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5 protocols using dnase 1 and dnase 2

1

Quantitative Lipid Analysis Protocol

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Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA). Solvents (HPLC-grade) were purchased from Fisher Scientific. 2′-Deoxyadenosine monohydrate were purchased from Berry & Associates Inc. (Dexter, NY, USA). Isotopic labeled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG and 8-oxo-dA (see Supplementary Materials) were prepared according to the previously reported procedures [21 (link)]. Ultrapure water (18.3 MΩ.cm) distilled and deionized water (Milli-Q water) were purified by a Milli-Q system (Merck-Millipore, Bedford, OH, USA). Chloroform, methanol and n-hexane were purchased from Merck (HPLC-grade). Anhydrous sodium sulfate (Na2SO4) was purchased from Carlo Erba (Val de Reuil Cedex, France). All fatty acid methyl esters (FAME) used as reference standard for GC analyses were purchased from Sigma-Aldrich or Fluka (Steinheim, Germany) without further purification. Analytical silica gel thin-layer chromatography was performed on Merck silica gel 60 plates.
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2

Quantitative Analysis of Oxidative DNA Damage

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Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA). Chemicals for the synthesis of oligonucleotides were purchased from Sigma Aldrich, Fluka and Link Technologies. CuCl2, L-methionine, L-ascorbic acid and alkaline phosphatase were purchased from Sigma-Aldrich. Hydrogen peroxide (30%) and solvents (HPLC-grade) were purchased from Fisher Scientific. 2′-deoxyadenosine monohydrate and 2′-deoxyaguanosine were purchased from Berry & Associates Inc. (Dexter, USA). Isotopic labeled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG, 8-oxo-dG and 8-oxo-dA (see Supporting Information) were prepared according to the previously reported procedures [31 (link)]. Ultrapure water (18.3 MΩcm) distilled and deionized water (Milli-Q water) were purified by a Milli-Q system (Merck-Millipore, Bedford, OH, USA).
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3

Quantification of Nucleoside Modifications

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Nuclease P1 from Penicilliumcitrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxaminemesylate and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). 2′-Deoxyadenosine monohydrate and 2′-Deoxyguanosine monohydrate were purchased from Berry & Associates Inc. (Dexter, NY, USA). Isotopically labelled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG, 8-oxo-dA and 8-oxo-dG were prepared according to the previously reported procedures [40 (link)]. Solvents (HPLC-grade) were purchased from Fisher Scientific (Waltham, MA, USA). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA). Written consent was obtained from all participants. The approval by the ethics committee was obtained for IBD and obese patients (Istituto Superiore di Sanità, PROT. PRE 173/16; University of Rome Tor Vergata Register 169/15).
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4

Quantification of DNA Modifications

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Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate, and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). 2′-Deoxyadenosine monohydrate and 2′-Deoxyguanosine monohydrate were purchased from Berry & Associates Inc. (Dexter, NY, USA). Isotopic labelled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG, 8-oxo-dA, and 8-oxo-dG were prepared according to the previously reported procedures [38 (link)]. Solvents (HPLC-grade) were purchased from Fisher Scientific (Waltham, MA, USA). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA).
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5

Comprehensive Glioblastoma Cell Culture Protocol

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All reagents were obtained from Sigma–Aldrich (Steinheim, Germany) and solvents (chloroform, methanol, n-hexane) were purchased from Fisher Scientific (HPLC grade). Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate, and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was obtained from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A was obtained from Roche Diagnostic GmbH (Mannheim, Germany). 2′-Deoxyadenosine monohydrate were purchased from Berry & Associates Inc. (Dexter, NY, USA). All fatty acid methyl esters (FAME) used as references were commercially available from Supelco (Bellefonte, PA, USA) or Sigma–Aldrich. U87MG brain glioblastoma was obtained from the American Type Culture Collection (ATCC). High glucose Dulbecco’s modified Eagle Medium (DMEM) was purchased from Sigma. Trypsin-EDTA, L-glutamine, penicillin–streptomycin solution, and heat inactivated fetal bovine serum (FBS) were obtained from Biochrom KG. Ultrapure water (18.3 MΩ·cm) and deionized water (Milli-Q water) were purified by a Milli-Q system (Merck–Millipore, Bedford, PA, USA).
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