Nucleospin tissue dna kit

Manufactured by Macherey-Nagel

Sourced in Germany

The Nucleospin Tissue DNA Kit is a DNA extraction kit designed for the isolation of genomic DNA from various biological samples, such as animal tissues, cultured cells, and bacteria. The kit utilizes a silica-membrane-based technology to efficiently bind, wash, and elute high-quality DNA.

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Lab products found in correlation

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Spelling variants of this product

NucleoSpin Tissue kit (780 citations) NucleoSpin Tissue (164 citations) NucleoSpin kit (156 citations) NucleoSpin® DNA Tissue (6 citations)

12 protocols using nucleospin tissue dna kit

Fecal Nematode Egg Isolation and Larval Culture

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Upon arrival to the laboratory, feces were investigated for GIN strongyle eggs. The number of nematode eggs was first counted using a modified McMaster technology based on 3 g of feces and with a minimum diagnostic sensitivity of 50 nematode eggs per gram feces (EPG) as described previously [23 (link)].
Thereafter, subsamples of approximately 2 g feces were collected from each animal in all groups and pooled in separate plastic containers. After blending the pooled fresh feces with Vermiculite, the eggs were cultured for approximately 10 days in a moistened chamber at ≈20 °C [24 (link)]. Infective third-stage larvae (L3) were harvested using the Petri dish method, concentrated in a Falcon tube and collected into an Eppendorf tube before storage in a freezer at approximately − 18 °C [14 (link)]. DNA was then extracted from the larval cultures (one per group and sampling occasion) using the Nucleospin® DNA tissue kit (Macherey-Nagel, Düren, Germany) following the manufacturer's guidelines.

Halvarsson P, & Höglund J. (2021). Sheep nemabiome diversity and its response to anthelmintic treatment in Swedish sheep herds. Parasites & Vectors, 14, 114.

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Cloning of Invertebrate Regulatory Promoters

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The En2, Hox, Otp and Ubx putative promoters (En2p, Hoxp, Otpp and Ubxp respectively) were amplified by PCR from genomic DNA (100 ng, Nucleospin DNA tissue kit, Macherey Nagel) using Phusion DNA polymerase (New England Biolabs). When necessary, nested PCRs were performed with primers containing restriction enzyme sites used for downstream subcloning (SacI for En2p and XmaI for Otpp). PCR products (En2p, 841 bp; Hoxp, 1158 bp; Otpp, 551 bp; Ubxp, 737 bp), were resolved by agarose gel electrophoresis stained with ethidium bromide (EtBr-AGE) and purified by affinity chromatography (Nucleospin Gel and PCR Cleanup, Macherey Nagel). The purified fragments were digested using SacI (En2p), XmaI (Otpp) or SmaI (Hoxp and Ubxp) and respectively subcloned using T4 DNA ligase (Promega) into the SacI, XmaI or EcoICRI site of the pGL2' vector (p3TP-lux plasmid [2] after PAI-1 promoter excision using SacI). The resulting constructs containing the firefly luciferase coding sequence under the control of the C. gigas Engrailed2, HoxC11, Orthopedia and Ultrabithorax genes putative proximal promoter, named pEn2p, pHoxp, pOtpp and pUbxp vectors, respectively (see Fig. 1) were assessed by PCR, restriction enzyme digestions and sequencing. Large amounts of the constructs were purified free of endotoxins from bacterial cultures using affinity chromatography (Nucleobond Xtra Maxi EF, Macherey Nagel).

Saint-Carlier E, & Riviere G. (2015). Regulation of Hox orthologues in the oyster Crassostrea gigas evidences a functional role for promoter DNA methylation in an invertebrate. FEBS letters, 589(13).

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Quantification of KSHV Gene Expression

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Cellular RNA was extracted using a NucleoSpin RNA extraction kit (Macherey Nagel, Düren, Germany). RNA was reverse transcribed to cDNA using oligo dT primers and Multiscribe reverse transcriptase (Thermo Fischer Scientific). cDNA and target primers were mixed with 2X SYBR (Thermo Fischer Scientific). The genomic DNA was isolated using a Nucleospin Tissue DNA kit (Macherey Nagel, Düren, Germany). The DNA was mixed with 2X SYBR and primers binding to the genomic actin and the viral genome at K8.1. The qPCR reactions were conducted in a Light Cycler 480 qPCR system (Roche, Basel, Switzerland). The primer sequences are listed below:

