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Mung bean nuclease

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Mung bean nuclease is an enzyme derived from mung bean sprouts. It is a single-stranded DNA and RNA-specific endonuclease that catalyzes the hydrolysis of phosphodiester bonds in single-stranded nucleic acids.

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Lab products found in correlation

Qiaquick gel extraction kit, by Qiagen (3 mentions) T4 ligase, by New England Biolabs (3 mentions) Anti mouse igg hrpo, by GE Healthcare (2 mentions) Dsdna, by Merck Group (2 mentions) Minelute column, by Qiagen (2 mentions) Ampure xp bead, by Beckman Coulter (2 mentions) Rnase a, by Merck Group (2 mentions) Megascript t7 kit, by Thermo Fisher Scientific (2 mentions) Dnase 1, by Promega (2 mentions) Xbai, by New England Biolabs (2 mentions) Odyssey fc imaging system, by LI COR (2 mentions) Hindiii, by Roche (2 mentions) Pcr dig probe synthesis kit, by Roche (2 mentions) Cdp star, by Roche (2 mentions) Nucleotide sequencing, by Eurofins (1 mentions) Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Dnase 1, by Sangon (1 mentions) Calf intestinal phosphatase, by New England Biolabs (1 mentions) Ligase, by New England Biolabs (1 mentions) T7 ribomax express large scale rna production system, by Promega (1 mentions)

39 protocols using mung bean nuclease

1

Functional Expression of Human nAChR α7

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Functional expression of insect only nAChRs in heterologous systems has proven technically difficult. In contrast, the vertebrate nAChR α7 readily expresses as a functional receptor in vitro and so human nAChR α7 in pSP64GL (Broadbent et al. 2011) was used to study the effect of exon 3 skipping on receptor functionality. Two BglII sites were introduced into hα7 flanking exon 3 using the QuikChange II XL site‐directed mutagenesis kit (Agilent Technologies). Exon 3 was excised and plasmid DNA treated with Mung bean nuclease (New England Biolabs) to remove the 5′ and 3′ overhangs prior to religation using T4 ligase. The absence of exon 3 and the preservation of the reading frame were verified by nucleotide sequencing (Eurofins Genomics).
Berger M., Puinean A.M., Randall E., Zimmer C.T., Silva W.M., Bielza P., Field L.M., Hughes D., Mellor I., Hassani‐Pak K., Siqueira H.A., Williamson M.S, & Bass C. (2016). Insecticide resistance mediated by an exon skipping event. Molecular Ecology, 25(22), 5692-5704.
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2

HBV Core DNA Extraction and Analysis

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HBV core DNA was extracted as previously described, with minor modifications [20 (link)]. Briefly, cells were lysed in 200 µL lysis buffer containing 0.5% NP-40, then incubated with Mung Bean Nuclease (M0250S, New England Biolabs, Ipswich, MA, USA) and DNase I (EN0521, Sangon Biotech, Shanghai, China) to remove the input plasmid DNA. Viral DNA was ethanol precipitated after protease K (Calbiochem, San Diego, CA, USA) digestion in the presence of 0.5% SDS at 55 °C overnight. Purified DNA was dissolved in 16 µL double-distilled water and separated by 1% agarose gel. Following overnight transfer to nylon membrane, the blot was hybridized with a Digoxigenin-labeled HBV-specific probe.
Zheng Y., Yang L., Yu L., Zhu Y., Wu Y., Zhang Z., Xia T, & Deng Q. (2023). Canocapavir Is a Novel Capsid Assembly Modulator Inducing a Conformational Change of the Linker Region of HBV Core Protein. Viruses, 15(5), 1195.
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3

Microsatellite DNA Isolation and Adapter Ligation

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Microsatellite DNA was isolated as described previously [21 (link)]. The extracted gDNA (1 μg) was digested with RsaI (New England Biolabs, Ipswich, MA, USA) for 10 s according to the manufacturer’s instructions. The resulting DNA fragments were treated with mung bean nuclease (New England Biolabs) for 30 min to obtain blunt ends and dephosphorylated using calf intestinal phosphatase (New England Biolabs). DNA fragments of 200–800 bp were separated using electrophoresis on 1.5% agarose gels and recovered using a QIAquick gel extraction kit (Qiagen, Germantown, MD, USA). The recovered DNA fragments were ligated to adapters (SNX/SNX reverse linker) by combining with 60 μM SNX adapter, 5 μL of NEB #2 buffer, 0.5 μL of 100× BSA, 1 μL each of NheI (New England Biolabs) and XmnI (New England Biolabs), 50 mM rATP (Promega, Madison, WI, USA), and 2000 units of ligase (New England Biolabs) in a total volume of 50 μL.
Hong Y.K., Kim K.R., Kim K.S, & Bang I.C. (2023). The Impact of Weir Construction in Korea’s Nakdong River on the Population Genetic Variability of the Endangered Fish Species, Rapid Small Gudgeon (Microphysogobio rapidus). Genes, 14(8), 1611.
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4

