nla iii  (New England Biolabs)


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    Name:
    NlaIII
    Description:
    NlaIII 2 500 units
    Catalog Number:
    r0125l
    Price:
    282
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    New England Biolabs nla iii
    NlaIII
    NlaIII 2 500 units
    https://www.bioz.com/result/nla iii/product/New England Biolabs
    Average 99 stars, based on 12871 article reviews
    Price from $9.99 to $1999.99
    nla iii - by Bioz Stars, 2020-03
    99/100 stars

    Images

    1) Product Images from "CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning"

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning

    Journal: Cell

    doi: 10.1016/j.cell.2018.04.035

    V H 81X-CBE Promotes Interactions of Its Flanking V H with the DJ H RC (A) Schematic representation of the 3C-HTGTS method for studying chromosomal looping interactions of a bait region of interest with the rest of Igh locus (see text and STAR Methods for details). (B) Schematic of the Nla III restriction fragment (indicated by a blue asterisk) and the relative positions of the biotinylated (cayenne arrow) and nested (blue arrow) PCR primers used for 3C-HTGTS from V H 81X bait in (C). (C) Top panel: schematic representation of chromosome interactions of V H 81X-CBE containing Nla III fragment with other Igh locales. Bottom two panels: 3C-HTGTS profiles of Rag2 −/− derivatives of control, V H 81X-CBE del , and V H 81X-CBE inv D H FL16.1J H 4 v-Abl lines using V H 81X-CBE locale as bait (blue asterisk). Owing to a D H FL16.1 to J H 4 rearrangement in the lines, the region spanning IGCR1, DJ H substrate and iEm appears as a broad interaction peak. As v-Abl lines lack locus contraction, we detected few substantial interactions with the upstream Igh locus beyond the most proximal V H ). Two independent datasets are shown from libraries normalized to 105,638 total junctions. .
    Figure Legend Snippet: V H 81X-CBE Promotes Interactions of Its Flanking V H with the DJ H RC (A) Schematic representation of the 3C-HTGTS method for studying chromosomal looping interactions of a bait region of interest with the rest of Igh locus (see text and STAR Methods for details). (B) Schematic of the Nla III restriction fragment (indicated by a blue asterisk) and the relative positions of the biotinylated (cayenne arrow) and nested (blue arrow) PCR primers used for 3C-HTGTS from V H 81X bait in (C). (C) Top panel: schematic representation of chromosome interactions of V H 81X-CBE containing Nla III fragment with other Igh locales. Bottom two panels: 3C-HTGTS profiles of Rag2 −/− derivatives of control, V H 81X-CBE del , and V H 81X-CBE inv D H FL16.1J H 4 v-Abl lines using V H 81X-CBE locale as bait (blue asterisk). Owing to a D H FL16.1 to J H 4 rearrangement in the lines, the region spanning IGCR1, DJ H substrate and iEm appears as a broad interaction peak. As v-Abl lines lack locus contraction, we detected few substantial interactions with the upstream Igh locus beyond the most proximal V H ). Two independent datasets are shown from libraries normalized to 105,638 total junctions. .

    Techniques Used: Polymerase Chain Reaction

    2) Product Images from "Enhancer-driven chromatin interactions during development promote escape from silencing by a long non-coding RNA"

    Article Title: Enhancer-driven chromatin interactions during development promote escape from silencing by a long non-coding RNA

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-4-21

    (A) Developmental imprinting pattern of Kcnq1 . Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with Nla III on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.
    Figure Legend Snippet: (A) Developmental imprinting pattern of Kcnq1 . Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with Nla III on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Amplification, Positive Control, Negative Control

    3) Product Images from "Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing"

    Article Title: Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-72

    Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.
    Figure Legend Snippet: Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.

    Techniques Used: Fractionation, In Silico, Magnetic Beads

    Related Articles

    Clone Assay:

    Article Title: A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers
    Article Snippet: Cloning of PCR-products was via TOPO TA Cloning® kit (Life Technologies Corp, Invitrogen) as recommended by manufacturer. .. EIL2 PCR products were purified with QIAquick PCR purification Kit (Qiagen) and restriction analyses using Nla III were done as supplier recommends (New England Biolabs).

