Gelgreen dna stain

The participants were genotyped for DRD2 Taq1A polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis described previously by Olasore et al.^{39 (link)}
However, with some modifications. The sequences of the primer set used are as follows: forward: 5′-CCG TCG ACG GCT GGC CAA GTT GTC TA-3′ and reverse: 5′-CCG TCG ACC CTT CCT GAG TGT CAT CA-3′. PCR was carried out in a reaction volume of 20 µl, containing 10 µl of 2× master mix with standard buffer (New England Biolabs, USA), 2 µl each of forward and reverse primers, 2 µl of nuclease-free water, and 4 µl of genomic DNA. The PCR cycling profile was initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 1 min, and extension at 72°C for 2 min, and then a final extension at 72°C for 2 min.
The PCR products (15 µl) were digested for 12 h at 65°C with five units of Taq1 restriction enzyme (New England Biolabs, USA). The products were electrophoresed on 2.5% gels stained with GelGreen^® DNA stain (Biotium, USA). The expected fragments were 310 bp for the whole amplified gene region, which meant the Taq1 restriction site was absent (A1 allele), while the presence of 180 and 130 bp fragments meant the site was present (A2 allele).

Genomic DNA was extracted from blood samples using a Blood DNA Preparation Kit
(Jena Bioscience, Germany). Genotyping was carried out as previously described
by Bhaskar et al^{12 (link)} with some modifications, using the following pair of
primers: forward—5′-TGTGGTGTAGGGAACGGCCTGAG-3ʹ and
reverse—5ʹ-CTTCCTGGAGGTCACGGCTCAAGG-3ʹ. The PCR reaction was performed with 5 μl
of 5× Red Load Taq Master (Jena Bioscience, Germany) and 50 ng of template DNA,
2 μl each of reverse and forward primers. The reaction mixture volume was made
up to 25 μl with PCR-grade water (Jena Bioscience, Germany). The PCR consisted
of an initial 2 cycles of touchdown PCR cycles with a denaturation temperature
of 94°C and 2 annealing temperatures of 66°C and 64°C each. These were then
followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 62°C
for 1 minute, extension at 72°C for 1 minute, and final extension at 72°C for
2 minutes. The PCR product was afterward resolved on 2.5% agarose gel stained
with GelGreen^® DNA stain (Biotium, USA). The expected fragments were
480 and 440 bp for 10 and 9 repeats, respectively.