Measurement of Autoantibody Titers by ELISA

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Titers of serum anti-nucleosome antibodies were determined by ELISA as described previously [16 (link), 17 (link)]. In brief, met-BSA-precoated Immunolon plated were coated overnight with dsDAN and then with total histone solution. Samples were incubated on plates in various dilutions between 1:600 and 1:1,200, and then plates were washed, and autoantibodies were detected with anti-mouse IgG-HRPO (GE Healthcare).
Autoantibody titer was expressed as ELISA unit, comparing OD values of samples with a standard curve prepared with serial dilutions of ANA-positive NZM2410 serum pool. Antichromatin and anti-dsDNA titers were determined as for the antinucleosome levels. UV-irradiated Immunolon plates were incubated overnight with 3μg/ml chicken chromatin [24 ] or mung bean nuclease (New England Biolabs, Ins.)-treated dsDNA (Sigma-Aldrich. Anti-single-stranded DNA (ssDNA) was determined as describe previously [25 (link)]
Wang N., Keszei M., Halibozek P., Yigit B., Engel P, & Terhorst C. (2016). Slamf6 negatively regulates autoimmunity. Clinical immunology (Orlando, Fla.), 173, 19-26.
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5

Genome-length Jad cDNA Transcription and Transfection

Cited 1 time
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Genome-length Jad and intergenotypic Jad/C recombinant cDNAs were linearized with XbaI and treated with mung bean nuclease (New England BioLabs, Evry, France) prior to in vitro transcription using T7 RiboMAX Express Large Scale RNA production system (Promega) and purification of resulting synthetic RNAs, as described previously49 (link). Huh-7.5 cells (2 × 106 cells) were transfected by electroporation with 5 µg of synthetic, genome-length RNAs, in 4-mm-gap-width cuvettes by applying one pulse at 240 V at 900 F using EasyjecT Plus instrument (Equibio, Lancashire, United Kingdom). Electroporated cells were then immediately resuspended in complete medium and seeded at 1.6 × 106 cells per 75 cm2 flask.
Aicher S., Kakkanas A., Cohen L., Blumen B., Oprisan G., Njouom R., Meurs E.F., Mavromara P, & Martin A. (2018). Differential regulation of the Wnt/β-catenin pathway by hepatitis C virus recombinants expressing core from various genotypes. Scientific Reports, 8, 11185.
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6

Exonuclease and Mung Bean Nuclease Treatment of DNA Bands

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S1 nuclease-untreated DNA bands excised from PFGE gels was used for exonuclease treatment. After washing the excised plugs with TE buffer, exonuclease treatment was performed using a modified procedure described previously (Kinashi and Shimaji-Murayama, 1991 (link); Kalkus et al., 1993 (link); Overhage et al., 2005 (link); Rose and Fetzner, 2006 (link); Dib et al., 2010 (link)). The DNA bands were digested with 100 U of exonuclease III (New England BioLabs) at 37°C for 1, 2, or 3 h. In addition, the DNA bands were digested with 10 U of lambda exonuclease (New England BioLabs) at 37°C for 15 h.
To remove the single-stranded DNA, 1 U mung bean nuclease (New England BioLabs) was used at 30°C for 0.5 or 1 h. S1 nuclease-treated pMG2200 was used as a control for linearized circular plasmid (Zheng et al., 2009 (link)).
Hashimoto Y., Taniguchi M., Uesaka K., Nomura T., Hirakawa H., Tanimoto K., Tamai K., Ruan G., Zheng B, & Tomita H. (2019). Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate. Frontiers in Microbiology, 10, 2568.
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7