Actin-Fw; Rv (5′-tcacccacactgtgccatctacga-3′; 5′-cagcggaaccgctcattgccaatgg-3′);

ORF50-Fw; -Rv (5′-cacaaaaatggcgcaagatga-3′; 5′-tggtagagttgggccttcagtt-3′);

ORF45-Fw; Rv (5′-cctcgtcgtctgaaggtga-3′; 5′-gggatgggttagtcaggatg-3′);

ORF57-Fw; -Rv (5′-tggacattatgaagggcatccta-3′; 5′-cgggttcggacaattgct-3′);

K8.1-Fw; -Rv (5′-aaagcgtccaggccaccacaga-3′; 5′-ggcagaaaatggcacacggttac-3′);

Genomic_Actin-Fw; -Rv (5′-agaaaatctggcaccacacc-3′; 5′-aacggcagaagagagaacca-3′).

Elbasani E., Falasco F., Gramolelli S., Nurminen V., Günther T., Weltner J., Balboa D., Grundhoff A., Otonkoski T, & Ojala P.M. (2020). Kaposi’s Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter. Viruses, 12(9), 952.

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Tissue DNA Extraction Protocol

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About 25 mg of tissue was homogenized and DNA was isolated using the Nucleospin Tissue DNA Kit (Macherey Nagel, GmbH, Düren, Germany) according to the manufacturer’s instructions. The isolated DNA was quantified with Nanodrop ND-2000 and stored at −20 °C until further use.

Ramasamy D., Rao A.K., Balaiah M., Vittal Rangan A., Sundersingh S., Veluswami S., Thangarajan R, & Mani S. (2022). Locus-Specific Enrichment Analysis of 5-Hydroxymethylcytosine Reveals Novel Genes Associated with Breast Carcinogenesis. Cells, 11(19), 2939.

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Quantitative Telomere Length Analysis

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Quantitative RT-PCR was used to determine changes in the telomere length of hiDFPs and hiPSCs relative to their corresponding HDFs. Total DNA was isolated from HDFs and hiDFPs from each line at the end of reprogramming using the NucleoSpin Tissue DNA kit (Macherey Nagel).
Relative Human Telomere Length Quantification qPCR assay (ScienCell) was used to determine relative telomere length. Two separate PCR reactions were performed in triplicate for each sample using telomere or SCR primer sets and PowerTrack^TM SYBR Green Master Mix. An equivalent of 4 ng genomic DNA per reaction was used, in a total of 20 μl per well. Amplification was performed under the following conditions: denaturation for 10 min at 95°C, followed by 32 cycles of denaturation for 20 s at 95°C, annealing for 20 s at 52°C and extension for 45 s at 72°C. The relative telomere length was calculated according to the instructions in the manufacturer’s protocol.

McCaughey-Chapman A., Tarczyluk-Wells M., Combrinck C., Edwards N., Jones K, & Connor B. (2023). Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures. Frontiers in Cellular Neuroscience, 17, 1003188.

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Parvovirus B19 Detection in Myocardium

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DNA was isolated from two frozen myocardial biopsy samples (20–50 µg of quick-frozen tissue) with the NucleoSpin Tissue DNA kit (Macherey-Nagel, Dueren, Germany). Primers and a dual-labeled probe, specific for VP1 gene of the parvovirus B19, were used for amplification. All PCR reactions were performed with primers and probe specific for human glucose-6-phosphate dehydrogenase for the confirmation of the DNA isolation from the tissue and for the normalization of viral DNA copy number. A detailed description of the method used for B19V detection and quantitation has been published previously [15 (link)].

Pawlak A., Gewartowska M., Przybylski M., Kuffner M., Wiligórska D., Gil R., Król Z, & Frontczak-Baniewicz M. (2022). Ultrastructural Changes in Mitochondria in Patients with Dilated Cardiomyopathy and Parvovirus B19 Detected in Heart Tissue without Myocarditis. Journal of Personalized Medicine, 12(2), 177.