    Centrifugation:

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei
    Article Snippet: Isolated nuclei are collected via centrifugation at 600 g (4°C, 5 min), gently resuspended in 500 μl of ice‐cold PB/0.4% NP‐40, and transferred to 2‐ml round‐bottom, low‐retention tubes. .. Next, chromatin is digested with 500 units of Apo I or Nla III (New England Biolabs; 33°C, 30–45 min) without shaking.

    Amplification:

    Article Title: A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment
    Article Snippet: PCR amplification was performed as described above. .. After verification of the PCR products on agarose gel, 3 μL was used to digest with 7.5 U of Nla III (New England Biolabs) in a final volume of 10 μL and fragments were separated on a 4% agarose gel.

    Whole Genome Amplification:

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: Paragraph title: RCA–RCA whole genome amplification ... Genomic DNA or cDNA were digested with 0.5 μL Nla-III (10 units/μL, New England Biolabs) at 37°C for 2 h in 10 μL of 1 × T4 DNA ligase buffer (New England Biolabs).

    Synthesized:

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Double-stranded cDNA was synthesized using the SuperScript Double Stranded cDNA Synthesis Kit (Invitrogen) and a biotinylated oligo(dT) primer (QIAGEN, Valencia, CA, USA). .. Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA).

    TA Cloning:

    Article Title: A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers
    Article Snippet: Cloning of PCR-products was via TOPO TA Cloning® kit (Life Technologies Corp, Invitrogen) as recommended by manufacturer. .. EIL2 PCR products were purified with QIAquick PCR purification Kit (Qiagen) and restriction analyses using Nla III were done as supplier recommends (New England Biolabs).

    Electrophoresis:

    Article Title: Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region
    Article Snippet: Amplicons were resolved by electrophoresis in 1% agarose gels in TAE buffer (40 mM Tris-acetate [pH 8.0], 1 mM disodium EDTA). .. Restriction reactions were done at 37°C with 50μl of the PCR product adjusted to recommended endonuclease buffer and 10 U of Nla III, Aci I, Nci I, or Sau 3A (New England Biolabs, Inc., Beverly, Mass.).

    Incubation:

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination
    Article Snippet: Electron microscopy Low molecular weight gel-extracted DNA from stn1-M1 and stn1-M1 ter1- Δ cells was incubated with 20 µg/ml T4 gene 32 protein (gift of Nancy Nossal, NIH, Bethesda, MD) for 5 min in a buffer containing 10 mM HEPES pH 7.5 and 1 mM EDTA. .. For examination of high molecular weight telomeric DNA, isolated genomic DNA was digested with Alu I, Hpa II and Nla III (NEB Beverly, MA), at enzyme concentration of 1 U/mg for 2 h, and then supplemented with an equal amount of each enzyme for an additional 2 h. The telomere restriction fragments were then separated by size exclusion chromatography and the eluted fractions monitored for DNA concentration and telomeric DNA abundance.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Determination of UPV230 restriction enzyme activity and its inhibitory activity of UPV229 To study the restriction enzyme activity in Ureaplasma cells, cultured OMC-P162 and C43 (DE3) strains in the mid-log growth phase were centrifuged at 17,800 × g for 20 min at 4°C, gently washed once with ice-cold PBS, suspended in PBS containing 1% Triton X-100, and incubated for 10 min on ice. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: To study the restriction enzyme activity in Ureaplasma cells, cultured OMC-P162 and C43 (DE3) strains in the mid-log growth phase were centrifuged at 17,800 × g for 20 min at 4°C, gently washed once with ice-cold PBS, suspended in PBS containing 1% Triton X-100, and incubated for 10 min on ice. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Activity Assay:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Paragraph title: Determination of UPV230 restriction enzyme activity and its inhibitory activity of UPV229 ... Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Expressing:

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Paragraph title: Expression tag library construction and high-throughput tag sequencing by Illumina sequencing technology ... Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA).

