cutsmart  (New England Biolabs)


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    Name:
    CutSmart Buffer
    Description:
    CutSmart Buffer 5 0 ml
    Catalog Number:
    b7204s
    Price:
    24
    Size:
    5 0 ml
    Category:
    Buffers
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    Structured Review

    New England Biolabs cutsmart
    CutSmart Buffer
    CutSmart Buffer 5 0 ml
    https://www.bioz.com/result/cutsmart/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cutsmart - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: Paragraph title: Barcoded guide-donor library cloning ... 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C.

    Centrifugation:

    Article Title: Long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele‐specific MYC expression in HeLa cells
    Article Snippet: .. The cytoplasmic components were removed by centrifugation and the nuclear pellets were dissolved in 1× cutsmart buffer (NEB) containing 0.3% SDS. .. Then, 2% Triton X‐100 was added and incubated for an hour.

    Amplification:

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: The guide-donor oligos were amplified using KAPA HiFi polymerase as directed by the manufacturer in 50 uL total reaction volume with an initial denaturation of 98°C for 1 min, and then 15 cycles of 98°C 10s, 60°C 20s, and 72°C 30s. .. 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C.

    Article Title: Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases
    Article Snippet: Amplification was performed in a 25-μl reaction mixture consisting of 0.75 μl 10 mM AMRE74L forward primer, 0.75 μl 10 mM AMRE59 reverse primer, 19.85 μl distilled water (dH2 O), 2.25 μl 11.1× buffer (for the recipe, see reference ), 0.2 μl Kapa Taq (5 U/μl) (Kapa Biosystems, UK), 0.15 μl Tris (pH 8.8), 0.05 μl Pfu (2.5 U/μl), and 1 μl resuspended cells or 1 single colony as the template. .. A total of 10 to 15 μl of the PCR product was digested according to the manufacturer’s instructions, using 1 U DraI (New England Biolabs, UK), 2 U PleI (New England Biolabs, UK), and 1× CutSmart buffer (New England Biolabs, UK) in a total volume of 20 μl.

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: .. Suspension testing of beads coupled to amplification probes Five µL of bead preparations ASP-7RV-polyG::polyC-HRP and ARV-7SP-polyG::polyC-HRP (at 2 mg/mL concentrations) were placed into individual 0.5 mL Eppendorf tubes, and washed twice with 10 µL of CutSmart buffer (New England Biolabs). .. The first preparation, ASP-7RV-polyG::polyC-HRP, was used to add 5 µL of the target solution: either CutSmart buffer supplemented at various concentrations with the SP trigger oligonucleotide (Table ), or a negative control of the same buffer with no targets added.

    Article Title: Genetic variants of major genes contributing to phosphate and calcium homeostasis and their association with serum parameters in pigs
    Article Snippet: .. Digestion of amplification products was performed using 10 μl PCR product, 2 μl CutSmart Buffer (New England BioLabs), and 1 U of restriction enzyme and filled up with aqua dest to a final volume of 20 μl. .. The reaction conditions were 16 h (for PTH1R and TRAFD1 ) or 2 h (for STC1 ) at 37 °C for incubation and 20 min at 65 °C for enzyme inactivation.

    Article Title: Cryptic Plutella species show deep divergence despite the capacity to hybridize
    Article Snippet: Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGxxxxxxTGCA- 3′ , bottom strand 5′ -[P]xxxxxxCTGTCTCTTATACACATCTGACGCTGCCGACGA- 3′ , x represents sites for MIDs) were then added using 0.5 µL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer. .. Libraries were amplified using KAPA HiFi Hotstart Readymix (Kapa Biosystems) and Nextera i7 and i5 indexed primers with PCR conditions: 95 °C for 3 min, two cycles of 98 °C for 20 s, 54 °C for 15 s, 72 °C for 1 min, then 15 cycles of 98 °C for 20 s, 65 °C for 15 s, 72 °C for 1 min followed by a final extension of 72 °C for 5 min.

