A widely accepted sensitive marker of oxidative DNA damage and oxidative stress is 8-OHdG. The determination of DNA damage using 8-OHdG began with the isolation of genomic DNA using a DNeasy kit (Qiagen Inc.) following the manufacturer's instructions. The concentration was determined using Nanodrop. DNA was briefly digested then 5 µg/µl (total DNA 200 µg) was incubated with 100 units of DNase I (Bio Basic, Inc) in 40 µl Tris/hydrochloric acid 10 mM and 10 µl of 0.5 M MgCl2 (final concentration of 20 mM) at 37°C for 1 h. The pH of the reaction mixture was then lowered with the addition of 15 µl of 0.5 M sodium acetate (pH 5.1), 10 µl of nuclease P1 (Bio Basic, Inc.) and 30 µl of 10 mM ZnSO4, and the mixture was incubated for 1 h. After readjusting the pH with 100 µl of 0.4 M Tris/HCl (pH 7.8) and 20 µl alkaline phosphatase (Bio Basic, Inc.), the samples were incubated at 37°C for 30 min then boiled for 10 min. The resultant DNA hydrolysate was used for experimentation using the 8-OH-dG ELISA kit (cat. no. ab201734; Abcam) according to the manufacturer's protocol.
Dnase 1
Manufactured by Bio Basic
Sourced in Canada, United States
DNase I is an enzyme that catalyzes the hydrolytic cleavage of DNA. It cleaves the phosphodiester bonds in the DNA backbone, effectively degrading DNA molecules. The enzyme is commonly used in molecular biology and biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Rnase a, by Bio Basic (2 mentions)
Total rna mini preps kit, by Bio Basic (2 mentions)
Rg 3000a r, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Dneasy kit, by Qiagen (1 mentions)
Antarctic phosphatase, by New England Biolabs (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
Lysozyme, by Bio Basic (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Lymphocyte separation medium, by MP Biomedicals (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Potassium phosphate, by Merck Group (1 mentions)
15n5 2 deoxyguanosine, by Cambridge Isotopes (1 mentions)
15n5 2 deoxyadenosine, by Cambridge Isotopes (1 mentions)
10 protocols using dnase 1
1
Quantifying Oxidative DNA Damage via 8-OHdG
2
Microbial Metabolite Synthesis Protocols
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Chemicals used in this study were of analytical grade and were commercially purchased. DNase I, RNase A, and lysozyme were purchased from Bio Basic Canada. Restriction endonucleases, T4 DNA ligase, Antarctic phosphatase, and Phusion DNA polymerase were from New England Biolabs. ε-Rhodomycinone was provided by Dr Kristiina Ylihonko.
3
Lung Cell Isolation and Separation
4
Isolation of Intestinal Lymphocytes
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Large intestines were cut into 1 cm slices, and epithelium was removed by stirring in RPMI-1640 containing 1mM EDTA (Gibco) for 30 min and 2% FBS at 37°C (twice). After washing the gut pieces with pre-warmed PBS at least five times, they were cut into 1–2 mm and stirred into RPMI-1640 containing 2% FBS, 10 U/mL collagenase IV (Gibco), and 5 U/mL DNase I (Bio Basic Inc., Amherst NY, USA) for 30 min at 37°C (twice). After incubation, the suspension was filtered through a 100 μm-pore nylon mesh (Small Parts Inc., FL, USA). The lymphocytes were purified by a 44%/70% Percoll (Pharmacia, Uppsala, Sweden) gradient.
5
Quantitative DNA Adduct Analysis
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All the chemicals employed here were of the highest purity grade commercially available. Chromatography grade acetonitrile and methanol, isopropyl alcohol, chloroform, hydrochloric acid, ethanol, magnesium chloride, and tris(hydroxymethyl)-aminomethane were obtained from Carlo Erba Reagents (Milan, Italy). Sodium hydroxide, potassium phosphate, and ammonium acetate were acquired from Merck (Darmstadt, Germany). DNA extraction solutions were obtained from QIAGEN (Valencia, CA). DNase I was acquired from Bio Basic Inc. (Ontario, Canada). [15N5]-2′-deoxyguanosine and [15N5]-2′-deoxyadenosine were provided by Cambridge Isotope Laboratories (Andover, MA). All the other reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO). The catalogue numbers of the reagents are described in Additional file 1 : Table S1. Water was purified in a Milli-Q system (Millipore, Bedford, MA).
