Genomic dna buffer set kit

High-molecular-weight DNA is essential in order to efficiently amplify long genomic targets. We have found that the DNA purified using the QIAGEN Genomic Tip and Genomic DNA Buffer Set Kit (QIAGEN, cat, nos. 10323 and 19060, respectively) is of high quality and quite reproducible from sample to sample. In addition, the purified DNA is very stable, yielding comparable amplification over long periods of storage.
DNA template integrity is essential for the reliable amplification of long PCR targets [80 (link)]. Although various kits are commercially available for DNA isolations, procedures that involve phenol extraction should be avoided due to potential introduction of artifactual DNA oxidation. As mentioned above, we use a DNA extraction kit from QIAGEN, which, in our hands, gives rise to templates of relatively high molecular weight and highly reproducible yield. The protocol for DNA isolation is followed as suggested by the manufacturer. Note that when using the manual genomic-tip protocol, the tissue protocol is used irrespective of whether tissue or cells are being studied, since the protocol for DNA extraction of cultured cells involves isolation of nuclei and hence loss of mtDNA. Samples that cannot be processed immediately after experiments should be stored at −80 °C until DNA is extracted. See additional information in ^{Note 2}.

The PCR assay was a modification of Santos et al. (2006) . Total DNA was purified from cell samples using the Qiagen Genomic Tip and Genomic DNA Buffer Set Kit (Qiagen, Valencia, CA, USA). For SC27 cells, a small mitochondrial fragment (221-bp) was amplified and standardized to a 13.5-kb fragment from the nuclear-encoded gene β-globin. A mitochondrial fragment (235-bp) was amplified and standardized to a 12.5-kb fragment from the nuclear-encoded gene, Clusterin (TRPM-2), in rat hippocampal neurons and NSC. PCR products were normalized to control levels. The sequences for human β-globin (13.5-kb) and mtDNA fragment (221-bp) primer sets used are: β-globin forward 5′-CGAGTAAGAGACCATTGTGGCAG-3′, reverse 5′-GCACTGGCTTTAGGAGTTGGACT-3′; mtDNA fragment forward 5′-CCCCACAAACCCCATTACTAAACCCA-3′ and reverse 5′-TTTCATCATGCGGAGATGTTGGA-3′. The sequences for rat TRPM-2 (12.5-kb) and mtDNA fragment (235-bp) primer sets used are: TRPM-2 forward 5′-AGACGGGTGAGACAGCTGCACCTTTTC-3′, reverse 5′-CGAGAGCATCAAGTGCAGGCATTAGAG-3′; mtDNA fragment forward 5′-CCTCCCATTCATTATCGCCGCCCTTGC-3′, reverse 5′-GTCTGGGTCTCCTAGTAGGTCTGGGAA-3′.

Cells were seeded in 6-well plates at 1.5 × 10⁶ cells per well. After being treated, cells were trypsinized and washed once with PBS. Cell pellets were frozen at −80°C. Mitochondrial DNA was isolated using the Genomic-tip 20/G and the Genomic DNA Buffer Set Kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. Quantification of mtDNA lesions was carried out using mitochondrial-specific primers and the QPCR technique described by Santos et al. (Santos et al., 2006 (link)).

Quantitative PCR (qPCR) was used to assay _mtDNA damage as described previously³³ . Briefly, total DNA was isolated using Genomic-tips and the Genomic DNA Buffer Set Kit (Qiagen, Valencia, CA). The purified DNA was quantified fluorometrically using Pico Green dsDNA reagent (Molecular Probes, Life Technologies). The Platinum PCR Super Mix (Invitrogen) was used to amplify 20 ng genomic DNA. Specific primers were used to amplify a long fragment of the _mtDNA (8.9 kb) to determine its integrity, as well as a short fragment (139 bp) to monitor changes in _mtDNA copy number and to normalize the data obtained from amplification of the 8.9-kb fragment. Ratios of relative amplification were calculated to compare _mtDNA damage in irradiated ECs to that in non-irradiated ECs; these values were used to express the number of lesions present in DNA, assuming a Poisson distribution as previously described ³³ .