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: .. To start the signal amplification cascade, 20 µL of CutSmart buffer supplemented with a particular concentration of the SP trigger oligonucleotide, and 20 µL of CutSmart buffer containing two REases, EcoRV and SspI (0.8 U/µL each) were added to the top of the second filter. .. The resultant microplate was incubated for various time intervals at 37 °C with shaking (90 rpm).

    Reporter Assay:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: Paragraph title: Lamprey reporter assay ... Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo.

    Construct:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo. .. For each injected construct, the tissue-specific GFP expression domains were noted, along with the number and proportion of screened embryos exhibiting GFP expression in each of those domains.

    Electrophoresis:

    Article Title: Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases
    Article Snippet: A total of 10 to 15 μl of the PCR product was digested according to the manufacturer’s instructions, using 1 U DraI (New England Biolabs, UK), 2 U PleI (New England Biolabs, UK), and 1× CutSmart buffer (New England Biolabs, UK) in a total volume of 20 μl. .. Following digestion, each 6-carboxyfluorescein (FAM)-labeled SpnIII variant has a unique size (Table S3) that can be distinguished by capillary electrophoresis on an ABI prism gene analyzer (Applied Biosystems, USA).

    Incubation:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: .. Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo. .. Embryos were screened for fluorescent reporter expression using a Zeiss SteREO Discovery V12 microscope.

    Article Title: A method for measuring the distribution of the shortest telomeres in cells and tissues
    Article Snippet: To ligate TeSLA-T (TeSLA-T 1–6) to each telomere overhang, 1000 units of T4 DNA ligase (New England Biolabs), 1 mM ATP, and 10−3 μM of TeSLA-Ts were added to a final volume of 20 μl in 1× CutSmart buffer (New England Biolabs) with 50 ng of isolated genomic DNA (without RE digestion). .. The inactivated mixture including two units of Cvi AII in 10 μl 1× CutSmart buffer was incubated at 25 °C for 2 h to generate genomic DNA fragments with 5′ AT overhangs.

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: .. 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C. .. Reactions were column-cleaned and 400 ng of each insert was ligated with T4 DNA ligase into 1 ug of recipient vector ( > 7:1 insert:vector) treated with NotI, AscI, as well as CIP (NEB) – either pKR216 (SEC14 library) or pKR348 (natural variants library) – in a total volume of 20 uL.

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: Suspension testing of beads coupled to amplification probes Five µL of bead preparations ASP-7RV-polyG::polyC-HRP and ARV-7SP-polyG::polyC-HRP (at 2 mg/mL concentrations) were placed into individual 0.5 mL Eppendorf tubes, and washed twice with 10 µL of CutSmart buffer (New England Biolabs). .. Finally, the two bead preparations were mixed together to start the REase-driven amplification cascade, and incubated at 37 °C in a rotisserie for a given time.

    Article Title: Long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele‐specific MYC expression in HeLa cells
    Article Snippet: Chromosome conformation capture Adherent cultured cells were made single‐cell suspension and fixed by adding formaldehyde to a final concentration of 2% followed by incubation for 10 min at room temperature. .. The cytoplasmic components were removed by centrifugation and the nuclear pellets were dissolved in 1× cutsmart buffer (NEB) containing 0.3% SDS.

    Article Title: Genetic variants of major genes contributing to phosphate and calcium homeostasis and their association with serum parameters in pigs
    Article Snippet: Digestion of amplification products was performed using 10 μl PCR product, 2 μl CutSmart Buffer (New England BioLabs), and 1 U of restriction enzyme and filled up with aqua dest to a final volume of 20 μl. .. The reaction conditions were 16 h (for PTH1R and TRAFD1 ) or 2 h (for STC1 ) at 37 °C for incubation and 20 min at 65 °C for enzyme inactivation.

    Article Title: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia
    Article Snippet: .. The DNA (2 μg) was incubated with 3 μl of Nla III restriction enzyme (10,000 units/ml) 37 °C overnight in the CutSmart™ Buffer (NEB Inc., Ipswich, MA), and the enzyme was inactivated by incubation at 65 °C for 20 min. .. The fragmented DNA was then incubated with anti-5-mC and anti-5-hmC antibodies in IP buffer for 6 h at 4 °C.

    Expressing:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo. .. Embryos were screened for fluorescent reporter expression using a Zeiss SteREO Discovery V12 microscope.

