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β agarase

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β-agarase is an enzyme that hydrolyzes the β-1,4-glycosidic bonds in agarose, a polysaccharide derived from certain red algae. It is used to break down agarose for various applications, such as the extraction of nucleic acids from agarose gels.

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Lab products found in correlation

Quant it dsdna assay kit, by Thermo Fisher Scientific (2 mentions) Qubit fluorometer, by Thermo Fisher Scientific (2 mentions) Invisorb spin plant mini kit, by Stratec (2 mentions) Mab3034, by Merck Group (2 mentions) Ab6946, by Abcam (2 mentions) Bal31 nuclease, by New England Biolabs (2 mentions) Attovision, by BD (1 mentions) Oligonucleotide, by Integrated DNA Technologies (1 mentions) Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) Gtg low melting temperature agarose for in gel cloning, by Lonza (1 mentions) Igg2a, by Merck Group (1 mentions) Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions) Nusieve gtg, by Lonza (1 mentions) Penicillin streptomycin amphotericin b, by Lonza (1 mentions) Obt0030, by Merck Group (1 mentions) Agarose, by Bio-Rad (1 mentions) Viscolase, by A&A Biotechnology (1 mentions) Amicon filter, by Merck Group (1 mentions) λ phage dna, by Thermo Fisher Scientific (1 mentions) S adenosylmethionine, by New England Biolabs (1 mentions)

Close spelling variants of this product

β-Agarase I (10 citations) Restriction enzymes and β-agarase (3 citations) Agarase (2 citations)

19 protocols using β agarase

1

Generation of Slc25a46 Transgenic Mice

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Human BAC clone RP11-671K7 (ImaGenes, GmbH) was digested with NotI and products were analyzed by Pulsed Field Electrophoresis. A 202.693 bp fragment containing the human Slc25a46 gene was isolated upon electrophoresis in a 4% low melting agarose gel and digestion with β-agarase (New England Biolabs). This fragment was microinjected into fertilized (C57BL/6J x CBA/J) F2 oocytes as previously described [18 (link),38 (link),39 (link)]. Microinjections and embryo implantations were carried out by Transgenics & Gene Targeting Facility at B.S.R.C. 'Alexander Fleming'. Screening of transgenic mice was performed by PCR analysis on genomic DNA with primers that hybridize a sequence of the human but not the mouse gene as follows: F, 5'-AAT CAC GTG CTC CGA AGA CT-3'; R, 5'-TAA CCC CTC ATC CCT GTG TC-3'.
Terzenidou M.E., Segklia A., Kano T., Papastefanaki F., Karakostas A., Charalambous M., Ioakeimidis F., Papadaki M., Kloukina I., Chrysanthou-Piterou M., Samiotaki M., Panayotou G., Matsas R, & Douni E. (2017). Novel insights into SLC25A46-related pathologies in a genetic mouse model. PLoS Genetics, 13(4), e1006656.
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2

Molecular Combing of DNA Replication Tracts

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Molecular combing was performed as described (6 (link)). Briefly, at the end of the CldU pulse, trypsinized cells were embedded in low-melting agarose. After digestion with β-agarase (New England Biolabs), DNA was combed on silanized surfaces (Microsurfaces, Inc.) and replicons were detected with anti-IdU and anti-CldU antibodies. Images were captured with the software Attovision using the epifluorescence microscope Pathway (Becton Dickinson). Signals were measured using ImageJ (open source from National Cancer Institute, NIH) with custom-made modifications.
Jossé R., Martin S.E., Guha R., Ormanoglu P., Pfister T.D., Reaper P.M., Barnes C.S., Jones J., Charlton P., Pollard J.R., Morris J., Doroshow J.H, & Pommier Y. (2014). ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase I inhibitors by disabling DNA replication initiation and fork elongation responses. Cancer research, 74(23), 6968-6979.
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3