    Modification:

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning
    Article Snippet: Nuclei were digested with 700 units of Nla III (NEB, #R0125) or Mse I (NEB, #R0525) restriction enzyme at 37°C overnight, followed by ligation under dilute conditions at 16°C overnight. .. LAM-HTGTS libraries were then prepared and analyzed as described in “HTGTS-Rep-Seq to determine VH utilization frequencies” section (see also ) and data was aligned to /mm9 hybrid genome as described in with an additional modification in which Chr12 coordinates from 114671120 to 114734564 in the /mm9 hybrid genome were replaced with CCCCT to incorporate the DH FL16.1 to JH 4 rearrangement for aligning data obtained from the DH FL16.1JH 4 rearranged v-Abl pro-B lines.

    Electron Microscopy:

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination
    Article Snippet: Paragraph title: Electron microscopy ... For examination of high molecular weight telomeric DNA, isolated genomic DNA was digested with Alu I, Hpa II and Nla III (NEB Beverly, MA), at enzyme concentration of 1 U/mg for 2 h, and then supplemented with an equal amount of each enzyme for an additional 2 h. The telomere restriction fragments were then separated by size exclusion chromatography and the eluted fractions monitored for DNA concentration and telomeric DNA abundance.

    Ligation:

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA). .. The bound cDNA was washed and subjected to ligation with adaptor 1_XX.

    Article Title: Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer
    Article Snippet: Paragraph title: Digestion and ligation ... The ds-cDNA was digested with Nla III (New England Biolabs, Beverly, MA) at 37°C for 1 h. As digestion is a critical step, it was thoroughly checked by means of control amplifications.

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning
    Article Snippet: .. Nuclei were digested with 700 units of Nla III (NEB, #R0125) or Mse I (NEB, #R0525) restriction enzyme at 37°C overnight, followed by ligation under dilute conditions at 16°C overnight. .. Crosslinks were reversed and samples were treated with Proteinase K (Roche, #03115852001) and RNase A (Invitrogen, #8003089) prior to DNA precipitation.

    Magnetic Beads:

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA). .. A suspension of streptavidin magnetic beads (Promega, Madison, WI, USA) was added to the digested reaction product, and 3′ end fragments of the cDNA were bound to streptavidin magnetic beads.

    Cell Culture:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Determination of UPV230 restriction enzyme activity and its inhibitory activity of UPV229 To study the restriction enzyme activity in Ureaplasma cells, cultured OMC-P162 and C43 (DE3) strains in the mid-log growth phase were centrifuged at 17,800 × g for 20 min at 4°C, gently washed once with ice-cold PBS, suspended in PBS containing 1% Triton X-100, and incubated for 10 min on ice. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region
    Article Snippet: In standard PCRs 0.5 μg of each oligonucleotide of the primer pair VL2N (5′-ACATGCATGCCAGGAC) and VL33 (5′-ACCATTACAAACATTATCC), plus 50 ng of template DNA extracted from virion or cell culture were used. .. Restriction reactions were done at 37°C with 50μl of the PCR product adjusted to recommended endonuclease buffer and 10 U of Nla III, Aci I, Nci I, or Sau 3A (New England Biolabs, Inc., Beverly, Mass.).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: To study the restriction enzyme activity in Ureaplasma cells, cultured OMC-P162 and C43 (DE3) strains in the mid-log growth phase were centrifuged at 17,800 × g for 20 min at 4°C, gently washed once with ice-cold PBS, suspended in PBS containing 1% Triton X-100, and incubated for 10 min on ice. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Generated:

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning
    Article Snippet: 3C libraries were generated as previously described ( ; ). .. Nuclei were digested with 700 units of Nla III (NEB, #R0125) or Mse I (NEB, #R0525) restriction enzyme at 37°C overnight, followed by ligation under dilute conditions at 16°C overnight.

    other:

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: Each sample was heated at 65°C for 20 min to inactivate Nla-III.

    Article Title: Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA
    Article Snippet: The values in brackets are the position of the Nla III tagging site in the Y. pestis chromosome.

    Sequencing:

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Paragraph title: Expression tag library construction and high-throughput tag sequencing by Illumina sequencing technology ... Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA).