    Modification:

    Article Title: Cryptic Plutella species show deep divergence despite the capacity to hybridize
    Article Snippet: RADseq library preparation and sequencing Libraries were prepared for restriction-site-associated DNA sequencing (RADseq) according to a protocol modified from Baird et al. [ ]. .. Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGxxxxxxTGCA- 3′ , bottom strand 5′ -[P]xxxxxxCTGTCTCTTATACACATCTGACGCTGCCGACGA- 3′ , x represents sites for MIDs) were then added using 0.5 µL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer.

    Article Title: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia
    Article Snippet: Paragraph title: Crosslink-assistant DNA modification immunoprecipitation assay ... The DNA (2 μg) was incubated with 3 μl of Nla III restriction enzyme (10,000 units/ml) 37 °C overnight in the CutSmart™ Buffer (NEB Inc., Ipswich, MA), and the enzyme was inactivated by incubation at 65 °C for 20 min.

    Transformation Assay:

    Article Title: Entirely enzymatic nanofabrication of DNA–protein conjugates
    Article Snippet: MOSIC oligonucleotides production The plasmids enclosing the oligonucleotide pseudogenes (GeneArt, Thermo Fisher) were transformed by heat shock in subcloning efficient Dh5α (Invitrogen) and grown overnight in 10 ml LB media supplemented with 100 μg/ml ampicillin. .. 20 ug of the obtained plasmid were digested for 4 h with BsaI-HF (NEB) in 100 μl with the addition of 10 μl of CutSmart buffer (NEB) and the psudogenes extracted from a 0.7% agarose gel, 0.5× TAE, using illustra GFX PCR DNA and gel band purification kit (GE Healthcare).

    Electroporation:

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C. .. 1 uL of each reaction was then electroporated into 20 uL NEB 10-beta in 0.1 cm-gap electroporation cuvettes (Bio-Rad) with the Bio-Rad GenePulser electroporator using the settings 1.7 kV, 200 Omega, and 25 μF.

    Ligation:

    Article Title: A method for measuring the distribution of the shortest telomeres in cells and tissues
    Article Snippet: TeSLA for TL measurement Before starting the TeSLA procedure, we make stocks of short double-stranded 5′ AT and TA overhang adapters for ligation at genomic and subtelomeric regions. .. To ligate TeSLA-T (TeSLA-T 1–6) to each telomere overhang, 1000 units of T4 DNA ligase (New England Biolabs), 1 mM ATP, and 10−3 μM of TeSLA-Ts were added to a final volume of 20 μl in 1× CutSmart buffer (New England Biolabs) with 50 ng of isolated genomic DNA (without RE digestion).

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C. .. Ligation reactions were ethanol precipitated by adding 80 uL 100% EtOH and 2 uL of 5M NaOAc pH 5.2 with 1 uL of glycoblue (Ambion), incubated on ice for 10 minutes and spun at 13.2 krpm for 5 min, washed with 70% ethanol, and then re-suspended in 3 uL of nuclease-free water (IDT).

    Cell Culture:

    Article Title: Long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele‐specific MYC expression in HeLa cells
    Article Snippet: Chromosome conformation capture Adherent cultured cells were made single‐cell suspension and fixed by adding formaldehyde to a final concentration of 2% followed by incubation for 10 min at room temperature. .. The cytoplasmic components were removed by centrifugation and the nuclear pellets were dissolved in 1× cutsmart buffer (NEB) containing 0.3% SDS.

    Article Title: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia
    Article Snippet: DNA was isolated from cultured cells or mononuclear bone marrow cells using a Qiagen DNAeasy kit (catalogue # 69506, Valencia, CA). .. The DNA (2 μg) was incubated with 3 μl of Nla III restriction enzyme (10,000 units/ml) 37 °C overnight in the CutSmart™ Buffer (NEB Inc., Ipswich, MA), and the enzyme was inactivated by incubation at 65 °C for 20 min.

    Generated:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: .. Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo. .. Embryos were screened for fluorescent reporter expression using a Zeiss SteREO Discovery V12 microscope.

