Peripheral blood samples were obtained from each participant. Mononuclear cells were isolated by Ficoll-Paque separation. Genomic DNA was extracted using the Axygen DNA isolation kit (Axygen). The rs2057482 in the gene of HIF-1α were detected by polymerase chain reaction (PCR) using a Mastercycler®gradient thermal cycler (Eppendorf®, Germany). The PCR mix (25 μl) contained genomic DNA (500 ng), MgCl2 (2.5 mM), dNTP (100 mM), Taq polymerase (1.0 U/ml) and primers (forward 5′-TAC AAG GCA GCA GAA ACC-3′ and reverse 5′-TAA ACT CCC TAG CCA AAA-3′, 50 pmol/ml for each). After a 10 min denaturation at 94°C, the mix underwent 30 cycles of denaturation (94°C, 45 sec), annealing (60°C, 45 sec) and extension (72°C, 45 sec). PCR products were verified by agarose gel electrophoresis. DNA fragments were sequenced in Sangon Biological Engineering Technology and Services (Shanghai, China) using 5′-GTG GAT AGT GAT ATG GTC AAT G-3′ as the primer. Sequence analysis was performed using the program of BioEdit. When C/T genotypes were found, the analysis was repeated to double-check the results.
Axygen dna isolation kit
Manufactured by Corning
Sourced in United States
The Axygen DNA isolation kit is a laboratory product designed for the efficient extraction and purification of DNA from various sample types. It utilizes a simple and optimized protocol to isolate high-quality DNA that can be used in downstream applications such as PCR, sequencing, and other molecular biology techniques.
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Lab products found in correlation
2 protocols using axygen dna isolation kit
1
HIF-1α Gene Polymorphism Analysis
2
DNA Polymorphism Detection Protocol
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The blood samples were collected from each enrolled subjects. The genomic DNA was extracted from peripheral venous blood using the Axygen DNA isolation kit (Axygen, USA). DNA fragments containing the polymorphism were amplified with the forward primer 5'-CGCGGGAGTTCAGGGTAAAG-3' and 5'-AGC-TGGAGACAAGTCAGGACTTAAC-3'. The PCR reaction was carried out in a 20 ml reaction mixture containing 1× Phusion High-Fidelity PCR Master Mix (Thermo Scientific, Finland) and 0.25 mM of each primer. The PCR cycle consisted of an initial denaturation step at 98 ℃ for 30 s, followed by 35 cycles of denaturation (98 ℃ for 5 s), annealing (65 ℃ for 5 s) and extension (72 ℃ for 5 s), and a final extension at 72 ℃ for 5 min. The 237 bp amplified product was digested overnight with 1 U of MspA1I (BioLaps, New England) at 37°C. The wild-type allele T was identified by the presence of 237 bp band, while the mutant allele G was represented by 189 and 48 bp bands.
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