Rnase cocktail

Manufactured by Thermo Fisher Scientific

Sourced in United States

The RNase cocktail is a laboratory reagent designed to inactivate ribonuclease (RNase) enzymes. RNases are ubiquitous enzymes that can degrade RNA, posing a risk of sample degradation during RNA-based experiments. The RNase cocktail offers a convenient solution to prevent this by effectively inhibiting RNase activity.

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Lab products found in correlation

Rnase a, by Thermo Fisher Scientific (5 mentions) Turbo dnase, by Thermo Fisher Scientific (3 mentions) Rnase t1, by Thermo Fisher Scientific (3 mentions) Dnase rnaase free water, by Thermo Fisher Scientific (2 mentions) Rnase a, by Merck Group (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Proteinase k, by New England Biolabs (2 mentions) Agencourt xp spri beads, by Beckman Coulter (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Dneasy blood and tissue kit, by Qiagen (2 mentions) Proteinase k, by Merck Group (2 mentions) Rnase if, by New England Biolabs (2 mentions) Lysozyme, by Merck Group (2 mentions) Dextran sulfate, by Merck Group (2 mentions) Formamide, by Merck Group (2 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Fastap, by Thermo Fisher Scientific (1 mentions) Pronase, by Roche (1 mentions) Microtip sonifier 450, by Emerson (1 mentions)

Close spelling variants of this product

RNase Cocktail Enzyme Mix (25 citations) RNase A/T1 Cocktail (11 citations) RNAse A + T cocktail (5 citations) Protease Inhibitor Cocktail and RNase inhibitor (3 citations) RNase cocktail mix (2 citations) RNase/T1 cocktail (2 citations)

60 protocols using rnase cocktail

Simultaneous RNA and DNA Interaction Mapping

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To map RNA and DNA interactions simultaneously, the SPRITE
protocol was performed with the following modifications: (i) Upon
coupling of lysate to NHS beads, RNA overhangs caused by fragmentation
are repaired by a combination of treatment with FastAP (Thermo) and T4
Polynucleoide Kinase (NEB) with no ATP at 37°C for 15 minutes
and 1 hour, respectively. RNA was subsequently ligated with a
“RNA Phosphate Modified” (RPM) tag using T4 RNA Ligase 1
(ssRNA Ligase), High Concentration (NEB) at 20°C for 1 hour
(Shishkin et al., 2015 (link)). The
RPM tag is designed with a 5′ ssRNA overhang and 3′
dsDNA sticky end for sequential ligation of DNA tags to the RNA (see
SPRITE tag design). (ii) RNA was converted to cDNA
using Superscript III (Thermo) using a manganese reverse transcriptase
protocol (Siegfried et al., 2014 (link))
to promote reverse-transcription through formaldehyde crosslinks on RNA.
After cDNA synthesis, cDNA was selectively eluted from NHS beads using
RNaseH (NEB) and RNAse Cocktail (Ambion). cDNA was ligated with a unique
cDNA tag as previously described (Shishkin et al., 2015 (link)), which serves as a RNA-specific
identifier during sequencing.

Quinodoz S.A., Ollikainen N., Tabak B., Palla A., Schmidt J.M., Detmar E., Lai M.M., Shishkin A.A., Bhat P., Takei Y., Trinh V., Aznauryan E., Russell P., Cheng C., Jovanovic M., Chow A., Cai L., McDonel P., Garber M, & Guttman M. (2018). Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus. Cell, 174(3), 744-757.e24.

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Multi-Omics Chromatin Profiling

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ChIP was performed as described previously^{33 (link)} with some modifications. Cells were treated with ethanol or tamoxifen (1 µM) for 24 h and then cross-linked using ethylene glycol bis (succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS) mixture (2.5 µM each) for 45 min at room temperature. After this initial crosslinking, cells were further fixed using 1% formaldehyde for 20 min at room temperature and then quenched by glycine (0.125 M). Chromatin in sonication buffer (50 mM HEPES, pH7.5, 140 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton-100, and 0.4% SDS) was sheared using Branson Microtip Sonifier 450 (12 cycles of 15 s at a sonication setting of output 4 and duty cycle 60%) to a size mostly between 100–150 bp. The sonicated chromatin solution was diluted to 0.085% SDS and immunoprecipitated with antibodies against H3K4me3 (ab8580), H3K27ac (ab4729), H3K4me1 (ab8895), H3K36me3 (ab9050), H3K27me3 (ab6002), and H3K9me3 (ab8898). Immunoprecipitated chromatin was decrosslinked using RNase Cocktail (Ambion, AM2286) and Pronase (Roche, 10165921001). ChIP DNA was end repaired, addition of “A” and adapters ligation and PCR amplification to produce ChIP-seq libraries. The DNA concentration was measured by Bioanalyzer before sequencing using Hiseq 2000 at the Bauer Core Facility, Harvard.