    DNA Sequencing:

    Article Title: Cryptic Plutella species show deep divergence despite the capacity to hybridize
    Article Snippet: RADseq library preparation and sequencing Libraries were prepared for restriction-site-associated DNA sequencing (RADseq) according to a protocol modified from Baird et al. [ ]. .. Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGxxxxxxTGCA- 3′ , bottom strand 5′ -[P]xxxxxxCTGTCTCTTATACACATCTGACGCTGCCGACGA- 3′ , x represents sites for MIDs) were then added using 0.5 µL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer.

    Sequencing:

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: We amplified subpools with a forward primer harboring an AscI restriction site at its 3′-end and a reverse primer with a NotI site at its 5′-end followed by a degenerate barcode encoding a pseudo-random sequence (either NNNVHTGNNNVHTGNNNVHTGNNNVHTGNNN or NNNTGVHNNNTGVHNNNTGVHNNNTGVHNNN) that excludes illegal restriction sites (NotI, AscI, and BspQI), followed by subpool-specific priming sequence. .. 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C.

    Article Title: Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing
    Article Snippet: As such the RMS analysis included 13 of the 109 women in the combined 16S rRNA gene sequencing analysis and was augmented with another 3 women who also had high levels of these OTUs in two sample types, but where their third sample type was not available ( ). .. Previously isolated DNA (~10 ng) was digested using 8 U of EcoRI (New England Biolabs, UK) and 4 U of MseI (New England Biolabs, UK) with addition of 1× CutSmart Buffer (New England Biolabs, UK) at 37 °C for 1 h and AFLP adapters (EcoRI-F: 5′-CTCGTAGACTGCGTACC-3′; EcoRI-R: 5′-AATTGGTACGCAGTCTAC-3′; MseI-F: 5′-GACGATGAGTCCTGAG-3′; MseI-R: 5′-TACTCAGGACTCAT-3′) were ligated.

    Article Title: Cryptic Plutella species show deep divergence despite the capacity to hybridize
    Article Snippet: Paragraph title: RADseq library preparation and sequencing ... Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGxxxxxxTGCA- 3′ , bottom strand 5′ -[P]xxxxxxCTGTCTCTTATACACATCTGACGCTGCCGACGA- 3′ , x represents sites for MIDs) were then added using 0.5 µL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer.

    Injection:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: .. Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo. .. Embryos were screened for fluorescent reporter expression using a Zeiss SteREO Discovery V12 microscope.

    Methylation:

    Article Title: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia
    Article Snippet: Crosslink-assistant DNA modification immunoprecipitation assay Crosslink-assisted DNA modification IP assay was modified from the previously published methylated DNA IP protocol . .. The DNA (2 μg) was incubated with 3 μl of Nla III restriction enzyme (10,000 units/ml) 37 °C overnight in the CutSmart™ Buffer (NEB Inc., Ipswich, MA), and the enzyme was inactivated by incubation at 65 °C for 20 min.

    Isolation:

    Article Title: A method for measuring the distribution of the shortest telomeres in cells and tissues
    Article Snippet: .. To ligate TeSLA-T (TeSLA-T 1–6) to each telomere overhang, 1000 units of T4 DNA ligase (New England Biolabs), 1 mM ATP, and 10−3 μM of TeSLA-Ts were added to a final volume of 20 μl in 1× CutSmart buffer (New England Biolabs) with 50 ng of isolated genomic DNA (without RE digestion). .. The inactivated mixture including two units of Cvi AII in 10 μl 1× CutSmart buffer was incubated at 25 °C for 2 h to generate genomic DNA fragments with 5′ AT overhangs.

    Article Title: Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing
    Article Snippet: .. Previously isolated DNA (~10 ng) was digested using 8 U of EcoRI (New England Biolabs, UK) and 4 U of MseI (New England Biolabs, UK) with addition of 1× CutSmart Buffer (New England Biolabs, UK) at 37 °C for 1 h and AFLP adapters (EcoRI-F: 5′-CTCGTAGACTGCGTACC-3′; EcoRI-R: 5′-AATTGGTACGCAGTCTAC-3′; MseI-F: 5′-GACGATGAGTCCTGAG-3′; MseI-R: 5′-TACTCAGGACTCAT-3′) were ligated. .. Resulting fragments were enriched in PCR reaction with EcoRI (5′-GACTGCGTACCAATTC-3′) and MseI (5′-GATGAGTCCTGAGTAA-3′) primers [ ].

