Purelink genomic dna extraction mini kit

Cerebellar samples were genotyped for the SNP rs6971 using the PureLink™ Genomic DNA Extraction Mini Kit (ThermoFisher, K182001) to extract DNA as per manufacturers protocol. Purified genomic DNA concentration was established using the NanoDrop™ ND-1000 Microvolume Spectrometer and diluted to a final concentration of 0.9ng/µl in DNAse free water. The TaqMan^® SNP Genotyping assay kit (ThermoFisher, C_2512465_20), which contained forward/reverse primers and fluorescent VIC/FAM probes to correspond to A/G DNA bases, was used along with the 2X TaqMan^® Genotyping Master Mix (ThermoFisher, 4371353). The following cycle program was performed on the Applied Biosystems StepOnePlus™ Real-Time PCR system: 95 °C for 10min (HOLD) then 40 cycles of 95 °C × 15secs, 60 °C × 1min.

To genotype the MAOBrs1799836 SNP, we used a commercially available Taqman PCR assay on genomic DNA extracted from frozen cerebellar samples. Briefly, we purified genomic DNA from ≈ 25 mg of frozen cerebellum using the PureLink Genomic DNA Extraction Mini Kit (ThermoFisher Scientific, K182002) following manufacturer’s instructions. We measured the DNA concentration in a DS-11 spectrophotometer (DeNovix Inc) and prepared 1.8 ng/μL working dilutions for the Taqman PCR assay. The latter reaction volume was 25 μL comprising 1.25 μL of 20 × TaqMan MAOBrs1799836 genotyping assay (ThermoFisher Scientific, Assay ID C—8878790_10), 12.50 μL of 2 × TaqMan Fast Universal PCR Master Mix, no AmpErase UNG (Thermo Scientific, 4352042), and 11.25 μL of DNA sample (20 ng). DNA samples were run in duplicates in 96-well plates (Bio-Rad) in a Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following protocol: 95 °C × 10 min (ramp 1 °C/s), 95 °C × 15 s, and 60 °C × 1 min, for 45 cycles. Principal component analysis of VIC versus FAM fluorescence [corresponding to base A (major allele) vs. G (minor allele), respectively] enabled allele discrimination and genotype assignment (AA, AG, or GG).