Genomic dna buffer

Tissues were homogenized in a glass homogenizer and DNA was isolated using the QIAGEN Genomic-tip Kit and Genomic DNA Buffer (QIAGEN, Hilden, Germany). The amount of DNA in all cases was determined by its reaction with the PicoGreen reagent according to the manufacturer’s protocol (Molecular Probes Inc., Eugene, OR, USA) and fluorescence was registered on an NanoQuant Infinite M200 instrument (Tecan Group Ltd., Grödig/Salzburg, Austria). DNA samples for mitochondrial genome PCR-analysis were incubated within 20 min at 25 °C in TE buffer with XhoI restriction endonuclease (New England Biolabs, Ipswich, MA, USA). XhoI endonuclease initiates a break at the site of the CTCGAG hexamer of the supercoiled mtDNA outside the amplified region and leads to relaxation of the mtDNA, making the selected region available for PCR [16 (link)].

Frozen tissue samples from three brain regions were thawed at room temperature before isolation of total DNA (nDNA and mtDNA), and then placed on ice. Then, brain tissue samples were disrupted with the glass homogenizer and DNA isolated using the QIAGEN Genomic Tip Kit and Genomic DNA Buffer (Qiagen, Hilden, Germany). The DNA quantity in all cases was determined by reaction with the PicoGreen reagent according to the manufacturer’s protocol (Molecular Probes, USA), with the registration of fluorescence on an Infinite 200 NanoQuant device (Tecan Group Ltd., Austria). DNA samples intended for analysis of the mitochondrial genome were incubated for 20 min at 25 °C, in TE buffer with restriction endonuclease XhoI (New England Biolabs, cat.№. R0146S). XhoI endonuclease initiated a break at the site of the CTCGAG hexamer of the super-helical mtDNA of the rat, outside of the amplified fragment, leading to relaxation of mtDNA, making the selected region accessible for PCR amplification [48 (link)].

Genomic DNA was purified using Qiagen Genomic-Tip and Qiagen Genomic DNA buffer set and was digested with 0.5 U Escherichia coli RNase HII (New England Biolabs) in 50 µl at 37°C for 2.5 h. After precipitation in 0.3 m sodium acetate, pH 7, and ethanol, DNA was resuspended in TE 0, 1%. Nick translation was performed using 5 U of E. coli DNA polymerase I (New England Biolabs), 20 µm of unlabeled dA/-T/-GTP, and 5 µC α³²PdCTP in a final volume of 20 µl. The reaction was incubated at 16°C for 30 min. Labeled DNA was separated from unincorporated nucleotides by electrophoresis on a 1% TAE agarose gel. After drying, the radioactive gel was analyzed using a Typhoon. The relevant ³²P-signal of each sample was normalized on total genomic DNA measured by ethidium bromide staining. The ratio between E. coli RNHII-treated and untreated sample was expressed as fold change respect to the control sample.