    Subcloning:

    Article Title: Entirely enzymatic nanofabrication of DNA–protein conjugates
    Article Snippet: MOSIC oligonucleotides production The plasmids enclosing the oligonucleotide pseudogenes (GeneArt, Thermo Fisher) were transformed by heat shock in subcloning efficient Dh5α (Invitrogen) and grown overnight in 10 ml LB media supplemented with 100 μg/ml ampicillin. .. 20 ug of the obtained plasmid were digested for 4 h with BsaI-HF (NEB) in 100 μl with the addition of 10 μl of CutSmart buffer (NEB) and the psudogenes extracted from a 0.7% agarose gel, 0.5× TAE, using illustra GFX PCR DNA and gel band purification kit (GE Healthcare).

    Microscopy:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo. .. Embryos were screened for fluorescent reporter expression using a Zeiss SteREO Discovery V12 microscope.

    Purification:

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: Reactions were column-cleaned with the Qiagen QIAquick PCR purification kit. .. 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C.

    Article Title: Cryptic Plutella species show deep divergence despite the capacity to hybridize
    Article Snippet: Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGxxxxxxTGCA- 3′ , bottom strand 5′ -[P]xxxxxxCTGTCTCTTATACACATCTGACGCTGCCGACGA- 3′ , x represents sites for MIDs) were then added using 0.5 µL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer. .. DNA purification between steps was performed using a MinElute PCR purification kit (Qiagen).

    Article Title: Entirely enzymatic nanofabrication of DNA–protein conjugates
    Article Snippet: .. 20 ug of the obtained plasmid were digested for 4 h with BsaI-HF (NEB) in 100 μl with the addition of 10 μl of CutSmart buffer (NEB) and the psudogenes extracted from a 0.7% agarose gel, 0.5× TAE, using illustra GFX PCR DNA and gel band purification kit (GE Healthcare). ..

    Polymerase Chain Reaction:

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: .. 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C. .. Reactions were column-cleaned and 400 ng of each insert was ligated with T4 DNA ligase into 1 ug of recipient vector ( > 7:1 insert:vector) treated with NotI, AscI, as well as CIP (NEB) – either pKR216 (SEC14 library) or pKR348 (natural variants library) – in a total volume of 20 uL.

    Article Title: Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases
    Article Snippet: .. A total of 10 to 15 μl of the PCR product was digested according to the manufacturer’s instructions, using 1 U DraI (New England Biolabs, UK), 2 U PleI (New England Biolabs, UK), and 1× CutSmart buffer (New England Biolabs, UK) in a total volume of 20 μl. .. Following digestion, each 6-carboxyfluorescein (FAM)-labeled SpnIII variant has a unique size (Table S3) that can be distinguished by capillary electrophoresis on an ABI prism gene analyzer (Applied Biosystems, USA).

    Article Title: Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing
    Article Snippet: Previously isolated DNA (~10 ng) was digested using 8 U of EcoRI (New England Biolabs, UK) and 4 U of MseI (New England Biolabs, UK) with addition of 1× CutSmart Buffer (New England Biolabs, UK) at 37 °C for 1 h and AFLP adapters (EcoRI-F: 5′-CTCGTAGACTGCGTACC-3′; EcoRI-R: 5′-AATTGGTACGCAGTCTAC-3′; MseI-F: 5′-GACGATGAGTCCTGAG-3′; MseI-R: 5′-TACTCAGGACTCAT-3′) were ligated. .. Resulting fragments were enriched in PCR reaction with EcoRI (5′-GACTGCGTACCAATTC-3′) and MseI (5′-GATGAGTCCTGAGTAA-3′) primers [ ].

