Ambion nuclease free water

Manufactured by Thermo Fisher Scientific

Sourced in Germany

Ambion Nuclease-free water is a high-quality, purified water product designed for use in sensitive molecular biology applications. It is free of DNase, RNase, and other nuclease contaminants, ensuring the integrity of nucleic acids during experiments.

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Lab products found in correlation

In fusion hd kit, by Takara Bio (4 mentions) Turbo dna free kit, by Thermo Fisher Scientific (3 mentions) Qubit fluorometer, by Thermo Fisher Scientific (3 mentions) Agilent eukaryotic total rna 6000, by Agilent Technologies (3 mentions) Cp01 200, by PNA Bio (3 mentions) Quant it rna assay kit, by Thermo Fisher Scientific (2 mentions) Endofree plasmid maxi kit, by Qiagen (2 mentions) Hiscribe t7 kit, by New England Biolabs (2 mentions) Nebnext high fidelity polymerase, by New England Biolabs (2 mentions) Nucleobond xtra midi ef kit, by Macherey-Nagel (2 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Genelute mammalian genomic dna miniprep kit, by Merck Group (1 mentions) Ethanol, by Carl Roth (1 mentions) 3 m sodium acetate, by Carl Roth (1 mentions) Nucleospin gdna clean up kit, by Macherey-Nagel (1 mentions) Trizol ls reagent, by Thermo Fisher Scientific (1 mentions) Wizard genomic dna purification kit, by Promega (1 mentions) Triton x 100, by Merck Group (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Blunt ta ligase master mix, by New England Biolabs (1 mentions)

Spelling variants of this product

Nuclease-free water (659 citations)

18 protocols using ambion nuclease free water

High-quality RNA Extraction from Cell Samples

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TRIzol-chloroform extraction was used to isolate total RNA from flow sorted cell pellets. Extracted RNA samples were resuspended in Ambion Nuclease-free water (Life Technologies Corporation), snap frozen, and stored at −80°C. An optimized proteinase K/acid phenol protocol was used to extract RNA from LCM samples (Khodosevich et al. 2007 (link)). Briefly, cells collected in lysis buffer were incubated at 55°C overnight, and the RNA was isolated in 1:1 phenol (pH 4.2) and chloroform. Total RNA was resuspended in Ambion Nuclease-free water (Life Technologies Corporation), and treated with DNAse prior to storage at −80°C and quality assessment.
RNA was treated with TURBO DNA-free kit (Invitrogen, Grand Island, NY) to eliminate residual DNA prior to quality and yield analysis using the Agilent Eukaryotic Total RNA 6000. RNA quantification was performed using the Quant-iT™ RNA assay kit on a Qubit™ Fluorometer (Life Technologies Corporation).

Solga A.C., Pong W.W., Walker J., Wylie T., Magrini V., Apicelli A.J., Griffith M., Griffith O.L., Kohsaka S., Wu G.F., Brody D.L., Mardis E.R, & Gutmann D.H. (2014). RNA-sequencing reveals oligodendrocyte and neuronal transcripts in microglia relevant to central nervous system disease. Glia, 63(4), 531-548.

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DNA Extraction from Insect Larvae

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DNA was extracted from two replicates of five F0/F1 third-instar female/male larvae representing the control, E. coli and S. entomophila treatment groups using the GenElute^TM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). For each sample, the larvae were homogenized in liquid nitrogen and 150–200 μg of the resulting powder was used for DNA isolation, with a final elution volume of 100 μl. The DNA was precipitated by adding 10 μl 3 M sodium acetate (Carl Roth) and 200 μl ice-cold 100% (v/v) ethanol (Carl Roth), incubating at −20°C for at least for 2 h, and centrifuging at 4°C at 13,000 × g in a microfuge for 15 min (Sambrook and Russell, 2000 ). The pellet was washed with 20 μl ice-cold 70% (v/v) ethanol in Ambion nuclease-free water (Thermo Fisher Scientific) and dried at room temperature for 15 min before dissolving in 50 μl nuclease-free water on ice for 30 min. The DNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). If the A₂₆₀/A₂₃₀ ratio was less than 1.5, the DNA was purified using the NucleoSpin^® gDNA Clean-up kit (Macherey-Nagel) according to the manufacturer’s instructions.

Gegner J., Baudach A., Mukherjee K., Halitschke R., Vogel H, & Vilcinskas A. (2019). Epigenetic Mechanisms Are Involved in Sex-Specific Trans-Generational Immune Priming in the Lepidopteran Model Host Manduca sexta. Frontiers in Physiology, 10, 137.

