Micrococcal nuclease mnase

Manufactured by New England Biolabs

Sourced in United States

Micrococcal nuclease (MNase) is an enzyme derived from the bacterium Staphylococcus aureus. It is a single-stranded DNA and RNA-specific endonuclease that cleaves phosphodiester bonds in nucleic acid molecules.

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Lab products found in correlation

Protease inhibitor cocktail, by Merck Group (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Proteinase k, by Thermo Fisher Scientific (2 mentions) Ab1791, by Abcam (1 mentions) Anti myc, by Merck Group (1 mentions) Hiseq 2500 sequencer, by Illumina (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Sequencing grade trypsin, by Promega (1 mentions) Yeast extract, by Thermo Fisher Scientific (1 mentions) Tryptone, by Thermo Fisher Scientific (1 mentions) Rnase free dnase, by Promega (1 mentions) Density gradient fractionation system, by BioComp Instruments (1 mentions) Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Human histone h4, by Merck Group (1 mentions) Goat anti rabbit igg, by Merck Group (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Wizard sv gel and pcr clean up system, by Promega (1 mentions) Mnase reaction buffer, by New England Biolabs (1 mentions)

16 protocols using micrococcal nuclease mnase

Chromatin Immunoprecipitation Assay in Arabidopsis

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The chromatin immunoprecipitation was conducted using 2-week-old seedlings of the wild type Col-0, DTF1-MYC/clsy1234, and DTF1-MYC/Col-0 grown in 1/2 strength MS solid medium as previously described^{43 (link)}. The antibody used for DTF1 ChIP assays was anti-MYC (16–219, Millipore). The DNA was diluted in 50 μl of TE, and a 1.5 μl volume was used for each qPCR reaction. The relative enrichment to input was calculated from three biological replicates.
Nuclei were extracted from 2-week-old Arabidopsis seedlings grown in 1/2 strength MS medium as previously described^{38 (link)} and were digested with Micrococcal Nuclease (MNase; NEB) before chromatin immunoprecipitation was conducted using antibodies against H3 (Abcam, ab1791) as previously reported^{44 (link)}.

Yang D.L., Zhang G., Wang L., Li J., Xu D., Di C., Tang K., Yang L., Zeng L., Miki D., Duan C.G., Zhang H, & Zhu J.K. (2018). Four putative SWI2/SNF2 chromatin remodelers have dual roles in regulating DNA methylation in Arabidopsis. Cell Discovery, 4, 55.

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Ultra-low input native ChIP-seq of H3K27me3

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Ultra-low input native ChIP-seq (ULI-NChIP-seq) using H3K27me3 antibody was performed as described previously (Brind’Amour et al., 2015 (link); Inoue et al., 2017b (link)). In brief, SCNT embryos were generated from Sertoli cells of Xist heterozygous knockout pups in BDF1 × PWK hybrid genetic background through Kdm4d mRNA injection. IVF embryos were generated using BDF1 oocytes and PWK sperm. Fifty to sixty IVF or SCNT embryos at the morula stage were collected at 72 hpa and pooled as a single sample with two biological replicates. After digestion of zona pellucida by acid Tyrode’s solution, polar bodies were removed by brief treatment with 0.25% trypsin 1 mM EDTA followed by several times of quick pipetting. The embryos were then washed three times with PBS containing 0.2% BSA (Merck # 12657) and processed for chromatin isolation and fragmentation by Micrococcal Nuclease (MNase: New England Biolabs # M0247S). 10% of MNase-treated chromatin solution was used as input. Rabbit anti-H3K27me3 antibody (Diagenode # C15410069) was used for immunoprecipitation. Sequencing libraries were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs # E7645S) following manufacturer’s instructions. Single-end 150 bp sequencing was performed on a HiSeq 2500 sequencer (Illumina).

Matoba S., Wang H., Jiang L., Lu F., Iwabuchi K.A., Wu X., Inoue K., Yang L., Press W., Lee J.T., Ogura A., Shen L, & Zhang Y. (2018). Loss of H3K27me3 Imprinting in Somatic Cell Nuclear Transfer Embryos Disrupts Post-Implantation Development. Cell stem cell, 23(3), 343-354.e5.

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Protein Purification and Mass Spectrometry

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Tryptone and yeast extract were obtained
from Fisher Scientific (Fairlawn, NJ). Trypsin (sequencing grade)
and RNase-free DNase were purchased from Promega (Madison, WI). Chymotrypsin
(sequencing grade) was obtained from Protea Biosciences (Morgantown,
WV). Micrococcal nuclease (MNase) was purchased from New England Biolabs
(Ipswich, MA). Phosphorylase b (MassPrep Standard) and Fibrinopeptide
b (Glu-Fib) were obtained from Waters (Milford, MA). Protease Inhibitor
Cocktail, endoproteinases Asp-N and Glu-C, chloramphenicol (CAM),
2-mercaptoethanol, dithiothreitol (DTT), iodoacetamide (IA), and molecular-grade
sucrose were obtained from Sigma (St. Louis, MO). Acids and organic
solvents were HPLC grade or better.

