RNA was isolated from cell pellets using kit (catalogue no. 74104, Qiagen, USA), and then using a cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific, Inc.), cDNA was synthesized. Quantitative PCR was performed using HotStart-IT® FideliTaq™ PCR Master Mix (2X) (catalog no. 71156, Affymetrix, USA). The effect of Aps on the expression of isolated genes families involved in the oxidative stress [Cat, SOD1 (Cu-Zn-SOD), and SOD2 (Mn-SOD)], inflammation [TNF-α and IL-6], apoptosis [Cas-3, Bax, and Bcl2] was investigated. The used primers are shown in Table 1 . PCR conditions were adjusted according to Huang et al.[32 (link)] CFX96 real-time system (Bio-Rad Laboratories, Inc.) was used for the reaction. Transcript levels were calculated and normalized of the expression of the targeted genes to the expression of GAPDH (internal reference gene). Experiments were conducted in triplicates.
Hotstart it fidelitaq pcr master mix 2x
Manufactured by Thermo Fisher Scientific
Sourced in United States
HotStart-IT® FideliTaq™ PCR Master Mix (2X) is a ready-to-use, optimized PCR reaction mix that includes all the necessary components for performing PCR amplifications, except for the template DNA and primers. The mix contains a chemically-modified DNA polymerase that is inactive at lower temperatures, preventing non-specific amplification during setup.
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4 protocols using hotstart it fidelitaq pcr master mix 2x
1
Quantitative Analysis of Oxidative Stress, Inflammation, and Apoptosis Genes
2
CASR Gene Polymorphism PCR Amplification
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For a 25 µl PCR reaction, 2 µl genomic DNA (100 ng/µl), 12.5 µl HotStart-IT® FideliTaq™ PCR Master Mix (2X) (catalog no. 71156, Affymetrix, USA), 8.5 µl RNase free water, and 1 µl of (100 pmol/ul) of forward primer: 5’-CAAGGACCTCTGGACCTCCCTTTGC-3’ and reverse primer: 5’-GACCAAGCCCTGCACAGTGCCCAAG-3’) were used. The tubes that contain the PCR mixture were centrifuged at 5000 rpm for 10 min. The PCR thermocycler conditions were as follows: initial denaturation at 94°C for 5 minutes. Followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 68°C for 30 seconds, and finally an extension at 72°C for one minute. A final extension step was performed at 72°C for 5 minutes.12 (link) The samples were then run on 2% agarose gel to visualize the amplified PCR products (320 bp). The genotyping examination of all PCR samples for intron 4 polymorphism (rs3804594) in CASR gene was done by DNA sequencing technique using the sequencer (3130 Genetic Analyzer, serial number: 313001026). The sequencing was done at Centre of Excellence in Genomic Medicine Research (CEGMR) in King Fahd Medical Research Centre (KFMRC), KAU, Jeddah, Saudi Arabia.
3
Genotyping Genetic Variants by RFLP
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Extracted genomic DNA (100 ng/ml) was amplified in a 25 µl polymerase chain reaction (PCR) with 12.5 µl HotStart-IT® FideliTaq™ PCR Master Mix (2X) (Affymetrix/USB™, 71156, USA), 9.5 µl RNase free water (Affymetrix/ USB™, 7732-18-5, USA), and 1 µl of each primer (0.2 µmol). The primers and the PCR thermocycler reactions were previously published in Hajj et al. [29 (link)] for ApaI and BsmI, whereas for TaqI and FokI in Rizk et al., [21 (link)]. The amplified PCR products were genotyped with restriction fragment length polymorphism (RFLP) using appropriate restriction endonucleases from Thermo Fisher Scientific (Waltham, MA, USA) [FastDigest ApaI (FD1414), FastDigest TaqI, (FD0674), FastDigest Mva1269I (FD0964), and FastDigest FokI (FD2144)] following the manufacturer's instructions. The different genotypes for each variant were confirmed with 2% agarose gel electrophoresis and 10% of the samples were selected randomly for further DNA sequencing confirmation.
4
PCR Amplification and RFLP Analysis of CASR Gene Exon 7
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For a 25 µl PCR reaction, 2 µl genomic DNA (100 ng/µl), 12.5 µl HotStart-IT® FideliTaq™ PCR Master Mix (2X) (catalog no. 71156, Affymetrix, USA), 8.5 µl RNase free water, and 1 µl of (100 pmol/ul) of forward primer:5’-CTGAGCTTTGATGAGCCTCAGAAGGAC-3’and reverse primer: 5’-CACTGATGACAAGCTCTGTGAACTGGA-3’)were used. The tubes that contain the PCR mixture were centrifuged at 5000 rpm for 10 min. The PCR thermocycler conditions were as follows: initial denaturation at 94°C for 5 minutes. Followed by 35 cycles of denaturation at 94°C for one minute, annealing at 63°C for one minute, and finally an extension at 72°C for one minute. A final extension step was performed at 72°C for 5 minutes.13 (link)The samples were then run on 2% agarose gel with ethidium bromide to visualize the amplified PCR products (269 bp).The genotype distributions of exon 7 polymorphism (rs1801725) in CASR gene were determined by restriction fragments length polymorphism (RFLP) procedure. In Eppendorf tube, 10µl of PCR product, 17µl nuclease-free water, 2µl 10x FastDigest green buffer and 1µl of Thermo Scientific FastDigest Hin1I (catalog no. FD0474, Thermo Scientific, USA) were added. Then the mixture was mixed by pipetting gently, and was spin down for few seconds. The incubation was done at 37°C in a heat block for 20 minutes with no inactivation step.
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