Selective Degradation of Viral DNA

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Viral DNA was extracted from a stock of PCV2 strain 1121, using QIAamp Cador Pathogen mini kit (Qiagen). Then, 1 µg of virus DNA was incubated with 100 units of the lysosomal DNase II (Sigma-Aldrich), in the presence of 50 mM sodium-acetate (pH 5.0) at 37 °C for 18 h to allow a complete reaction [24 (link)]. In addition, the Mung Bean Nuclease (New England BioLabs, Ipswich, MA, USA) which targets single-stranded DNA, and the Benzonase endonuclease (Millipore, Burlington, MA, USA) which degrades all forms of DNA and RNA, were used to control the degradation of PCV2 genome. Due to the small amount of DNA, instead of a direct visualization by agarose gel electrophoresis, the digested products were purified and evaluated by PCR with three sets of primers (Table 1). These primers amplified different regions of the PCV2 genome, as shown in Figure 3 [23 (link),25 (link)]. The PCR was performed with Herculase II fusion DNA polymerase as described in Section 2.6. The amplification products were examined by agarose gel electrophoresis.
Wei R., Trus I., Yang B., Huang L, & Nauwynck H.J. (2018). Breed Differences in PCV2 Uptake and Disintegration in Porcine Monocytes. Viruses, 10(10), 562.
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8

Generating HCV Genotype 2a Virus

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The plasmid pJFH1, containing the full‐length genomic cDNA sequence of the HCV genotype 2a strain and the N17/JFH117, 18 plasmid were linearized using XbaI enzyme (New England Biolabs) and then treated with Mung Bean Nuclease (NEB) prior purification. Linearized plasmids were used as a template to generate in vitro transcribed RNA using MEGAscript T7 (Life Technologies, Milan, Italy). The 10 μg of RNA were electroporated into Huh7 cells as previously described.19
Magri A., Barbaglia M.N., Foglia C.Z., Boccato E., Burlone M.E., Cole S., Giarda P., Grossini E., Patel A.H., Minisini R, & Pirisi M. (2016). 17,β‐estradiol inhibits hepatitis C virus mainly by interference with the release phase of its life cycle. Liver International, 37(5), 669-677.
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9

In Vitro RNA Transcription and Electroporation

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After linearization of the plasmids described in the former paragraph using XbaI and treatment with the Mung Bean Nuclease (New England Biolabs, Ipswich, MA, USA) as previously described [8 (link)], RNA was in vitro transcribed using MEGAscript® T7 transcription kit (Life Technologies, Carlsbad, CA, USA). Huh-7 cells were electroporated using 10 μg of RNA according to the method previously described [51 (link)], using 0.2 cm gap electroporation cuvettes (Biorad, Hercules, CA, USA).
Riva L., Spriet C., Barois N., Popescu C.I., Dubuisson J, & Rouillé Y. (2021). Comparative Analysis of Hepatitis C Virus NS5A Dynamics and Localization in Assembly-Deficient Mutants. Pathogens, 10(2), 172.
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10

Generation of Csf1r-mApple Transgenic Mice

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The 7.2 kb Csf1r reporter construct previously used to generate the Csf1r-EGFP mice (Sasmono 2003) was digested with ApaI and SalI (NEB) to remove EGFP before gel purification using the QIAquick gel extraction kit (Qiagen). Overhangs were removed with Mungbean nuclease (NEB), DNA purified using QIAGEN MinElute columns (Qiagen) and DNA then dephosphorylated using TSAP (Promega). A construct encoding the fluorescent protein Csf1r-mApple (34 (link)) was digested with SmaI and AflII and similarly purified and overhangs removed before both constructs were precipitated with EtOH/NaOAc and then ligated with T4 ligase (NEB) at 16°C overnight. The resulting Csf1r-mApple construct was transformed into DH5α competent cells. The Csf1r-rtTA-M2 construct utilizing the same 7.2 kb mouse Csf1r promoter region was used previously to generate Csf1r-rtTA transgenic mice (35 ) For generation of transgenic mice, plasmid backbones were removed by digestion with DrdI/PvuI (Csf1r-mApple, NEB) and SalI/MluI (Csf1r-rtTA, Promega/NEB) and then gel-purified using a QIAquick gel extraction kit. DNA was then further purified using AMPure XP beads (Agencourt) according to instructions.
Hawley C.A., Rojo R., Raper A., Sauter K.A., Lisowski Z.M., Grabert K., Bain C.C., Davis G.M., Louwe P.A., Ostrowski M.C., Hume D.A., Pridans C, & Jenkins S.J. (2018). Csf1r-mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System. The Journal of Immunology Author Choice, 200(6), 2209-2223.
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