Ab 2720 thermocycler

Genomic DNA was extracted from the peripheral blood leucocytes using magnetic bead technology (chemagic DNA Blood Kit special, Chemagen, Germany) on automated high throughput nucleic acid isolation platform (chemagic Magnetic Separation Module I, Chemagen, Germany).
The polymorphic regions of leptin and BMP4 genes were amplified by polymerase chain reaction (PCR) with primer sets previously designed by Mórocz et al. [20 (link)]. The PCR was carried out in a reaction mix of 20 μL containing 100 ng DNA and 10x Prime Taq buffer (Genet Bio, Korea), 10 mM dNTPs mixture (Genet Bio, Korea), 20 pmol forward and reverse primers (AlphaDNA, Canada), and 0.1 U Prime Taq DNA polymerase (Genet Bio, Korea). PCR amplifications were performed in an AB 2720 Thermocycler (Life Technologies, USA) with an initial denaturation at 94°C for five minutes and a final extension of seven minutes at 72°C. The following thermal cycle was repeated 30 times: denaturation at 94°C for 30 seconds, annealing at 55°C for leptin and 59°C for BMP4 for 30 seconds, and extension at 72°C for 30 seconds.
The PCR products were cleaved with the restriction enzymes: HhaI (NEB, USA) for leptin and MluCI (NEB, USA) for BMP4, according to the manufacturer's instructions, and the obtained fragments were separated on agarose 3% gel in VG-SYS Horizontal Electrophoresis System (Biochrom, UK).

Genomic DNA was extracted from the peripheral blood leucocytes using magnetic bead technology (chemagic DNA Blood Kit special, Chemagen) on automated high throughput nucleic acid isolation platform (chemagic Magnetic Separation Module I, Chemagen).
The polymorphic region of the IL-6 gene was amplified by polymerase chain reaction (PCR). The primer set [5 (link)] is listed in Table 1.
The PCR was carried out in a reaction mix of 20 μL containing 100 ng DNA and 10x Prime Taq buffer (Genet Bio), 10 mM dNTPs Mixture (Genet Bio), 20 pmol Forward and Reverse primers (AlphaDNA), and 0.1 U Prime Taq DNA Polymerase (Genet Bio). PCR amplification was performed in an AB 2720 Thermocycler (Life Technologies) with an initial denaturation at 94°C for five minutes and a final extension of seven minutes at 72°C. The following thermal cycle was repeated 30 times: denaturation at 94°C for 30 seconds, annealing for 30 seconds at temperature presented in Table 2, and extension at 72°C for 30 seconds.
The PCR product was cleaved with SfaNI restriction endonuclease (New England Biolabs), according to the manufacturer's instructions, and the restriction fragments were separated on agarose 3% gel in VG-SYS Horizontal Electrophoresis System (Biochrom). The lengths of the fragments representing the genotypes of IL-6 are presented in Table 2.

Genomic DNA was extracted from the peripheral blood leucocytes using magnetic bead technology (chemagic DNA Blood Kit special, Chemagen) on automated high throughput nucleic acid isolation platform (chemagic Magnetic Separation Module I, Chemagen).
The polymorphic regions of the genes were amplified by polymerase chain reaction (PCR). The primer sets are listed in Table 1.
The PCR was carried out in a reaction mix of 20 µL containing 100-ng DNA and 10x Prime Taq buffer (Genet Bio), 10 mM dNTPs Mixture (Genet Bio), 20 pmol Forward and Reverse primers (Alpha DNA), and 0.1 U Prime Taq DNA Polymerase (Genet Bio). PCR amplification was performed in an AB 2720 Thermocycler (Life Technologies) with an initial denaturation at 94°C for five minutes and a final extension of seven minutes at 72°C. The following thermal cycle was repeated 30 times: denaturation at 94°C for 30 seconds, annealing for 30 seconds at temperature presented in Table 2, and extension at 72°C for 30 seconds.
The PCR products were cleaved with the appropriate restriction enzymes (New England Biolabs), according to the manufacturer's instructions, and the restriction fragments were separated on agarose 3% gel in VG-SYS Horizontal Electrophoresis System (Biochrom). The restriction enzymes and the lengths of the fragments representing the genotypes are presented in Table 2.