Genomic DNA Isolation and Library Preparation

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An Invisorb Spin Plant Mini Kit (Stratec Molecular) was used to isolate genomic DNA and prepare short-read libraries for the Roche-454 and Illumina sequencing platforms. DNA concentrations were determined using a Quant-iT dsDNA Assay Kit (Life Technologies) and a Qubit Fluorometer (Invitrogen). We checked the integrity of the genomic DNA by agarose gel electrophoresis and pulsed-field gel electrophoresis. Agarose-embedded high-molecular weight (HMW) DNA was prepared as described previously48 , and modified as described previously49 (link), to construct Illumina TruSeq Synthetic Long Read (TSLR) libraries. Agarose gel plugs were washed three times in Tris EDTA buffer and subjected to digestion with 8 U of β-agarase (New England Biolabs) for 12–16 h at 42 °C. HMW DNA was then drop-dialysed for 2.5 h. DNA concentrations were quantified with the Quant-iT dsDNA Assay Kit. DNA quality was then checked using an Argus Qcard Kit (OpGen) and was estimated at 20–100 kb.
Plomion C., Aury J.M., Amselem J., Leroy T., Murat F., Duplessis S., Faye S., Francillonne N., Labadie K., Le Provost G., Lesur I., Bartholomé J., Faivre-Rampant P., Kohler A., Leplé J.C., Chantret N., Chen J., Diévart A., Alaeitabar T., Barbe V., Belser C., Bergès H., Bodénès C., Bogeat-Triboulot M.B., Bouffaud M.L., Brachi B., Chancerel E., Cohen D., Couloux A., Da Silva C., Dossat C., Ehrenmann F., Gaspin C., Grima-Pettenati J., Guichoux E., Hecker A., Herrmann S., Hugueney P., Hummel I., Klopp C., Lalanne C., Lascoux M., Lasserre E., Lemainque A., Desprez-Loustau M.L., Luyten I., Madoui M.A., Mangenot S., Marchal C., Maumus F., Mercier J., Michotey C., Panaud O., Picault N., Rouhier N., Rué O., Rustenholz C., Salin F., Soler M., Tarkka M., Velt A., Zanne A.E., Martin F., Wincker P., Quesneville H., Kremer A, & Salse J. (2018). Oak genome reveals facets of long lifespan. Nature Plants, 4(7), 440-452.
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4

Construction of Anti-MARV NP Single-Domain Antibodies

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Recombinant DNA methods were according to established procedures and employed commercially available reagents; Phusion High-Fidelity DNA Polymerase (Thermo Fisher, Waltham, MA, USA); restriction enzymes and β-agarase (New England BioLabs, Beverly, MA, USA); T4 DNA ligase, CIP and T4 PNK (Roche, Nutley, NJ, USA); GTG low melting temperature agarose for in gel cloning (Lonza, Walkersville, MD, USA); oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA); cloned synthetic DNA (Genscript, Piscataway, NJ, USA). Assemblies involving cloning and PCR amplification were sequenced through the inserts and junctions to verify the desired construct. Cloning was typically carried out in XL1-Blue cells unless otherwise stated. Parental sdAb genes employed in this work were anti-MARV NP–sdAb A, B, C, and D with GenBank accession numbers MF780583, MF780584, MF780585, and MF780586, respectively. Full details of cloning, oligonucleotides, maps, and sequences of resulting constructs are available on request.
Garza J.A., Taylor A.B., Sherwood L.J., Hart P.J, & Hayhurst A. (2017). Unveiling a Drift Resistant Cryptotope within Marburgvirus Nucleoprotein Recognized by Llama Single-Domain Antibodies. Frontiers in Immunology, 8, 1234.
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5