Ji Z., He L., Rotem A., Janzer A., Cheng C.S., Regev A, & Struhl K. (2018). Genome-scale identification of transcription factors that mediate an inflammatory network during breast cellular transformation. Nature Communications, 9, 2068.

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High-Purity DNA Extraction Protocol

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The DNA extractions were done in an ISO 8 cleanroom to reduce carbon contamination. Beforehand, the glass utensils were baked at 450 °C for 4 hours. To each sample, 1 ml of lysis buffer (100 mM Tris [pH 8.0], 200 mM NaCl, 1% SDS, and 5 mM EDTA) and 12 μl of Proteinase K (40 mg/ml) were added. The sample was then incubated overnight at 65 °C. RNase cocktail (6 μl, Ambion) was added to each sample, and the samples were then incubated at 65 °C for 1 hours. Each sample received half a volume of NaCl (5 M). After a 30-second vortex, the tubes were centrifuged at 13,000 rpm for 6 minutes. The pellet was discarded and the supernatant transferred to a glass tube with 3 volumes of absolute ethanol. The tubes were gently agitated for 30 seconds. The DNA pellet formed in the previous step was washed three times in buffer (70% Ethanol [v/v] and 0.5 M NaCl) and later dried at 65 °C overnight to evaporate all the ethanol. DNase/RNAase free water (500 ml, GIBCO/Invitrogen) was added to each vial and left overnight at 65 °C to resuspended the DNA. The concentration and purity were assessed by UV spectroscopy (NanoDrop).

Mold J.E., Réu P., Olin A., Bernard S., Michaëlsson J., Rane S., Yates A., Khosravi A., Salehpour M., Possnert G., Brodin P, & Frisén J. (2019). Cell generation dynamics underlying naive T-cell homeostasis in adult humans. PLoS Biology, 17(10), e3000383.

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Exosomal Small RNA RNase Assay

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To ascertain that small RNAs were encapsulated within the exosomes rather than in contaminants on the outside of exosomes, we treated samples with RNase before proceeding with further experiments. We diluted exosome pellets in 50 μL PBS and added Ambion RNase cocktail 2.5 μL at 37 °C for 15 min.

Liu Y.M., Chen H.C., Chen Y.C., Yu W.Y., Ho M.Y., Ho C.Y., Lai M.M, & Su W.C. (2020). miR-1975 serves as an indicator of clinical severity upon influenza infection. European Journal of Clinical Microbiology & Infectious Diseases, 40(1), 141-149.

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FLAG and GFP-Trap Immunoprecipitation Protocols

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FLAG immunoprecipitations were performed following the manufacturer’s protocol (FLAG M2 beads, Sigma Aldrich) as described (Thorne et al., 2012 (link)). In brief, protein concentration in lysates was normalised using BCA. Lysates were then diluted with 1 vol of wash buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA). Equal volumes of anti-FLAG affinity gel were dispensed into either WT infected cell lysates, or lysates of cells infected with NS1/2-FLAG or NS4-FLAG. Binding was carried out overnight at 4°C with rotation. After binding, beads were washed three times with wash buffer. All liquid was carefully removed from each tube, before boiling in SDS-PAGE loading buffer for 10 min. GFP-trap immunoprecipitation of GFP-tagged VPg was accomplished using GFP-trap beads (Chromotek) per the manufacturer’s protocol, as described (Emmott and Goodfellow, 2014 (link)). RNase cocktail (Ambion) was also included in the lysis buffer at a concentration of 5 μl/ml to prevent non-specific interactions mediated by RNA. In all cases, light, medium, and heavy-labelled proteins eluted from the beads for each experimental replicate were pooled together in a ratio of 1:1:1 before submission for mass spectrometry analysis at the University of Bristol Proteomics Facility.

Hosmillo M., Lu J., McAllaster M.R., Eaglesham J.B., Wang X., Emmott E., Domingues P., Chaudhry Y., Fitzmaurice T.J., Tung M.K., Panas M.D., McInerney G., Locker N., Wilen C.B, & Goodfellow I.G. (2019). Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation. eLife, 8, e46681.

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Viral RNA Extraction from Cell Lines

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The F9 strain of FCV and the CW1 isolate of MNV were grown in Crandell-Reese feline kidney cells (obtained from ATCC) and RAW264.7 cells (obtained from ATCC) respectively. For each preparation, at least five 170 cm² flasks were infected with a multiplicity of infection of 0.2 TCID50/cell. Infected cells were harvested at ∼15 h post infection. Cells were resuspended directly in lysis buffer and the total RNA was isolated using the GenElute mammalian total RNA miniprep kit as per the manufacturer’s instructions. Eluted samples were combined, further concentrated by ethanol precipitation and resuspended in nuclease free water. Where required, preparations of VPg-linked RNA were treated with RNase cocktail (Ambion) at 37 °C for 1 h prior to the addition of SDS-PAGE sample buffer and separation by 15% SDS-PAGE.