    Article Title: Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer
    Article Snippet: The ds-cDNA was digested with Nla III (New England Biolabs, Beverly, MA) at 37°C for 1 h. As digestion is a critical step, it was thoroughly checked by means of control amplifications. .. Each fragment was ligated to an adapter using T4 DNA ligase (Invitrogen) at 16°C for 1 h. The adapter sequence was designed according to one sequence used as a linker in SAGE protocols , and is as follows: 5′-GGATTTGCTGGTGCAGTACACATG-3′ (Ad primer).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The DNA nucleotide sequence of the restriction site was determined using the BigDye Terminator v3.1 kit on an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific, MA, USA).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The DNA nucleotide sequence of the restriction site was determined using the BigDye Terminator v3.1 kit on an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific, MA, USA).

    Sonication:

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning
    Article Snippet: Nuclei were digested with 700 units of Nla III (NEB, #R0125) or Mse I (NEB, #R0525) restriction enzyme at 37°C overnight, followed by ligation under dilute conditions at 16°C overnight. .. The 3C libraries were sonicated for 25 s ON and 60 s OFF for two cycles on a Diagenode Bioruptor Sonicator at low setting.

    Binding Assay:

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: Genomic DNA or cDNA were digested with 0.5 μL Nla-III (10 units/μL, New England Biolabs) at 37°C for 2 h in 10 μL of 1 × T4 DNA ligase buffer (New England Biolabs). .. Four microliters circular DNA was mixed with 0.5 μL hexamers (400 ng/μL, Sigma) and 0.5 μL binding buffer (400 mM Tris-HCl at pH 8.0, 160 mM KCl) and denatured at 95°C for 4 min. Alternatively, the denaturation step was omitted.

    Molecular Weight:

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination
    Article Snippet: .. For examination of high molecular weight telomeric DNA, isolated genomic DNA was digested with Alu I, Hpa II and Nla III (NEB Beverly, MA), at enzyme concentration of 1 U/mg for 2 h, and then supplemented with an equal amount of each enzyme for an additional 2 h. The telomere restriction fragments were then separated by size exclusion chromatography and the eluted fractions monitored for DNA concentration and telomeric DNA abundance. ..

    Nucleic Acid Electrophoresis:

    Article Title: Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region
    Article Snippet: Restriction reactions were done at 37°C with 50μl of the PCR product adjusted to recommended endonuclease buffer and 10 U of Nla III, Aci I, Nci I, or Sau 3A (New England Biolabs, Inc., Beverly, Mass.). .. Endonuclease-cleaved DNA products were resolved by gel electrophoresis in TAE buffer using a mixture of 3% NuSieve-GTG agarose gels and 1% SeaKem-GTG agarose (FMC Corp., Marine Colloids Division, Rockland, Maine) in TAE buffer.

    Methylation:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The inhibitory activity of UPV229 against the endonuclease activity of UPV230 was assessed by incubating purified UPV229 recombinant protein and 1 μg of closed circular pT7Blue plasmid or 250 ng of PCR products in a total volume of 20 μl containing methylation buffer (20 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 0.5 mM EDTA, 5% glycerol, and 4 μM S-adenosylmethionine) at 37°C for 1 h. The plasmid or PCR products was recovered by phenol extraction and ethanol precipitation and digested with UPV230 protein.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The inhibitory activity of UPV229 against the endonuclease activity of UPV230 was assessed by incubating purified UPV229 recombinant protein and 1 μg of closed circular pT7Blue plasmid or 250 ng of PCR products in a total volume of 20 μl containing methylation buffer (20 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 0.5 mM EDTA, 5% glycerol, and 4 μM S-adenosylmethionine) at 37°C for 1 h. The plasmid or PCR products was recovered by phenol extraction and ethanol precipitation and digested with UPV230 protein.

    Mutagenesis:

    Article Title: Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations
    Article Snippet: .. To summarize, Nla III distinguishes between the wild type and any mutant allele (Fig. , lane 2 versus lanes 3 and 4), whereas Hae III permits further discrimination of the mutants depending on the base mutated (first/A or third/G; Fig. , lane 6 versus lane 7). .. Thus, for all of the strains studied, the resulting PCR-RFLP data corroborated those generated by the MAS-PCR assay.

