Blood dna preparation kit

DNA extraction from peripheral blood will be performed using the commercial assay "Blood DNA Preparation Kit" (Jena Bioscience, PP205S) which is based on a cell lysis step to subsequently precipitate proteins, with subsequent hydration of the DNA obtained [89 ]. Once the DNA is extracted, it will be stored at -80°C until use. The determination of the Val66Met polymorphism of the BDNF gene will be studied by allele-specific PCR, for which specific primers will be designed to identify by conventional PCR each different type of alleles present (homozygous or heterozygous) [90 (link)].

From the preserved buffy coat, DNA was extracted utilizing the “Blood DNA Preparation Kit” from Jena Bioscience (PP205S) (Jena Bioscience, Jena, Germany). The Infinite^® 200 PRO NanoQuant assessed the DNA’s concentration and purity. The extracted DNA was then stored at −80 °C until needed.

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.