    Article Title: Genetic variants of major genes contributing to phosphate and calcium homeostasis and their association with serum parameters in pigs
    Article Snippet: .. Digestion of amplification products was performed using 10 μl PCR product, 2 μl CutSmart Buffer (New England BioLabs), and 1 U of restriction enzyme and filled up with aqua dest to a final volume of 20 μl. .. The reaction conditions were 16 h (for PTH1R and TRAFD1 ) or 2 h (for STC1 ) at 37 °C for incubation and 20 min at 65 °C for enzyme inactivation.

    Article Title: Cryptic Plutella species show deep divergence despite the capacity to hybridize
    Article Snippet: Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGxxxxxxTGCA- 3′ , bottom strand 5′ -[P]xxxxxxCTGTCTCTTATACACATCTGACGCTGCCGACGA- 3′ , x represents sites for MIDs) were then added using 0.5 µL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer. .. DNA purification between steps was performed using a MinElute PCR purification kit (Qiagen).

    Article Title: Entirely enzymatic nanofabrication of DNA–protein conjugates
    Article Snippet: .. 20 ug of the obtained plasmid were digested for 4 h with BsaI-HF (NEB) in 100 μl with the addition of 10 μl of CutSmart buffer (NEB) and the psudogenes extracted from a 0.7% agarose gel, 0.5× TAE, using illustra GFX PCR DNA and gel band purification kit (GE Healthcare). ..

    Concentration Assay:

    Article Title: Long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele‐specific MYC expression in HeLa cells
    Article Snippet: Cross‐linking was stopped by adding glycine to a final concentration of 1 M and incubating at room temperature for 5 min. .. The cytoplasmic components were removed by centrifugation and the nuclear pellets were dissolved in 1× cutsmart buffer (NEB) containing 0.3% SDS.

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: .. To start the signal amplification cascade, 20 µL of CutSmart buffer supplemented with a particular concentration of the SP trigger oligonucleotide, and 20 µL of CutSmart buffer containing two REases, EcoRV and SspI (0.8 U/µL each) were added to the top of the second filter. .. The resultant microplate was incubated for various time intervals at 37 °C with shaking (90 rpm).

    Plasmid Preparation:

    Article Title: A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates
    Article Snippet: .. Injection mixes containing 20 ng µl−1 reporter plasmid (generated by miniprep), 1× CutSmart buffer (NEB), and 0.5U µl−1 I-SceI enzyme (NEB) in water were incubated at 37 °C for 30 min prior to micro-injection at a volume of ~2 nl per embryo. .. Embryos were screened for fluorescent reporter expression using a Zeiss SteREO Discovery V12 microscope.

    Article Title: Multiplexed precision genome editing with trackable genomic barcodes in yeast
    Article Snippet: NotI and AscI sites enable sticky end cloning into a multi-copy recipient vector, with the AscI site at the 3′-end of the guide RNA promoter. .. 5 ug of each PCR-cleaned reaction was cut with 2 uL of AscI (NEB) and 2 uL of NotI (NEB), 10 uL of 10× CutSmart buffer (NEB), and incubated at 37°C for 1 hour followed by 20 minutes of heat inactivation at 80°C.

    Article Title: Triggering autophagic cell death with a di-manganese(II) developmental therapeutic
    Article Snippet: .. Salmon testes DNA (D1626) and synthetic double stranded alternating co-polymers, Poly[d(G-C)2 ] (P9389) and Poly[d(A-T)2 ] (P0883) used in CD studies were purchased from Sigma Aldrich. pUC19 plasmid DNA (N3041), CutSmart® buffer (B7204), 100X BSA (B9000) and topoisomerase I (E. coli) (M0301) were all purchased from New England Biolabs. .. LC3 isoform LC3A rabbit monoclonal antibody (Cell Signalling) was kindly donated by Dr. Joanne Keenan while goat anti-rabbit conjugated Alex Fluor-647 (ThermoFisher) was donated by Dr. Clair Gallagher.

    Article Title: Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination
    Article Snippet: .. Doxorubicin (Dox) hydrochloride (D2975000), dihydroethidium (D7008), salmon testes DNA (D1626), synthetic double stranded alternating co-polymers, poly[d(G⋅C)2 ] (P9389) and poly[d(A⋅T)2 ] (P0883) and Micrococcus Lysodeikticus (D8259) were purchased from Sigma-Aldrich. pUC19 plasmid DNA (N3041), CutSmart® buffer (B7204), 100× bovine serum albumin (BSA) (B9000) and topoisomerase I (E. coli) (M0301) were all purchased from New England Biolabs. ..