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RNA and DNA Extraction from Whole Blood

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The same volume of Ambion nuclease-free water (Thermo Fisher Scientific, Tokyo, Japan) was added to all whole blood samples (in EDTA) for dilution, followed by mixing with a triplicate volume of TRIzol LS Reagent (Thermo Fisher Scientific, Tokyo, Japan); the samples were stored at −80 °C until use. RNA was then extracted from the mixture of TRIzol LS Reagent and whole blood according to the manufacturer’s instructions. Briefly, we added 0.2 mL of chloroform into 0.75 mL of the mixture of TRIzol LS Reagent and whole blood samples for lysis. After centrifugation for 10 min at 12,000× g and 4 °C, the supernatant was discarded, and the pellet was resuspended in 1 mL of 75% ethanol to centrifuge again. After removing the supernatant, the sample was air-dried and 20–50 µL of Ambion nuclease-free water was added. DNA was extracted from the blood samples using Wizard Genomic DNA Purification Kit (Promega Corp., Tokyo, Japan) at each time point to measure the PVL using the BLV-CoCoMo-qPCR-2 method [32 (link),43 (link),44 (link)].

Bai L., Hirose T., Assi W., Wada S., Takeshima S.N, & Aida Y. (2020). Bovine Leukemia Virus Infection Affects Host Gene Expression Associated with DNA Mismatch Repair. Pathogens, 9(11), 909.

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Preparation of Unconjugated and Conjugated siRNAs

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Unconjugated siRNA 7 (Scheme 1) was prepared by mixing equimolar amounts of sense (4ss) and antisense strands (4as) and annealed by heating in a water bath at 95°C for 5 min followed by cooling to RT to obtain the duplex. The β-lactam linker-conjugated sense strands were annealed with their complementary antisense strands. For this, aqueous solutions of purified 4ss-BL, 6ss-BL, BL-5ss and BL-5ss-bio were analyzed by UV spectrophotometry to obtain an exact concentration. A subsequent equimolar amount of antisense strand (4as complementary to 4ss-BL, BL-5ss and BL-5ss-bio; 6as complementary to 6ss-BL) was added at a concentration of 20–100 mg/ml in water. The combined strands were vortexed for 30 s and centrifuged to the bottom of a conical tube. For low temperature annealing, the strands were frozen on dry ice and lyophilized to a powder. This afforded siRNAs 4, 6, 5 and 5-bio respectively (Scheme 1, Supplementary Table S2). All siRNAs were dissolved in Ambion nuclease-free water (Thermo Fisher Scientific) to a working concentration of 2.27 or 2.75 mM and stored as aliquots at –80°C. Quality control of the siRNAs included analyses for purity, endotoxins and osmolality.

Nanna A.R., Kel’in A.V., Theile C., Pierson J.M., Voo Z.X., Garg A., Nair J.K., Maier M.A., Fitzgerald K, & Rader C. (2020). Generation and validation of structurally defined antibody–siRNA conjugates. Nucleic Acids Research, 48(10), 5281-5293.

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Optimizing Taq DNA Ligase Reactions

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Blunt/TA Ligase Master Mix, T4 DNA ligase, 10x T4 DNA ligase buffer, T. thermophilus strain HB8 DNA Ligase (sold commercially as Taq DNA Ligase for historical reasons), 9°N DNA ligase, 2 M KCl, 1 M MgCl₂, 1 M DTT, 10 mM ATP and 50 mM NAD⁺ were obtained from New England BioLabs (NEB, Ipswich, MA). Tris-HCl (1 M) buffer stocks of pH 7.0, 7.5, 8.0, 8.5 and 9.0 (determined @ 25°C) were obtained from Amresco (Solon, OH). Triton X-100 (10%) was obtained from Sigma-Aldrich (St. Louis, MO). Ambion Nuclease-Free water was obtained from Life Technologies (Grand Island, NY). Taq DNA ligase reaction buffers (20 mM Tris-HCl, 25 mM KCl, 10 mM MgCl₂,1 mM NAD⁺, 10 mM DTT, 0.1% Triton® X-100) were prepared as 10X stocks at pH 7.0, 7.5, 8.0, 8.5 and 9.0 @ 25°C. Oligonucleotide annealing buffer (10 mM Tris pH 7.5 @ 25°C, 50 mM KCl, 0.1 mM EDTA) was prepared as a 10X stock.

Lohman G.J., Bauer R.J., Nichols N.M., Mazzola L., Bybee J., Rivizzigno D., Cantin E, & Evans TC J.r. (2015). A high-throughput assay for the comprehensive profiling of DNA ligase fidelity. Nucleic Acids Research, 44(2), e14.

