Dnase enzyme

Manufactured by Qiagen

Sourced in Germany

The DNase enzyme is a laboratory reagent used to degrade DNA molecules. It functions by catalyzing the hydrolysis of phosphodiester bonds within DNA, effectively breaking down the DNA strands. The DNase enzyme is commonly used in various molecular biology and biochemistry applications that require the removal of DNA from samples or reactions.

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5 protocols using dnase enzyme

Single-cell RNA-seq Library Preparation

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Individual embryos were lysed in 300 µl Trizol with a 5 mm stainless steel bead (Qiagen) for 2 min at 20 Hz in a tissue lyser (Qiagen). After mixing of 200 µl chloroform to the homogenate and incubation for 30 min at room temperature the upper phase was transferred to a new tube. One volume of fresh 70% ethanol was added and the RNA was purified over a spin column (Qiagen RNeasy MinElute). After quantification (Qubit RNA BR) the sample was treated with DNAse enzyme (Qiagen) and purified over a spin column again. Up to 500 ng total RNA was used for the generation of strand-specific RNA-seq libraries containing unique index sequences in the adapter. Libraries were pooled and sequenced on Illumina HiSeq.

Collins J.E., White R.J., Staudt N., Sealy I.M., Packham I., Wali N., Tudor C., Mazzeo C., Green A., Siragher E., Ryder E., White J.K., Papatheodoru I., Tang A., Füllgrabe A., Billis K., Geyer S.H., Weninger W.J., Galli A., Hemberger M., Stemple D.L., Robertson E., Smith J.C., Mohun T., Adams D.J, & Busch-Nentwich E.M. (2019). Common and distinct transcriptional signatures of mammalian embryonic lethality. Nature Communications, 10, 2792.

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Transcriptome Profiling of Soybean Meristem Tissues

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Total RNA from the meristem tissues of inoculated (Ss97009 and SsV89101) and mock-inoculated control plants at 2 dpi, 5 dpi, and 60 dpi (three biological replications per sample) were extracted using the RNeasy Plant Mini kit (Qiagen, Germany) with on-column digestion using DNase enzyme (Qiagen, Germany) according to manufacturer’s instructions. RNA quality, integrity, and concentrations were assessed by agarose gel electrophoresis, NanoDrop (ThermoScientific, USA), and Qubit (Invitrogen, USA). The quality of the samples was further verified by determining the RNA Integrity Number (RIN) using Agilent 2100 Bioanalyzer (Agilent Technologies, USA). High-quality RNA samples with RIN value >6.0 were used for the construction of cDNA libraries using the TruSeq RNA Sample Prep Kits (Illumina, USA) as described in the manufacturer’s instructions. A total of 27 libraries (3 libraries/sample) were sequenced on HiSeq 4000 (Illumina, USA) to generate paired-end reads (2x150 bp) of about 5-10 GB quality reads/library.

Agisha V.N., Ashwin N.M., Vinodhini R.T., Nalayeni K., Ramesh Sundar A., Malathi P, & Viswanathan R. (2022). Transcriptome analysis of sugarcane reveals differential switching of major defense signaling pathways in response to Sporisorium scitamineum isolates with varying virulent attributes. Frontiers in Plant Science, 13, 969826.

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RNA Extraction and Purification from NRVM Cells

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NRVM cells were placed directly in 1 ml TRIzol reagent prior to nucleic acid extraction. The TRIzol protocol was followed as recommended by the manufacturer (Invitrogen). Briefly, the total RNA was precipitated, purified and air-dried. The pellet was subsequently resuspended in a solution of water, DNase buffer and DNase enzyme (Qiagen, CA, USA), and incubated at room temperature for 10 min to remove any genomic DNA contamination. Total RNA was then purified on a Qiagen RNeasy^® column according to manufacturer’s instructions. Elution was carried out with RNase/DNase-free water in two parts, combined and concentrated using a Savant™ Integrated SpeedVac™ Vacuum System (Thermo Scientific Inc., MA, USA) until the total volume reached 15 µl. For RNA quantification, 1 µl of total RNA was used to measure the absorbance at 260 nm of each sample by means of the NanoDrop ND-1000 (NanoDrop Technologies, DE, USA). The samples were then transported to the UCSD BIO-GEM core for Illumina (CA, USA) Beadarray processing.

Paolini P., Pick D., Lapira J., Sannino G., Pasqualini L., Ludka C., Sprague L.J., Zhang X., Bartolotta E.A., Vazquez-Hidalgo E., Barba D.T., Bazan C, & Hardiman G. (2014). Developmental and extracellular matrix-remodeling processes in rosiglitazone-exposed neonatal rat cardiomyocytes. Pharmacogenomics, 15(6), 759-774.

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Transcriptomic Analysis of Larval Development

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For the molecular analyses, six larvae were sampled at T0, while six larvae were sampled per replicate, per treatment at T1 and T2 (n = 54). Larvae were immediately stored at −80 °C soon after the sampling procedure. Total RNA was extracted from each frozen single larval sample using the RNeasy Mini Kit^® (Qiagen, Hilden, Germany) following the manufacturer’s instructions with a final elution in 40 μL of RNase-free water. A double treatment with DNase enzyme (Qiagen, Hilden, Germany) was performed in order to remove any genomic DNA contamination, according to the manufacturer’s instructions. The quantification and purity of the extracted RNA was assessed using the Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Five hundred nanograms of RNA was retro-transcribed to cDNA using the Quantitect Reverse Transcription Kit^® (Qiagen, Hilden, Germany) following the manufacturer’s protocol. An additional reaction without retrotranscriptase enzyme was performed to verify the complete DNA removal. The cDNAs were stored at −80 °C until subsequent use.

Aidos L., Cafiso A., Lopez A., Vasconi M., Valente L.M., Bazzocchi C, & Di Giancamillo A. (2022). Rearing Environment during the Endogenous Feeding Stage of Acipenser baerii. Animals : an Open Access Journal from MDPI, 12(17), 2205.

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RNA Isolation from PBMCs Using Trizol and RNeasy

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For isolation of total RNA, the purified PBMCs were suspended in 1.0 ml Trizol reagent (Thermo Fisher Scientific, USA). After homogenization, the standard protocol based on chloroform and isopropanol extraction was followed to isolate the total RNA. The total RNA was further purified by employing silica-membrane RNeasy spin columns (Qiagen, Germany) along with on column digestion by DNase enzyme (Qiagen, Germany). The concentration and purity of extracted was measured using Nano view plus (Biohrome Spectros, USA). The integrity of each RNA sample was also confirmed by presence of 28S and 18S ribosomal bands on 1.5% agarose gel.

Tiwari M., Sodhi M., Verma P., Vivek P., Kataria R.S., Niranjan S.K., Bharti V.K., Masharing N., Gujar G., Chanda D., & Mukesh M. (2021). Selection of Species Specific Panel of Reference Genes Across Native Livestock Species Adapted to Trans-Himalayan Region of Leh-ladakh.

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