Each experiment was performed at least three times in triplicates.

Levin M., Stark M., Berman B, & Assaraf Y.G. (2019). Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine. Cell Death & Disease, 10(6), 390.

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RNA Isolation and Real-time PCR

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RNA isolation and purification were performed as previously described (Ramirez et al., 2015 (link)). A total of 1.0 μg of total RNA from HEK was reverse transcribed using a qScript cDNA kit and real-time PCR was performed in triplicate using the Bio-Rad CFX Connect thermal cycler and detection system (Bio-Rad Laboratories) and a PerfeCTa SYBR Green Supermix (QuantaBio, Beverly, MA). Relative expression was normalized for levels of HPRT1. The primer sequences are provided in Supplementary Materials. Statistical comparisons were performed using Student t test.

Jozic I., Vukelic S., Stojadinovic O., Liang L., Ramirez H.A., Pastar I, & Canic M.T. (2016). Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure. The Journal of investigative dermatology, 137(5), 1144-1154.

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Quantifying Bovine Cells and MAP Bacteria

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Genomic DNA was isolated from 3D enteroids, 2D monolayers and 3D apical-out enteroids infected with MAP, or uninfected controls, after the initial 1 h infection time point and 24 and 72 h post infection. In all instances the cells were treated with TrypLE express and incubated at 37°C for 10 min. Samples were suspended in 180 μL enzymatic lysis buffer containing 40 mg/mL lysozyme for 6 h at 37°C before being digested with proteinase K at 56°C for 2.5 h. The gDNA was then extracted using a Qiagen DNeasy Blood & Tissue Kit as described by the manufacturer's instructions.
To enumerate the number of bovine cells and MAP bacteria present in the samples, qPCR was performed using SYBR green Supermix (Quantabio, VWR international Ltd). Primers were designed against the bovine Spastin gene and the MAP F57 sequence element (Supplementary Table S1), both of which are single copy genes. Templates with a known concentration of Spastin and F57 were used to generate the standard curves, from which the number of bovine and MAP cells could be extrapolated. Negative controls (no cDNA) were included to verify the absence of contamination.

Blake R., Jensen K., Mabbott N., Hope J, & Stevens J. (2022). The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis. Frontiers in Veterinary Science, 9, 921160.

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Cell Viability Assay with DMEM/F12

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Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12, 1:1), phenol red-free DMEM, horse serum, penicillin/streptomycin, GlutaMAX, PrestoBlue® cell viability reagent, were obtained from Thermo Fisher Scientific (Rockford, IL, USA). All cell lines (MDA-MB-231, and MCF-10A), Eagle’s minimum essential medium (EMEM) and fetal bovine serum (FBS) were obtained from American Type Culture Collection (Manassas, VA, USA). Epidermal growth factor (EGF) was obtained from Peprotech (Rocky Hill, NJ, USA). Glucose, insulin, hydrocortisone, and cholera toxin were obtained from Millipore Sigma (Milwaukee, WI, USA). Alpha-difluoromethylornithine (DFMO) was obtained from Bachem Americas Inc (Torrance, CA, USA). One-step cDNA kit, and SYBR Green supermix were obtained from Quantabio (Beverly, MA, USA).

Capellen C.C., Ortega J.A., Morwitzer M.J., Tofilau H.M., Dunworth M., Casero RA J.r, & Chandra S. (2021). Hyperglycemic Conditions Proliferate Triple Negative Breast Cancer Cells: Role of Ornithine Decarboxylase. Breast cancer research and treatment, 190(2), 255-264.

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Quantitative RT-PCR Analysis of LUHMES Cells

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RNA was purified from 6–8 d differentiated LUHMES using TRIzol. One microgram of RNA was reverse-transcribed into cDNA using qScript cDNA SuperMix (Quantabio). qRT-PCR was performed on an ABI 7900HT using SYBR Green SuperMix (Quantabio). Relative expression levels were determined using the comparative threshold (ΔΔCT) method (Livak and Schmittgen 2001 (link)). Actin beta (ACTB) mRNA levels were used as a normalization control. qPCR primers sequences are provided in Supplemental Table S5.

Lalli M.A., Avey D., Dougherty J.D., Milbrandt J, & Mitra R.D. (2020). High-throughput single-cell functional elucidation of neurodevelopmental disease–associated genes reveals convergent mechanisms altering neuronal differentiation. Genome Research, 30(9), 1317-1331.

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RNA Extraction and RT-qPCR Analysis

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RNA was extracted using the DirectZol RNA kit (Zymo, R2062). The iScript Reverse Transcription SuperMix (Bio-RAD, 1708841BUN) was used to synthesize cDNA from 500ng RNA. The PerfecTa SYBR Green SuperMix (Quantabio, 95054-02K) was used in quantitative PCR reactions. Primers used in RT-qPCR were designed using primer-blast and listed in S4 Table.

Luo Z, & Farnham P.J. (2020). Genome-wide analysis of HOXC4 and HOXC6 regulated genes and binding sites in prostate cancer cells. PLoS ONE, 15(2), e0228590.

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Cell Culture and Viability Assay

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Dulbecco's Modi ed Eagle Medium/F12 (DMEM/F12, 1:1), phenol red-free DMEM, horse serum, penicillin/streptomycin, GlutaMAX, PrestoBlue® cell viability reagent, were obtained from Thermo Fisher Scienti c (Rockford, IL, USA). All cell lines (MDA-MB-231, and MCF-10A), Eagle's minimum essential medium (EMEM) and fetal bovine serum (FBS) were obtained from American Type Culture Collection (Manassas, VA, USA). Epidermal growth factor (EGF) was obtained from Peprotech (Rocky Hill, NJ, USA).
Glucose, insulin, hydrocortisone, and cholera toxin were obtained from Millipore Sigma (Milwaukee, WI, USA). Alpha-di uoromethylornithine (DFMO) was obtained from Bachem Americas Inc (Torrance, CA, USA). One-step cDNA kit, and SYBR Green supermix were obtained from Quantabio (Beverly, MA, USA).

Chandra S., Capellen C., Ortega J.Á.M., Morwitzer M.J., Tofilau H.M.N., Dunworth M., & Casero R.A. (2021). Hyperglycemic Conditions Proliferate Triple Negative Breast Cancer Cells: Role of Ornithine Decarboxylase.

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