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DNA Extraction and Sequencing of Culicoides Larvae

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Genomic DNA of larvae Culicoides was individually extracted using the NucleoSpin® Tissue DNA Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions and maintained at 20 °C until further use. PCR amplification reactions were performed in a 25 μl total reaction volume containing 1× buffer, 1 mM MgCl₂, 0.2 mM of each dNTP (dATP, dCTP, dGTP and dTTP), 0.2 μM forward primer LCO1490 (5'-GGT CAA CAA ATC ATA AAG ATATTG G-3'), 0.2 μM reverse primer HCO2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') [61 ], 1.25 U of Taq DNA Polymerase (Qiagen, Hilden, Germany) and 0.4 ng/μl genomic DNA. The PCR cycling conditions were as follows: an initial denaturation step at 94 °C for 5 min followed by 5 cycles of 94 °C for 30 s, 45 °C for 40 s, 72 °C for 1 min, 35 cycles of 94 °C for 30 s, 51 °C for 30 s, 72 °C for 1 min, and a final extension step at 72 °C for 10 min. Positive and negative controls for the amplification reactions were carried out at every PCR round. The PCR products were separated on 1.5% agarose gels and the products were sequenced using the same primers as used in PCR amplifications (https://www.genewiz.com). All generated sequences were deposited in GenBank and BOLD.

Bakhoum M.T., Sarr M., Fall A.G., Huber K., Fall M., Sembène M., Seck M.T., Labuschagne K., Gardès L., Ciss M., Gimonneau G., Bouyer J., Baldet T, & Garros C. (2018). DNA barcoding and molecular identification of field-collected Culicoides larvae in the Niayes area of Senegal. Parasites & Vectors, 11, 615.

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Quantification of DNA Methylation in Cells

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The amount of methylated DNA was quantified using the Methylated DNA Quantification Kit (Abcam) which specifically measures the level of 5-methylcytosine (5-mC) from total gDNA isolated from hiPSCs, HDFs, and hiDFPs using the NucleoSpin Tissue DNA kit (Macherey Nagel). The assay was conducted as per the manufacturer’s protocol. Briefly, 100 ng of DNA was loaded for each sample in a 96-well strip plate with standards and a negative control. After binding by incubation at 37°C for 90 min, a capture antibody was added to each well and incubated at room temperature for 60 min, followed by incubation for 30 min in the detection antibody. The signal was enhanced by incubation for 30 min in an enhancer solution, followed by a 10 min incubation in a developer solution. The enzymatic reaction was stopped by the addition of a stop solution and the absorbance in each well was then read on a microplate reader at 450 nm. DNA methylation was determined as measured by the production of 5-mC (ng) from total DNA.

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DNA Isolation and PCR Sequencing

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DNA isolation was performed using the Nucleospin Tissue DNA Kit (Macherey-Nagel, Germany) according to manufacturer's instructions. PCR was performed on a Veriti well thermal cycler (Applied Biosystems, the Netherlands) with an annealing temperature of 57°C for all combinations of primers and 35 cycles. The PCR products were sequenced using the Big Dye Sequencing Kit (ThermoFisher SCIENTIFIC, The Netherlands) according to manufacturers' instructions. All experiments were repeated at least 3 times.

Chteinberg E., Sauer C.M., Rennspiess D., Beumers L., Schiffelers L., Eben J., Haugg A., Winnepenninckx V., Kurz A.K., Speel E.J., Zenke M, & zur Hausen A. (2018). Neuroendocrine Key Regulator Gene Expression in Merkel Cell Carcinoma. Neoplasia (New York, N.Y.), 20(12), 1227-1235.

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Quantitative Serological and Molecular HBV Analysis

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HBeAg, HBsAg and anti-HBs were quantified using the Architect™ platform (Abbott Laboratories, Wiesbaden, Germany), anti-HBc using Enzygnost® Anti-HBc monoclonal test (Siemens Healthcare Diagnostics, Erlangen, Germany). Alanine aminotransferase (ALT) activity was determined after 1:4 dilution in PBS by Reflotron® GPT/ALT tests (Roche Diagnostics, Mannheim, Germany).
DNA was extracted from 50 μl mouse serum using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) or 20 mg of liver tissue using a NucleoSpin Tissue DNA Kit (Macherey-Nagel, Dueren, Germany) according to the manufacturer’s instructions. The quantification of HBV-DNA was performed through real-time PCR with SYBR green as previously described.²²

Sacherl J., Kosinska A.D., Kemter K., Kächele M., Laumen S.C., Kerth H.A., Öz E.A., Wolff L.S., Su J., Essbauer S., Sutter G., Scholz M., Singethan K., Altrichter J, & Protzer U. (2022). Efficient stabilization of therapeutic hepatitis B vaccine components by amino-acid formulation maintains its potential to break immune tolerance. JHEP Reports, 5(2), 100603.

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