The total reaction volume for HRM analysis was 20 μl, containing 10 ng of genomic DNA, 2.0 μl of 10 × Easy Taq buffer (TransGen Biotech, Beijing, China), 1.0 μl of 2.5 mM dNTP mixture (TransGen Biotech), 0.1 μl of Easy Taq DNA polymerase (Transgen Biotech), 1.0 μl of SYTO®9 green fluorescent nucleic acid stain (Life Technologies™, Carlsbad, CA, United States), 0.5 μl each of 10 pmol μl−1 of a pair of primers (Supplementary Table S1 ), and autoclaved distilled water for the remainder of the volume. On the Biometra TAdvanced (Biometra GmbH, Göttingen, Germany), the following PCR conditions were used: initial denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, followed by annealing and elongation at 60°C for 20 s, repeated 40 times; and final denaturation at 95°C for 10 s. HRM analysis was performed using the LightCycler® 96 Real-Time PCR System (Roche, Basel, Switzerland) at each temperature during a 0.3% rise from 60 to 90°C. The HRM graphs were drawn by LightCycler® 96 software ver. 1.1 (Roche).
Dntp mixture
Manufactured by Transgene
Sourced in China
The DNTP mixture is a laboratory product that provides a combination of the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) necessary for various DNA-based applications, such as DNA synthesis, amplification, and sequencing. The product ensures a consistent and reliable source of these essential components for researchers and scientists working in the field of molecular biology and genetics.
Automatically generated - may contain errors
Lab products found in correlation
Easytaq dna polymerase, by Transgene (1 mentions)
Syto 9 green fluorescent nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96 real time pcr system, by Roche (1 mentions)
Easytaq buffer, by Transgene (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Rna extraction kit, by Promega (1 mentions)
Benz a anthracene, by Merck Group (1 mentions)
Pcr thermocycler, by Thermo Fisher Scientific (1 mentions)
Dna polymerase, by Transgene (1 mentions)
Pgl3 basic, by Promega (1 mentions)
3 protocols using dntp mixture
1
High-Resolution Melting Analysis of DNA
2
Benz[a]anthracene Extraction and Analysis
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Benz[a]anthracene was purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropyl-d-1-thiogalactopyranoside (IPTG), T4 DNA ligase, and the restriction enzymes were purchased from TaKaRa. An RNA Extraction Kit was purchased from Promega (Promega Corporation, Madison, WI, USA). Random primers, ribonuclease inhibitor, dNTP mixture, recombinant DNase I, and SYBR® Premix Ex Taq™ (Tli RNaseH Plus) were purchased from TransGen Biotech (TransGen Biotech, Beijing, China).
3
Cloning of ADFP Gene Promoter
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The promoter region of the ADFP gene was amplified from human genomic DNA using polymerase chain reaction (PCR) and cloned into a luciferase reporter vector (pGL3-Basic; Promega Corporation, Madison, WI, USA). Briefly, human genomic DNA was extracted using a Quick Genomic DNA Extraction kit (Guangzhou Dongsheng Biotech Co., Ltd., Guangzhou, China) according to manufacturer's instructions. A total of 50 ng genomic DNA was then used as a template to amplify the promoter region of the ADFP gene in 20 µl reaction system containing 1 µl of 10 µM primers (Sangon Biotech Co., Ltd. Shanghai, China), 1 µl of 25 µM dNTP mixture (Beijing TransGen Biotech Co., Ltd., Beijing, China) and 1 µl DNA polymerase (Beijing TransGen Biotech Co., Ltd.). The following primer sequences were used: ADFP, forward 5′-agacgcgtCATGCCTGGCTATTTAGTG-3′ and reverse 5′-ccctcgagCTCATGCCGGTAATCCCAGCA-3′. The PCR reaction was performed in a PCR Thermocycler (Thermo Fisher Scientific, Inc.) with the following reaction conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 1 min. As a positive control, the nucleotide sequence containing the identified HRE of the erythropoietin (EPO) gene (14 (link)) was also cloned into the pGL3-promoter.
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