6
Isolation of Porcine Leydig Stem Cells
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An enzymatic digestion method was used for obtaining porcine SLCs. Testes were first washed and minced after the epididymis and tunica albuginea were removed. Then the testicular fragments were suspended in 0.75 mg/mL collagenase type IV (Invitrogen) containing 5% (v/v) fetal bovine serum (FBS, Gibco, UK) plus DNase I (100 μg/mL; Bio Basic, Markham, Canada) and incubated, with constant shaking at 34°C for 90 min [21 (link)]. The 160 and 59 μm monofilament nylon meshes (Solarbio, Beijing, China) were then used to filter the cell suspension [21 (link)]. The isolated cells were treated with 1 mg/mL hyaluronidase (Invitrogen) and centrifugation at 500g for 5 min at 20°C. After 5 min stilling, the upper side of the suspensions was cultured in media for another 15 min stilling. The cells on the upper side of the suspensions were then collected. Finally, the isolated LCs were cultured in Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12, Invitrogen) medium.
7
qPCR Analysis of Enterobacterial Cluster Genes
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Total RNA from E. coli NC101 grown in LB for 7 h was isolated, and contaminating DNA was removed using DNase I (Biobasic, Ontario, Canada) for 30 min at 37 °C, followed by RNA extraction using the Total RNA Mini-Preps Kit (Biobasic). Reverse transcription PCR was performed on cDNA reverse transcribed from 50 ng RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Real time PCR was performed using the enzyme PowerUp™ SYBR™ Green Master Mix (Thermo Fisher) using the RG 3000A R (Qiagen, Québec, Canada). Primers used are presented in Additional Table 1 . Relative quantitation was performed using standard curves constructed from serial dilutions of PCR products [22 ]. mRNA expression for each gene was determined by direct comparison with the standard curve of the specific target generated in each PCR run. Expression levels of clbA, clbB, clbQ and clbR were normalized to 16S rRNA.
8
Quantitative Gene Expression Analysis
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Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA), digested with DNase I (Biobasic, Amherst, NY, USA), and reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Amplification of the cDNA was performed using a LightCycler 480 II (Roche, Basel, Switzerland) and LightCycler 480 SYBR Green I Master mix (Roche, Basel, Switzerland), according to the manufacturer’s recommended conditions30 (link),31 (link). The PCR primer sequences are given in Table S2 .
9
Quantifying clbA Expression in E. coli
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Total RNA from E. coli NC101 grown in LB for 7 h was isolated, and contaminating DNA was removed using DNase I (Biobasic, Ontario, Canada) for 30 min at 37 °C, followed by RNA extraction using the Total RNA Mini-Preps Kit (Biobasic). Reverse transcription PCR was performed on cDNA reverse transcribed from 50 ng RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher). For determination of the clbA transcript levels the primer pair RT-4711up/RT-4711lp was used (7) ; and as an internal standard the transcript level of the 16S rRNA gene was amplified with primer pair 16s1114F/16s1275R (Supplementary Table 1). Real time PCR was performed using the enzyme PowerUp™ SYBR™ Green Master Mix (Thermo Fisher) using the RG 3000A R (Qiagen, Quebec, Canada).
10
Nuclease-mediated dsRNA Characterization
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The RNA elements were treated with DNase I, RNase A or S1 nuclease. Each sample of 200 ng of dsRNA was treated with 1 µg/mL of RNase A (Bio Basic; Canada) in 0.1× SSC (15 mM NaCl, 1.5 mM sodium citrate) and 2× SSC (300 mM NaCl, 30 mM sodium citrate) for treatments as low salt and high salt conditions, respectively, and then incubated at room temperature for 10 min. S1 nuclease 0.1 U (Promega; USA) or DNase I 0.1 U (Bio Basic; Canada) was added in the 20 µL reaction comprising 500 ng of dsRNA and buffers and then incubated at 37ºC for 30 min. The nuclease-treated reactions were cleaned up as described by Das et al. (2014) and determined using electrophoresis on 1% agarose gel in TAE buffer, stained with ethidium bromide and visualized under UV illumination.
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