    Article Title: Entirely enzymatic nanofabrication of DNA–protein conjugates
    Article Snippet: .. 20 ug of the obtained plasmid were digested for 4 h with BsaI-HF (NEB) in 100 μl with the addition of 10 μl of CutSmart buffer (NEB) and the psudogenes extracted from a 0.7% agarose gel, 0.5× TAE, using illustra GFX PCR DNA and gel band purification kit (GE Healthcare). ..

    Software:

    Article Title: Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases
    Article Snippet: A total of 10 to 15 μl of the PCR product was digested according to the manufacturer’s instructions, using 1 U DraI (New England Biolabs, UK), 2 U PleI (New England Biolabs, UK), and 1× CutSmart buffer (New England Biolabs, UK) in a total volume of 20 μl. .. Data received from the ABI Prism gene analyzer were analyzed using Peak Scanner v1.0 software.

    Negative Control:

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: Suspension testing of beads coupled to amplification probes Five µL of bead preparations ASP-7RV-polyG::polyC-HRP and ARV-7SP-polyG::polyC-HRP (at 2 mg/mL concentrations) were placed into individual 0.5 mL Eppendorf tubes, and washed twice with 10 µL of CutSmart buffer (New England Biolabs). .. The first preparation, ASP-7RV-polyG::polyC-HRP, was used to add 5 µL of the target solution: either CutSmart buffer supplemented at various concentrations with the SP trigger oligonucleotide (Table ), or a negative control of the same buffer with no targets added.

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: .. The first preparation, ASP-7RV-polyG::polyC-HRP, was used to add 5 µL of the target solution: either CutSmart buffer supplemented at various concentrations with the SP trigger oligonucleotide (Table ), or a negative control of the same buffer with no targets added. .. The second preparation, ARV-7SP-polyG::polyC-HRP, was used to add two REases, EcoRV and SspI, at 0.8 U/µL each, in 5 µL of CutSmart buffer.

    Agarose Gel Electrophoresis:

    Article Title: Entirely enzymatic nanofabrication of DNA–protein conjugates
    Article Snippet: .. 20 ug of the obtained plasmid were digested for 4 h with BsaI-HF (NEB) in 100 μl with the addition of 10 μl of CutSmart buffer (NEB) and the psudogenes extracted from a 0.7% agarose gel, 0.5× TAE, using illustra GFX PCR DNA and gel band purification kit (GE Healthcare). ..

    Activation Assay:

    Article Title: Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing
    Article Snippet: Previously isolated DNA (~10 ng) was digested using 8 U of EcoRI (New England Biolabs, UK) and 4 U of MseI (New England Biolabs, UK) with addition of 1× CutSmart Buffer (New England Biolabs, UK) at 37 °C for 1 h and AFLP adapters (EcoRI-F: 5′-CTCGTAGACTGCGTACC-3′; EcoRI-R: 5′-AATTGGTACGCAGTCTAC-3′; MseI-F: 5′-GACGATGAGTCCTGAG-3′; MseI-R: 5′-TACTCAGGACTCAT-3′) were ligated. .. Cycling protocol comprised of polymerase activation step of 95 °C for 15 min followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 1 min and elongation at 72 °C for 30 s with a final elongation step at 72 °C for 7 min. Fragments of around 500 bp were selected using AmPure XP beads (Beckman Coulter, Brea, CA, USA), quantified, normalized and then sequenced on HiSeq platform (Illumina, San Diego, CA, USA) at the Norwegian Sequencing Centre (Oslo, Norway).

    Immunoprecipitation:

    Article Title: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia
    Article Snippet: Paragraph title: Crosslink-assistant DNA modification immunoprecipitation assay ... The DNA (2 μg) was incubated with 3 μl of Nla III restriction enzyme (10,000 units/ml) 37 °C overnight in the CutSmart™ Buffer (NEB Inc., Ipswich, MA), and the enzyme was inactivated by incubation at 65 °C for 20 min.