Soil Metagenome Library Construction

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Extraction of soil DNA for the construction of a metagenomic library was performed using a modification of a previous protocol (Van Elsas et al. 2008 (link)). Briefly, 10 g of soil were suspended in 10 ml buffer (100 mM Tris-HCl, 100 mM NaEDTA, 100 mM NaPO4, 1.5 % NaCl, 1 % CTBA, pH 8.0), shortly Vortex-mixed and sonicated (water bath) for 15 min. After sonication, 100 μl of proteinase K (10 mg/ml) were added, which was followed by incubation at 37 °C (2 h) with shaking at 200 rpm. DNA was gently extracted with phenol/chloroform/iso-amylalcohol (25:24:1) at 60 °C for 30 min. The DNA was then precipitated with 2-propanol, dissolved and embedded in plugs of (1 %) low-melting-point agarose. Then, 30–40 kb DNA fragments were separated by pulsed-field gel electrophoresis (PFGE) at 14 °C on 1 % agarose gels supplemented (upper part) with 2 % polyvinyl pyrrolidone (PVP), in 0.5 strength Tris-borate-EDTA (TBE). A PFGE DRIII system (BioRad, CA, USA) was used with gradient of 6 V/cm, included angle 120o, initial switch time 0.5 s, final switch time 8.5 s, linear ramping factor 20 h. Agarose fragments (2 cm) containing the 30–40 kb DNA fragments were then excised from gel, after which a β-agarase (New England Biolabs, MA, USA) treatment allowed recovery of the DNA.
Cretoiu M.S., Berini F., Kielak A.M., Marinelli F, & van Elsas J.D. (2015). A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil. Applied Microbiology and Biotechnology, 99(19), 8199-8215.
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6

Measuring Replication Dynamics in Cells

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As previously described (25 ), asynchronous DMS114 and H524 cells were sequentially labelled with 20 μM IdU for 20 min and 50 μM CldU for 20 min. To preserve long genomic DNA fibers, cells were embedded in low melting point agarose plugs and incubated in cell lysis buffer with proteinase K at 50°C for overnight. Washed plugs with TE buffer, and then melted plugs in 0.1M MES (pH 6.5) at 70°C for 20 min. Agarose was subsequently degraded by adding 2 μl of β-agarase (New England Biolabs). DNA fibers were then stretched onto salinized coverslips (Genomic Vision, cov-002-RUO) using an in-house combing machine. Combed DNA on coverslips was then baked at 60 °C for 2 hours and denatured in 0.5 N NaOH for 20 min. IdU, CldU and single-strand DNA were detected using a mouse antibody directed against BrdU (IgG1, Becton Dickinson, 347580, 1:25 dilution), a rat antibody directed against BrdU (Accurate chemical, OBT0030, 1:200 dilution) and a mouse antibody directed against single-stranded DNA (ssDNA) (IgG 2a, Millipore, MAB3034, 1:100), respectively. The secondary antibodies used were goat anti-mouse Cy3 (Abcam ab6946), goat anti-rat Cy5 (Abcam, ab6565), and goat anti-mouse BV480 (Jackson ImmunoResearch, 115–685-166) for ssDNA. Slides were scanned with a FiberVision Automated Scanner (Genomic Vision). Replication signals on single DNA fibers were analyzed using FiberStudio (Genomic Vision).
Takahashi N., Kim S., Schultz C.W., Rajapakse V.N., Zhang Y., Redon C.E., Fu H., Pongor L., Kumar S., Pommier Y., Aladjem M.I, & Thomas A. (2022). Replication Stress Defines Distinct Molecular Subtypes Across Cancers. Cancer Research Communications, 2(6), 503-517.
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7

Isolation and Purification of Micronuclear DNA from Ciliate Strains

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We maintain two previously characterized strains of C. uncinata, Pol = ATCC PRA-256, USA = USA-SC2, following protocols in Katz et al. (2011) (link). To isolate DNA, cultures were treated overnight with antibiotics and cells were pelleted by spinning at 5,000 rpm for 20 min. Genomic DNA was extracted using phenol/chloroform following standard protocols (Ausubel et al. 1993 ). Micronuclear DNA was isolated according to Katz and Kovner (2010) . Briefly, micronuclear DNA was isolated by gel electrophoresis using Low Melt UltraClean™ Agarose (Mobio15005-50, Carlsbad, CA) after digesting with Bal-31 Nuclease (New England Biolabs M02135, Ispwich, MA) to enrich micronuclear DNA. Gel isolated micronuclear DNA was purified using β-agarase (New England Biolabs M03925).
Gao F., Song W, & Katz L.A. (2014). Genome structure drives patterns of gene family evolution in ciliates, a case study using Chilodonella uncinata (Protista, Ciliophora, Phyllopharyngea). Evolution; international journal of organic evolution, 68(8), 2287-2295.
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8

