Extraction:Article Title: Analysis of DNA damage and repair in nuclear and mitochondrial DNA of animal cells using quantitative PCR
Article Snippet: .. Materials for manual method of extraction (Qiagen Genomic-tip protocol) Cell pellets of 1x10 6 cells/pellet or tissue sample QIAGEN Genomic-tips: 20/G for small pellets and tissues <20 mg (QIAGEN, cat. no. 10223), 100/G for tissue samples <100 mg (QIAGEN, cat. no. 10243) Associated buffers with Genomic-tip kit (QIAGEN, cat. no. 19060)(Buffers G2, QBT, QC and QF, RNase A and Proteinase K) Isopropanol Cold 70% ethanol in H 2 O 1X Tris-EDTA, pH 8.0 (Fisher; cat. no. BP2473-500) 15-mL conical tubes (four per sample, BD Falcon, BD Biosciences, Bedford, MA) 2.2 mL microcentrifuge tubes (two per sample) Water bath that can be set to 50°C Refrigerated microcentrifuge (Thermo Scientific) 2.1.2. .. Materials for automated method of extraction (QIAcube) Cell pellets of 1x10 6 cells/pellet or tissue sample <25 mg 1X PBS (Cellgro) QIAcube (QIAGEN, cat. no. 9001292) QIAcube materials (pipette tips, 2 mL screw-cap sample tubes, rotor adapters, 1.5 mL elution tubes)(QIAcube starter kit, QIAGEN, cat. no. 990395) For human samples, associated buffers and spin columns with the QIAamp DNA mini kit (QIAGEN, cat. no. 51304), and for animal samples, associated buffers and spin columns with the DNeasy blood and tissue kit (QIAGEN, cat. no. 69504) Ethanol, 200 proof 2.2 Pre-PCR DNA quantitation and post-PCR analysis PicoGreen dsDNA quantitation reagent from Molecular Probes (cat. no. P-7581) 1X Tris-EDTA, pH 8.0 (Fisher; cat. no. BP2473-500) Lambda (λ)/HindIII DNA (Gibco; cat. no. 15612-013) to generate a standard curve A fluorescence reader capable of measuring fluorescence with 485 nm excitation and 528 nm emission (BioTek Synergy 2 Multi-Mode microplate reader, BioTek Instruments), black-bottomed 96-well plates (Costar, Corning Inc., Corning, NY, cat. no. 3015), single-channel p10 and p200 pipettes, multichannel pipette and pipet basin 15-mL conical tube (four per sample, BD Falcon, BD Biosciences, Bedford, MA) 0.5-mL autoclaved microcentrifuge tubes (to dilute DNA for PicoGreen reads) 2.3 Restriction digest 225 ng purified DNA Restriction enzyme (mouse: HaeII, New England Biolabs, cat. no. R0107S; human: PvuII, New England Biolabs, cat. no. R3151S; rat: XhoI, New England Biolabs, cat. no. R0146S) and associated components (10X NEBuffer, 100X BSA) ddH 2 O 0.2 mL PCR tubes (USA Scientific) Thermocycler or another method of incubating samples at 37°C/56°C 2.4 QPCR GeneAmp XL PCR Kit (Applied Biosystems, cat. no. N8080193) Bovine Serum Albumin (Roche, cat. no. 10-711-454-001) dNTPs (GeneAmp dNTP Mix with dTTP, Applied Biosystems, cat. no. N8080261) Primer oligos 0.2 mL PCR tubes (USA Scientific) A 96-well format thermocycler (Biometra Professional Standard Thermocycler 96, Biometra) 2.5 Dedicated equipment and workstations (see Notes section for additional information) PCR enclosure with UV sterilization and, if possible, an air purifier (Labconco) Distinct, dedicated workstations (preferably in separate laboratories) for: (1) DNA isolation; (2) setting-up the PCR reactions; and (3) post-PCR analysis Pipettes and tips exclusively used for QPCR setup kept apart from pipettes and tips used for pre- and post-QPCR DNA analysis
|
Qiagen is a verified supplier 
Qiagen manufactures this product 