Dator R.P., Gaston K.W, & Limbach P.A. (2014). Multiple Enzymatic Digestions and Ion Mobility Separation Improve Quantification of Bacterial Ribosomal Proteins by Data Independent Acquisition Liquid Chromatography−Mass Spectrometry. Analytical Chemistry, 86(9), 4264-4270.

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Chromatin Accessibility Assay by CHART-PCR

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The chromatin accessibility assay was performed by chromatin accessibility by real-time PCR (CHART-PCR) method as previously described [20 (link)]. Cell pellets were resuspended in ice-cold Nonidet P-40 lysis buffer (10 mM Tris (pH 7.4), 10 mM Nacl, 3 mM MgCl₂, 0.15 mM spermine, 0.5 mM spermidine, and 0.5% Nonidet P-40) and incubated on ice for 5 min. The suspension was centrifuged at 3000 rpm for 5 min to pellet the nuclei. And then, the nuclei were suspended and digested with 5 units of micrococcal nuclease, MNase (New England BioLabs) or mock for 15 min. Purified genomic DNA was subjected to qPCR and the relative level of MNase resistance was calculated after normalization to mock-digested DNA. Primers for qPCR are following: PAX5BS (F: 5′-AAGGAGCAGGGAAGGAGGGGGAGG-3′, R: 5′-AGCGGCTAAAGGACCCCCGAGCTCGG-3′), PAX5BS-100 bp (F: 5′-CTGCGCAGGCGCGCTAGGGGGCTGC-3′, R: 5′-CAGGCCCGTTCTCCGCTGCCG-3′), and PAX5BS + 100 bp (F: 5′-GCAGCCTCTGCAGCTGGGTTTCCC-3′, R: 5′-GGGAAGAGGAGGGGGAAAGACAGGA-3′).

Xu S., Jiang C., Lin R., Wang X., Hu X., Chen W., Chen X, & Chen T. (2021). Epigenetic activation of the elongator complex sensitizes gallbladder cancer to gemcitabine therapy. Journal of Experimental & Clinical Cancer Research : CR, 40, 373.

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Cytosolic Lysate Preparation and Ribosome Profiling

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After cells were exposed to various treatments, cytosolic lysates were prepared using 20 mM Hepes pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 100 μg/ml digitonin, 100 μg/ml cycloheximide, 1X protease inhibitor cocktail (Sigma, #P2714) and 200 U RiboLock RNase Inhibitor (Thermo Fisher Scientific, #EO0382). Extracts were passed 10 times through a 26 G needle and incubated on ice for 5 min prior to centrifugation at 17,000 g for 5 min at 4 °C. After adding calcium chloride to a final concentration of 1 mM, lysates were optionally digested with 500 U micrococcal nuclease (MNase) (New England Biolabs, #M0247) for 30 min at 22 °C. Digestion was terminated by adding 2 mM EGTA. Equivalent amounts of lysate (100–120 μg of undigested RNA or 150–180 μg of MNase-digested RNA) were resolved on 15–50% sucrose gradients. Gradients were centrifuged at 38,000 rpm in a Sorvall TH64.1 rotor for 2.5 h at 4 °C. The gradients were passed through a Biocomp density gradient fractionation system with continuous monitoring of the absorbance at 260 nm.

Ryder L., Arendrup F.S., Martínez J.F., Snieckute G., Pecorari C., Shah R.A., Lund A.H., Blasius M, & Bekker-Jensen S. (2023). Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms. Cell Death & Disease, 14(7), 467.

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Histone H4 chromatin remodeling assay

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Micrococcal nuclease (MNase), New England Biolabs, Massachusetts, USA, Cat# M0247S; Human-Histone H4, Sigma-Aldrich, St. Louis, Missouri, USA, Cat# H2667; Human histone H4 antibodies, Proteintech, Rosemont, USA, Cat# 16047-1-AP; Goat anti-rabbit IgG, Sigma-Aldrich, St. Louis, Missouri, USA, Cat# A0545; TbUMSBP2 antibodies were prepared in rabbits by Adar Biotech, Rehovot, Israel. DNeasy Blood & Tissue Kits, Qiagen, Hilden, Germany, Cat# 69504; RNeasy Mini Kit, Qiagen, Hilden, Germany, Cat# 74104; RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher Scientific, Massachusetts, USA, Cat# K1621; KAPA SYBR® FAST qPCR Master Mix (2×) Kit, Merck, Darmstadt, Germany, Cat# KK4602.