DNA Combing Assay for Replication Dynamics

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DNA combing assay was performed as described(Josse et al., 2014 (link)). After drug treatments and CldU-IdU pulses, DMS114 cells were embedded in low-melting agarose for digestion with β-agarase (New England Biolabs). DNA was then combed on silanized surfaces (Microsurfaces, Inc.) and replicons were detected with anti-CIdU and anti-IdU antibodies. Signal was captured and analyzed with FiberVision automated scanner (Genomic Vision).
Thomas A., Takahashi N., Rajapakse V.N., Zhang X., Sun Y., Ceribelli M., Wilson K.M., Zhang Y., Beck E., Sciuto L., Nichols S., Elenbaas B., Puc J., Dahmen H., Zimmermann A., Varonin J., Schultz C.W., Kim S., Shimellis H., Desai P., Klumpp-Thomas C., Chen L., Travers J., McKnight C., Michael S., Itkin Z., Lee S., Yuno A., Lee M.J., Redon C.E., Kindrick J.D., Peer C.J., Wei J.S., Aladjem M.I., Figg W.D., Steinberg S.M., Trepel J.B., Zenke F.T., Pommier Y., Khan J, & Thomas C.J. (2021). Therapeutic targeting of ATR yields durable regressions in small cell lung cancers with high replication stress. Cancer cell, 39(4), 566-579.e7.
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9

Preparation of DNA Fiber Samples

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The washed plug was transferred to a 2 ml tube and melted for 20 minutes at 70°C in 0.1 M MES (pH 6.5). Melted plug was cooled for 5 minutes in a 42°C water bath. 3 units of β-agarase (New England BioLabs, M0392S) were added to cooled DNA solution and incubated overnight in a 42°C water bath. DNA solution was carefully poured into a 2 ml Teflon reservoir. A silanized coverslip was inserted into the DNA solution and incubated for 5 minutes. The coverslip was removed and adhered to a glass slide with cyanoacrylate glue. A test coverslip was pulled, stained with YOYO-1 (2:10,000 YOYO-1 in 0.1 M MES), and visualized with a microscope to ensure quality and density of DNA fibers. Coverslips were baked for 30 minutes at 60°C to crosslink DNA fibers to coverslip.
Warburton A., Redmond C.J., Dooley K.E., Fu H., Gillison M.L., Akagi K., Symer D.E., Aladjem M.I, & McBride A.A. (2018). HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression. PLoS Genetics, 14(1), e1007179.
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10

Genetic Sequence Verification of Transgenic Mice

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Selected regions of Tg4510 and rT2/T2 genomic DNA were amplified by PCR using Herculase II Fusion DNA polymerase (Agilent Technologies). For Tau transgene junctions in Tg4510 DNA, PCR and nested PCR reactions were run (Supplementary Tables 3–5). CaMKIIα-tTA transgene junctions were confirmed using rT2/T2 DNA (Supplementary Tables 6 and 7). Gel bands were isolated from a low melting point agarose gel (NuSieve GTG, Lonza) and DNA was purified following digestion with β-agarase (New England Biolabs). Gel-purified PCR products were sequenced with classical Sanger sequencing at the University of Minnesota Genomics Center, Minneapolis, MN.
Gamache J., Benzow K., Forster C., Kemper L., Hlynialuk C., Furrow E., Ashe K.H, & Koob M.D. (2019). Factors other than hTau overexpression that contribute to tauopathy-like phenotype in rTg4510 mice. Nature Communications, 10, 2479.
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