Human genomic DNA was separated from peripheral blood samples using a nucleic acid extraction automatic analyzer (Lab-Aid 820, Xiamen, China), and all DNA samples were stored in TE buffer. Genotyping was done by Tm-shift polymerase chain reaction (PCR) (17 (link)). The sequences of the primers were 5’-gcgggcagggcggcTCACCATCTGATGTACTGTTTTCCTc-3’ (AS1 primer), 5’-gattaccgTCACCATCTGATGTACTGTTTTCCTg-3’ (AS2 primer), and 5’-TTCATGGAACTTGAAGTCTAGTGGGGA-3’ (common primer). PCR amplification was performed on ABI GeneAmp® PCR System 9700 Dual 384-Well Sample Block Module (Applied Biosystems, Foster City, CA). The PCR process included an initial denaturation stage at 95°C for 30 s, followed by 40 cycles of 95°C for 30 s, 59°C for 30 s, 72°C for 30 s, and a final extension stage at 72°C for 5 min. Then, melting curve analysis was conducted on a Roche LightCycler 480® fluorescence quantitative PCR instrument (Roche, Basel, Switzerland). The process included using temperatures of 95°C of 15 s and 60°C of 30 s and then increasing the temperature by 0.11°C/s to 95°C. Melting curve data were acquired by Air borne software provided by Roche automatic clustering based on fluorescence intensity analysis (18 (link)).
Abi geneamp pcr system 9700 dual 384 well sample block module
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI GeneAmp® PCR System 9700 Dual 384-Well Sample Block Module is a laboratory instrument designed for conducting polymerase chain reaction (PCR) experiments. It features a dual 384-well sample block that allows for the simultaneous processing of two sets of 384 samples. The module is compatible with the ABI GeneAmp® PCR System 9700 platform.
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2 protocols using abi geneamp pcr system 9700 dual 384 well sample block module
1
Genotyping by Tm-shift PCR
2
SNP Genotyping from Blood Lymphocytes
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Genomic DNA was isolated from peripheral blood lymphocytes using a nucleic acid extraction automatic analyzer (Lab-Aid 820, Xiamen, China). PCR was performed on the ABI GeneAmp PCR System 9700 Dual 384-Well Sample Block Module (Applied Biosystems, Foster City, CA, USA). PCR conditions included an initial denaturation of 95°C for 2 min, followed by 45 cycles of 95°C for 30 sec, 56°C for 30 s, 72°C for 1 min and then a final extension at 72°C for 5 min. After purification by SAP Reaction, we proceeded with primer extension. The primer extension protocol included an initial denaturation at 94°C for 30 s, followed by 40 cycles of amplification (including 94°C for 5 s, 52°C for 5 s, 80°C for 5 s), 5 cycles of amplification (5 s at 52°C, 5 s at 80°C), a final extension at 72°C for 3 min after which samples were held at 4°C. Single nucleotide polymorphism genotyping was performed using the Sequenom Mass-ARRAY iPLEX platform per the manufacturer's instructions [28 ]. The primer sequences were 5’- ACGTTGGATGAGCATTTGGGCTTGCTCTCC-3’ (first primer), 5’-ACGTTGGATGTCTGAGCCCCAGGAACTGGA-3’ (second primer) and 5’- caGAACTGGAGCGAAAGT-3’ (extended primer).
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