cDNA was synthesized by the Tetro cDNA Synthesis Kit (Meridian, TN, USA) according to the manufacturer’s protocol in the T100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). A mixture of specific forward and reverse primers, SYBR® Green Supermixes for Real-Time PCR (Bio-Rad Laboratories or Meridian USA) and samples were prepared. Amplifications were performed according to the manufacturer’s instructions using LightCycler 480II (Roche, USA). Primers (Table S1 ) were commercially manufactured (Sigma-Aldrich, St. Louis, MO, USA). The mRNA quantity was calculated relative to the expression of the average value of GeNorm of Cyclo, RPLP0, HPRT, B2M, GUSB, YWHAZ.
Sybr green supermixes for real time pcr
Manufactured by Bio-Rad
Sourced in United States
SYBR® Green Supermixes for Real-Time PCR are optimized reagent mixes designed for quantitative real-time PCR (qPCR) applications. The mixes contain SYBR® Green I dye, which binds to double-stranded DNA and emits fluorescence upon excitation, enabling detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
T100 thermal cycler, by Bio-Rad (2 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Primers table s1, by Merck Group (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
T4 ligase, by Takara Bio (1 mentions)
Na3vo4, by Merck Group (1 mentions)
Trizol, by Takara Bio (1 mentions)
Pyruvic acid sodium salt, by Merck Group (1 mentions)
Flag antibody, by GenScript (1 mentions)
Xav 939, by MedChemExpress (1 mentions)
3 protocols using sybr green supermixes for real time pcr
1
Quantitative Real-Time PCR Analysis
2
Quantitative RT-PCR Analysis of Adipocytes
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cDNA was synthesized by an iScript TM advanced kit (Bio-Rad, Hercules, CA, USA) (adipocytes) according to the manufacturer’s protocol in the T100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). A mixture of specific forward and reverse primers, SYBR® Green Supermixes for Real-Time PCR (Bio-Rad Laboratories or Meridian USA) and samples were prepared, and amplifications were performed according to the manufacturer’s instructions using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Primers (Table S1 ) were commercially manufactured (Biolegio BV, Nijmegen, Netherlands). The specificity and efficiency of the primers were tested by a temperature gradient, the melting curve was evaluated, and the optimum annealing temperature was determined. The mRNA quantity was calculated relative to the expression of β-actin.
3
Proteasome and Wnt Signaling Pathway Inhibition
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Proteasome inhibitor MG132, β-catenin inhibitor ICG-001 and XAV939 were purchased from MedChemExpress. Phosphatase inhibitor cocktail I (#C002) was purchased from Target Molecule Corp. Leupeptin, Na3VO4, pyruvic acid sodium salt (PN) and WSB1 antibody (#HPA003293) was purchased from Sigma–Aldrich. Non-essential amino acid and l -glutamine was purchased from Invitrogen (Thermo Fisher Scientific) and polyethylenimine 40000 (PEI-40000) was purchased from Polysciences. TRIzol, T4 ligase and KOD PCR kit were purchased from Takara Bio. SYBR Green supermixes for real-time PCR was purchased from BIO-RAD. The BCA protein quantitative kit was purchased from Shanghai Yisheng Biotechnology Co., Ltd. And c-Myc (#db1667), HA (#db2603), GAPDH (#db106), ubiquitin (#db935), GSK3β (#db2953) and CK1 (#db3190) antibodies were purchased from Diagnostic Biosystems. c-Myc (#sc-40) antibody for immunohistochemistry was purchased from Santa Cruz Biotechnology. Phosphatase PP1/PP2A inhibitor calyculin A (#9902), as well as p-β-catenin (#9561S), β-catenin (#9562S), AXIN1 (#2087S), β-TRCP (#4394S) and phospho-(Ser/Thr) Phe (#9631) antibodies were purchased from Cell Signaling Technology. FLAG antibody was purchased from GenScript. PPP2CA (I3482-I-AP), lamin B (66095-I-Ig) antibodies were purchased from Proteintech Group. WNT 3A conditional medium was generated according to previous report25 (link).
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