Olspert A., Hosmillo M., Chaudhry Y., Peil L., Truve E, & Goodfellow I. (2016). Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins. PeerJ, 4, e2134.

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RNA and DNA Denaturation Protocol

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After fixation in 4% PFA and permeabilization in 0.5% Triton 100-X, cells were treated with 200 μg/mL RNaseA (Sigma), and 2.5 U/mL RNaseA and 100 U/mL RNaseT1 (RNase cocktail, Ambion) at 37°C for 1 hour. DNA was then denatured (1.5 M NaCl, 0.5 M NaOH) for 30 min at RT and neutralized (0.5 M Tris-HCl, pH 7.0, 3 M NaCl) twice for 5 min at RT. Samples were then washed three times with 1x PBS for 5 minutes at RT before proceeding with blocking and incubation of antibodies as described above.

Keller D., Stinus S., Umlauf D., Gourbeyre E., Biot E., Olivier N., Mahou P., Beaurepaire E., Andrey P, & Crabbe L. (2024). Non-random spatial organization of telomeres varies during the cell cycle and requires LAP2 and BAF. iScience, 27(4), 109343.

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RNA-Protein Interactions: A Pull-Down Assay

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Pull-down assay was performed using in vitro transcribed biotinylated VIM, FABP7 and 5′UTR msl2 RNA probes (negative control) according to Pierce™ Magnetic RNA-Protein Pull-Down Kit protocol with some modifications. Labeled RNA was captured using 100 μl of streptavidin magnetic beads in RNA Capture Buffer for 30 min at room temperature. Beads were washed twice in 20 mM Tris (pH 7.5), once in Protein-RNA Binding Buffer and 200 μl of H9 hESCs extract was added (8 μg μl⁻¹ of total protein). Samples were incubated for 30 min at room temperature, irradiated or not with 0.15 J cm − 2 at 254-nm UV light, washed three times with Wash Buffer and eluted after 30 min of incubation at 37 °C with alternative Elution Buffer (Tris–HCl pH 7.4 10 mM, MgCl2 1 mM, NaCl 40 mM) with 2 μl of RNAse cocktail (Ambion AM2286; RNases A and T1). RNA pull-down specificity was assessed by Western blotting using anti-CSDE1 antibody (Abcam, #96124, 1:1,000), anti-CIRBP antibody (Abcam, #94999, 1:500), anti-CELF1 (Santa Cruz, c-20003, 1:500) and anti-Actin (Sigma, #A2066, 1:1,000).

Ju Lee H., Bartsch D., Xiao C., Guerrero S., Ahuja G., Schindler C., Moresco J.J., Yates JR I.I.I., Gebauer F., Bazzi H., Dieterich C., Kurian L, & Vilchez D. (2017). A post-transcriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells. Nature Communications, 8, 1456.

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Genomic DNA Extraction from Marine Organisms

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Colonies were starved for at least 2 days before to DNA extraction. Tissue was harvested by scraping it from slide with a razor blade and transferring it into a 1.5 ml microcentrifuge tube, briefly centrifuging at max speed (> 21,000 g), pipetting off the seawater, and storing at − 80 °C until extraction. To extract genomic DNA, UEB1 buffer (7 M urea, 0.3125 M NaCl, 0.05 M Tris-HCl, 0.02 M EDTA, and 1% w:v N-lauroylsarcosine sodium salt) was added to the tissue, which was then ground with a pestle and incubated at 37 °C for 1 h. This was followed by a phenol/chloroform extraction and precipitation with 2.5 volumes 100% ethanol and 1/10 volume 5 M sodium acetate (pH 5.2). The precipitate was pelleted, washed with 70% ethanol, and resuspended with TE. RNA was removed by adding 1/100th volume of Ambion RNAse cocktail (Ambion #AM2286) and incubating at 37 °C for 15 min. This was followed by another ethanol precipitation. The precipitate was pelleted, washed with 70% EtOH, resuspended with TE, and diluted to a working stock concentration of 25 ng/μl.

Sanders S.M., Ma Z., Hughes J.M., Riscoe B.M., Gibson G.A., Watson A.M., Flici H., Frank U., Schnitzler C.E., Baxevanis A.D, & Nicotra M.L. (2018). CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus. BMC Genomics, 19, 649.

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EV RNA Isolation and RNase Treatment

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RNA isolation was performed using Ambion's Mirvana miRNA Isolation kit (cat. no. AM1560) following the manufacturer's protocol. Prior to RNA isolation, EVs were treated with Ambion RNase cocktail (cat. no. AM2286) at 37° for 15 min. One milliliter of lysis/binding buffer was immediately added to the RNase treated EVs to deactivate the RNase.

Chakrabortty S.K., Prakash A., Nechooshtan G., Hearn S, & Gingeras T.R. (2015). Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA. RNA, 21(11), 1966-1979.

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