Puregene genomic dna purification kit

DNA from whole blood was either isolated on the BioRobot Universal with the QIAamp DNA Blood BioRobot MDx Kit (Qiagen, Hilden, Germany) and eluted in Qiagen buffer AE (10 mM Tris-Cl 0.5 mM EDTA; pH 9.0), or with the Gentra Autopure LS machine using the Puregene Genomic DNA purification Kit (Gentra Systems, Minneapolis, MN 55441 USA), or manually using the MasterPure TM DNA Purification Kit for Blood Version II (Epicentre® Biotechnologies, Madison, WI, USA).
Fresh frozen tumor tissue was cut with scalpel, and one piece was used for DNA isolation using the Maxwell® 16 instrument (Promega, Madison, WI, USA) and the Maxwell® 16 tissue DNA Purification Kit (Promega). DNA was isolated according to the manufacturer’s protocol. The isolation procedure is automated, starting with sample lysis and tissue homogenization, followed by bead isolation of DNA, and finally the washing steps. The DNA was eluted in TE buffer (pH 8.5). DNA was stored at −20°C. DNA concentration and quality were measured using NanoDrop® ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).

Chromosomal DNA was extracted from cells growing exponentially by the Puregene Genomic DNA Purification kit (Gentra Systems, Minneapolis, MN). Plasmids were purified by a QIAprep Kit [QIAGEN Inc., Valencia, CA 91355]. PCR reactions were conducted in a MasterCycler Personal (Eppendorf AG, Hamburg, Germany). Reagents included Taq DNA polymerase from Roche (Branford, CT), deoxyribonucleotides from Sigma-Aldrich (St Louis, MO) for short sequences and the RangerMix from Bioline (Taunton, MA) for amplification of fragments longer than 6 kbp. Transformations were performed by standard methods [102 (link), 103 ].

Animal studies were designed and performed as per the statement issued by the Association for Research in Vision and Ophthalmology (ARVO) for the recommended use of animals in ophthalmic and vision research. The experimental protocols were approved by the University of Delaware Institutional Animal Care and Use Committee (IACUC). Tdrd7-targeted germline knockout (KO) mouse line (Tdrd7^tm1.1Chum, hereafter referred to as Tdrd7−/−) (32 (link)) was used in this study. Mice were housed in a 14-h light to 10-h dark cycle. Mouse-tail tissue genomic DNA was isolated using the Puregene® Genomic DNA Purification Kit (Gentra Systems, Minneapolis, MN). Genotyping was performed using the following PCR primer sets: Tdrd7-WT-g-F 5′-GAG TAA CTC TGG GCG CAG TC-3′, Tdrd7-WT-g-R 5′-GCC ATA GCA ATC AGT GAG CA-3′, expected product size 250 bp; and Tdrd7-KO-g-F 5′ GTC TAA CCC ATT CAG GGA TGA AGA 3′, Tdrd7-KO-g-R 5′ GAA TCC TCA CCA GTT AGC CTC ACC 3′, expected product size 500 bp, as previously described (32 (link)). Unless otherwise noted, Tdrd7+/− mice, which do not exhibit discernable ocular defects, were used as a control. Wild-type ICR outbred mice (Harlan Laboratories, Frederick, MD) were used for collecting lens tissue in RNA immunoprecipitation (RIP) assays.

Tumor tissues were manually microdissected from formalin-fixed paraffin embedded tissue sections obtained from the files of the Department of Pathology, Institute of Oncology and Radiology of Serbia.
DNA was extracted from four 10-µm sections using the Puregene Genomic DNA purification kit (Gentra Systems, Qiagen, Minneapolis, MN, USA). Total RNA was extracted from three 10-µm sections using Recover All Total Nucleic Acid Isolation Kit optimized for FFPE samples (Ambion, Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocols. DNA and RNA were quantified with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, USA).