    Isolation:

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Poly(A)+ RNA was purified with an mRNA isolation kit (GE Healthcare UK Ltd, Buckinghamshire, UK). .. Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA).

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination
    Article Snippet: .. For examination of high molecular weight telomeric DNA, isolated genomic DNA was digested with Alu I, Hpa II and Nla III (NEB Beverly, MA), at enzyme concentration of 1 U/mg for 2 h, and then supplemented with an equal amount of each enzyme for an additional 2 h. The telomere restriction fragments were then separated by size exclusion chromatography and the eluted fractions monitored for DNA concentration and telomeric DNA abundance. ..

    Article Title: A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers
    Article Snippet: Genomic DNA from Cp, Cf, and Cm was isolated from flowers with DNeasy Plant Mini Kit (Qiagen) using 300 mg of plant material following manufacturer’s recommendations. .. EIL2 PCR products were purified with QIAquick PCR purification Kit (Qiagen) and restriction analyses using Nla III were done as supplier recommends (New England Biolabs).

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei
    Article Snippet: Isolated nuclei are collected via centrifugation at 600 g (4°C, 5 min), gently resuspended in 500 μl of ice‐cold PB/0.4% NP‐40, and transferred to 2‐ml round‐bottom, low‐retention tubes. .. Next, chromatin is digested with 500 units of Apo I or Nla III (New England Biolabs; 33°C, 30–45 min) without shaking.

    Size-exclusion Chromatography:

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination
    Article Snippet: .. For examination of high molecular weight telomeric DNA, isolated genomic DNA was digested with Alu I, Hpa II and Nla III (NEB Beverly, MA), at enzyme concentration of 1 U/mg for 2 h, and then supplemented with an equal amount of each enzyme for an additional 2 h. The telomere restriction fragments were then separated by size exclusion chromatography and the eluted fractions monitored for DNA concentration and telomeric DNA abundance. ..

    Purification:

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: Genomic DNA or cDNA were digested with 0.5 μL Nla-III (10 units/μL, New England Biolabs) at 37°C for 2 h in 10 μL of 1 × T4 DNA ligase buffer (New England Biolabs). .. The fragmented DNAs were circularized with 0.5 μL T4 DNA ligase (2000 units/μL, New England Biolabs) in a volume of 15 μL at room temperature for 2 h. After inactivation of ligase at 65°C for 10 min, linear DNAs were eliminated with 1.2 μL Lamda Exonuclease (5 units/μL, New England Biolabs) and 0.3 μL Exonuclease I (20 units/μL, New England Biolabs) in a volume of 25 μL at 37°C for 1 h. The circularized DNAs were then purified using a QIAquick PCR Purification Kit (QIAGEN) and eluted in 35 μL of H2 O.

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Poly(A)+ RNA was purified with an mRNA isolation kit (GE Healthcare UK Ltd, Buckinghamshire, UK). .. Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The endonuclease activity of the purified protein from the OMC-P162 and UPV_ 230 expressed in C43 (DE3) strains was assessed by incubating the purified protein and 1 μg of closed circular pT7Blue plasmid in a total volume of 20 μl containing L buffer at 37°C for the various times indicated. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region
    Article Snippet: For clinical samples, PCRs employed the same amount of the primers plus a ∼1:50 portion of the DNA purified from a single crusted scab (∼5 mm in diameter). .. Restriction reactions were done at 37°C with 50μl of the PCR product adjusted to recommended endonuclease buffer and 10 U of Nla III, Aci I, Nci I, or Sau 3A (New England Biolabs, Inc., Beverly, Mass.).

    Article Title: Stabilizing mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients
    Article Snippet: .. Restriction Enzyme Digestion CTNNB1 exon 3 PCR fragments were purified using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Europe GmbH, Uppsala, Sweden) and cleaved with Xma I or Nla III according to instructions by the manufacturer (New England Biolabs, Inc., Beverly, MA). ..

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The endonuclease activity of the purified protein from the OMC-P162 and UPV_ 230 expressed in C43 (DE3) strains was assessed by incubating the purified protein and 1 μg of closed circular pT7Blue plasmid in a total volume of 20 μl containing L buffer at 37°C for the various times indicated. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers
    Article Snippet: .. EIL2 PCR products were purified with QIAquick PCR purification Kit (Qiagen) and restriction analyses using Nla III were done as supplier recommends (New England Biolabs). ..