Soni A., Klebanov-Akopyan O., Erben E., Plaschkes I., Benyamini H., Mitesser V., Harel A., Yamin K., Onn I, & Shlomai J. (2023). UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes. Nucleic Acids Research, 51(11), 5678-5698.

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Nuclei Isolation and MNase Digestion

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Briefly, 2 g of leaf tissues were ground into powder in liquid N₂. Afterwards, the nuclei are purified with a nuclei extraction buffer (0.25 M sucrose, 60 mM KCl, 15 mM MgCl₂, 1 mM CaCl₂, 15 mM PIPES (pH 6.8), 0.8% (v/v) Triton X-100, 1 mM PMSF), digested with 0.5 units/µL micrococcal nuclease (MNase) (NEB) for 10 min. Finally, purified nuclei are incubated with Proteinase K overnight at 37 °C.

Jabre I., Chaudhary S., Wilson C.M., Staiger D, & Syed N. (2022). Stochastic Variation in DNA Methylation Modulates Nucleosome Occupancy and Alternative Splicing in Arabidopsis thaliana. Plants, 11(9), 1105.

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MNase Digestion and DNA Purification

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Chromatin was digested by Micrococcal Nuclease (MNase) (New England Biolabs) following established procedure with modifications (30 ). 16 μg of DNA were digested for 10 min at room temperature following manufacturer's guidelines using 0.05 U/μl and 0.3 U/μl of MNase. Digestion was stopped by addition of EDTA to 100 mM final concentration. After the addition of 25 μg of Proteinase K (Thermofisher Scientific), the solution was incubated at 50°C for 30 min. The DNA was purified on a Wizard SV Gel and PCR clean-up system (Promega) and run on 10% polyacrylamide gel electrophoresis at 100 V at room temperature.

Zinchenko A., Berezhnoy N.V., Wang S., Rosencrans W.M., Korolev N., van der Maarel J.R, & Nordenskiöld L. (2017). Single-molecule compaction of megabase-long chromatin molecules by multivalent cations. Nucleic Acids Research, 46(2), 635-649.

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Chromatin Dissection and Nuclease Digestion

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Chromatin was dissected as described previously^{17 (link)}. Briefly, cells were grown to confluency on 8.5 cm dishes, UV-irradiated with the indicated UV doses and lysed on ice with NP-40 buffer [25 mM Tris-HCl (pH 8.0), 0.3 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) NP-40, 0.25 mM phenylmethylsulfonyl fluoride and EDTA-free protease inhibitor cocktail (Roche)]. Cell lysis was carried out for 30 min on a turning wheel. Free, non-chromatin bound proteins were recovered by centrifugation (10 min, 15’000 g). Micrococcal nuclease (MNase; New England Biolabs) digestion was then accomplished for 20 min at 37 °C. For that purpose, the chromatin-containing pellet was suspended in CS buffer [(20 mM Tris-HCl, pH 7.5, 100 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 0.3 M sucrose and 0.1% (v/v) Triton X-100)]^{55 (link)} and supplemented with 10× reaction buffer (500 mM Tris-HCl, pH 7.9, 50 mM CaCl₂), bovine serum albumin (1 mg·ml⁻¹) and MNase (4 U·μl⁻¹). Solubilised proteins were recovered by centrifugation (10 min, 15’000 g) after adding EDTA (5 mM) to stop the nuclease digestion.

Puumalainen M.R., Lessel D., Rüthemann P., Kaczmarek N., Bachmann K., Ramadan K, & Naegeli H. (2014). Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity. Nature communications, 5, 3695.

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Micrococcal Nuclease Digestion of NETs

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PMA-induced NETs were partially digested with 10 U/mL of Micrococcal nuclease (MNase; New England Biolabs [NEB]) in the presence of MNase reaction buffer (NEB) for 20 minutes at 37°C. The reaction was stopped by adding EDTA (MilliporeSigma) to a final concentration of 15 mM. NET protein concentration was determined using Bicinchoninic Acid Kit (Pierce) according to the manufacturer’s instructions.

Zuo Y., Yalavarthi S., Navaz S.A., Hoy C.K., Harbaugh A., Gockman K., Zuo M., Madison J.A., Shi H., Kanthi Y, & Knight J.S. (2021). Autoantibodies stabilize neutrophil extracellular traps in COVID-19. JCI Insight, 6(15), e150111.

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