    DNA Purification:

    Article Title: Cryptic Plutella species show deep divergence despite the capacity to hybridize
    Article Snippet: Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200 ng digested with 10 units of high fidelity SbfI in Cutsmart Buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID) (top strand 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGxxxxxxTGCA- 3′ , bottom strand 5′ -[P]xxxxxxCTGTCTCTTATACACATCTGACGCTGCCGACGA- 3′ , x represents sites for MIDs) were then added using 0.5 µL T4 DNA ligase (Promega), 1 nM ATP and Cutsmart buffer. .. DNA purification between steps was performed using a MinElute PCR purification kit (Qiagen).

    Lysis:

    Article Title: Long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele‐specific MYC expression in HeLa cells
    Article Snippet: Cell lysis was performed by adding 1 ml of cell lysis buffer (10 mM Tris‐HCl, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1 × protein inhibitor) and incubating for 30 min on ice. .. The cytoplasmic components were removed by centrifugation and the nuclear pellets were dissolved in 1× cutsmart buffer (NEB) containing 0.3% SDS.

    Variant Assay:

    Article Title: Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases
    Article Snippet: A total of 10 to 15 μl of the PCR product was digested according to the manufacturer’s instructions, using 1 U DraI (New England Biolabs, UK), 2 U PleI (New England Biolabs, UK), and 1× CutSmart buffer (New England Biolabs, UK) in a total volume of 20 μl. .. Following digestion, each 6-carboxyfluorescein (FAM)-labeled SpnIII variant has a unique size (Table S3) that can be distinguished by capillary electrophoresis on an ABI prism gene analyzer (Applied Biosystems, USA).

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    New England Biolabs cutsmart buffer
    Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with <t>CutSmart</t> reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).
    Cutsmart Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

    Journal: PLoS ONE

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    doi: 10.1371/journal.pone.0114208

    Figure Lengend Snippet: Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

    Article Snippet: Portions of lysates were further heat inactivated (HI) at 95°C for 10 min. One µg of the commercial plasmid pECFP-N1 (Clonetech, CA, USA) was subjected to either MS11 P+ lysate or HI lysate together with CutSmart buffer (New England Biolabs, Ipswich, MA, USA) for 1 h. As controls, circular/uncut pECFP-N1 was used as well as HindIII (Roche, Mannheim, Germany) linearized pECFP-N1.

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Purification, Negative Control, Incubation

    Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

    Journal: Journal of Virology

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    doi: 10.1128/JVI.00539-17

    Figure Lengend Snippet: Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

    Article Snippet: For exonuclease T5 (Exo T5) digestion, 20 μl PF DNA sample was treated with 0.5 μl (5 units) Exo T5 in 1× Cutsmart buffer (NEB) in a total volume of 23 μl ( ) at 37°C for 2 to 3 h. Phenol extraction was then used to remove the nucleases before qPCR.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Migration

    Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).

    Journal: Journal of Virology

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    doi: 10.1128/JVI.00539-17

    Figure Lengend Snippet: Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).

    Article Snippet: For exonuclease T5 (Exo T5) digestion, 20 μl PF DNA sample was treated with 0.5 μl (5 units) Exo T5 in 1× Cutsmart buffer (NEB) in a total volume of 23 μl ( ) at 37°C for 2 to 3 h. Phenol extraction was then used to remove the nucleases before qPCR.

    Techniques: Countercurrent Chromatography, Modification, Agarose Gel Electrophoresis, Southern Blot, Migration

    Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

    Journal: Journal of Virology

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    doi: 10.1128/JVI.00539-17

    Figure Lengend Snippet: Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

    Article Snippet: For exonuclease T5 (Exo T5) digestion, 20 μl PF DNA sample was treated with 0.5 μl (5 units) Exo T5 in 1× Cutsmart buffer (NEB) in a total volume of 23 μl ( ) at 37°C for 2 to 3 h. Phenol extraction was then used to remove the nucleases before qPCR.

    Techniques: Modification, Countercurrent Chromatography, Agarose Gel Electrophoresis, Southern Blot, Migration, Marker