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DNA Extraction and Dilution Protocol

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DNA was extracted from 40 whole blood samples using an Invitrogen^® PureLink™ Genomic DNA Mini kit (Life Technologies Inc.). Aliquots (20 µl) of the 40 DNA extracts (nos. 1-40) were diluted by adding nine volumes (180 µl) of Ambion^® Nuclease-Free Water (Life Technologies Inc.) to obtain 10-fold dilutions of the DNA solutions (final volume, 200 µl). The Promega^® stock solutions 9948 Male DNA and 2800M control DNA standards (10 ng/µl; 25 µl; Promega, Corp., Madison, WI, USA) were added with nine volumes (225 µl) of Ambion^® Nuclease-Free Water, to obtain 10-fold dilutions of the standard samples (final volume, 250 µl).

HU N., CONG B., GAO T., CHEN Y., SHEN J., LI S, & MA C. (2015). Application of mixsep software package: Performance verification of male-mixed DNA analysis. Molecular Medicine Reports, 12(2), 2431-2442.

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RNA-Seq Library Preparation and Analysis

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Total RNA from flow-sorted cells was isolated by TRIzol-chloroform extraction. RNA samples were resuspended in Ambion Nuclease-free water (Life Technologies), snap frozen, and stored at -80 °C. Prior to RNA sequencing, RNA was treated with TURBO DNA-free kit (Invitrogen | Thermo Fisher Scientific, Waltham, Massachusetts, USA) and assessed using the Agilent Eukaryotic Total RNA 6000 and Quant-iT™ RNA assay kit on a Qubit™ Fluorometer (Life Technologies). cDNA was synthesized using the Ovation® RNA-Seq method, and the Illumina paired-end LT indexing protocol used to construct an Illumina library from 500 ng cDNA [19 (link), 30 (link)]. Libraries were sequenced on an Illumina HiSeq, and15-22Mbp per lane of 100 basepair paired-end reads generated. RNA-Seq paired-end reads were processed using the TopHat suite [44 (link)] with Cufflinks [36 (link), 37 ]. A fold-change and significance (< 0.05 False Discovery Rate, FDR) for every gene was generated using cuffdiff [43 (link)].

Haage V., Semtner M., Vidal R.O., Hernandez D.P., Pong W.W., Chen Z., Hambardzumyan D., Magrini V., Ly A., Walker J., Mardis E., Mertins P., Sauer S., Kettenmann H, & Gutmann D.H. (2019). Comprehensive gene expression meta-analysis identifies signature genes that distinguish microglia from peripheral monocytes/macrophages in health and glioma. Acta Neuropathologica Communications, 7, 20.

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RNA Isolation and Quantification from Microglia

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TRIzol-chloroform extraction was used to isolate total RNA from flow-sorted microglia. Extracted RNA samples were resuspended in Ambion Nuclease-free water (Life Technologies), snap frozen, and stored at − 80°C. Before cDNA library construction and quantitative real-time polymerase chain reaction (qRT-PCR), residual DNA was eliminated with the TURBO DNA-free kit (Invitrogen). RNA quality was assayed using the Agilent Eukaryotic Total RNA 6000 and quantified using the Quant-iT RNA assay kit on a Qubit Fluorometer (Life Technologies).

Solga A.C., Pong W.W., Kim K.Y., Cimino P.J., Toonen J.A., Walker J., Wylie T., Magrini V., Griffith M., Griffith O.L., Ly A., Ellisman M.H., Mardis E.R, & Gutmann D.H. (2015). RNA Sequencing of Tumor-Associated Microglia Reveals Ccl5 as a Stromal Chemokine Critical for Neurofibromatosis-1 Glioma Growth. Neoplasia (New York, N.Y.), 17(10), 776-788.

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Generation of Syt1-GCaMP Transgenic Mice

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We prepared injection mixes for Syt1-T2A-QF2-QUAS-GCaMP6s, Syt1-T2A-3XGCaMP6s according to Kistler et al., 2015 (link). sgRNAs were generated as described above. We prepared donor plasmids using the InFusion HD Kit (Clontech, 638910) and EndoFree Plasmid Maxi Kit (Qiagen, 12362), and verified them via Sanger sequencing. We mixed donor plasmid (700 ng/uL) and sgRNA (80 ng/uL), purified the mixture via ethanol precipitation, and resuspended in Ambion nuclease-free water (Life Technologies, AM9937), before adding recombinant Cas9 protein (300 ng/μL; PNA Bio, CP01-200) for embryo injection.

Zhao Z., Tian D, & McBride C.S. (2021). Development of a pan-neuronal genetic driver in Aedes aegypti mosquitoes. Cell Reports Methods, 1(3), 100042.

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CRISPR Plasmid Preparation for Transgenic Mouse

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Zhao Z., Tian D, & McBride C.S. (2021). Development of a pan-neuronal genetic driver in Aedes aegypti mosquitoes. Cell Reports Methods, 1(3), 100042.

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