    Polymerase Chain Reaction:

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: Genomic DNA or cDNA were digested with 0.5 μL Nla-III (10 units/μL, New England Biolabs) at 37°C for 2 h in 10 μL of 1 × T4 DNA ligase buffer (New England Biolabs). .. The fragmented DNAs were circularized with 0.5 μL T4 DNA ligase (2000 units/μL, New England Biolabs) in a volume of 15 μL at room temperature for 2 h. After inactivation of ligase at 65°C for 10 min, linear DNAs were eliminated with 1.2 μL Lamda Exonuclease (5 units/μL, New England Biolabs) and 0.3 μL Exonuclease I (20 units/μL, New England Biolabs) in a volume of 25 μL at 37°C for 1 h. The circularized DNAs were then purified using a QIAquick PCR Purification Kit (QIAGEN) and eluted in 35 μL of H2 O.

    Article Title: A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment
    Article Snippet: .. After verification of the PCR products on agarose gel, 3 μL was used to digest with 7.5 U of Nla III (New England Biolabs) in a final volume of 10 μL and fragments were separated on a 4% agarose gel. ..

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: To analyze the endonuclease activity of UPV230 on PCR products and the Ureaplasma genome, the reaction pH was adjusted to 9.2. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region
    Article Snippet: .. Restriction reactions were done at 37°C with 50μl of the PCR product adjusted to recommended endonuclease buffer and 10 U of Nla III, Aci I, Nci I, or Sau 3A (New England Biolabs, Inc., Beverly, Mass.). .. Endonuclease-cleaved DNA products were resolved by gel electrophoresis in TAE buffer using a mixture of 3% NuSieve-GTG agarose gels and 1% SeaKem-GTG agarose (FMC Corp., Marine Colloids Division, Rockland, Maine) in TAE buffer.

    Article Title: Stabilizing mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients
    Article Snippet: .. Restriction Enzyme Digestion CTNNB1 exon 3 PCR fragments were purified using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Europe GmbH, Uppsala, Sweden) and cleaved with Xma I or Nla III according to instructions by the manufacturer (New England Biolabs, Inc., Beverly, MA). ..

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: To analyze the endonuclease activity of UPV230 on PCR products and the Ureaplasma genome, the reaction pH was adjusted to 9.2. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers
    Article Snippet: .. EIL2 PCR products were purified with QIAquick PCR purification Kit (Qiagen) and restriction analyses using Nla III were done as supplier recommends (New England Biolabs). ..

    Plasmid Preparation:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The endonuclease activity of the purified protein from the OMC-P162 and UPV_ 230 expressed in C43 (DE3) strains was assessed by incubating the purified protein and 1 μg of closed circular pT7Blue plasmid in a total volume of 20 μl containing L buffer at 37°C for the various times indicated. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The endonuclease activity of the purified protein from the OMC-P162 and UPV_ 230 expressed in C43 (DE3) strains was assessed by incubating the purified protein and 1 μg of closed circular pT7Blue plasmid in a total volume of 20 μl containing L buffer at 37°C for the various times indicated. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Software:

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination
    Article Snippet: For examination of high molecular weight telomeric DNA, isolated genomic DNA was digested with Alu I, Hpa II and Nla III (NEB Beverly, MA), at enzyme concentration of 1 U/mg for 2 h, and then supplemented with an equal amount of each enzyme for an additional 2 h. The telomere restriction fragments were then separated by size exclusion chromatography and the eluted fractions monitored for DNA concentration and telomeric DNA abundance. .. Images were captured using a Gatan Ultrascan US4000SP digital camera (Gatan, Pleasanton, CA) and molecule dimensions determined using Gatan Digital Micrograph 3.0 software.

    Recombinant:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The inhibitory activity of UPV229 against the endonuclease activity of UPV230 was assessed by incubating purified UPV229 recombinant protein and 1 μg of closed circular pT7Blue plasmid or 250 ng of PCR products in a total volume of 20 μl containing methylation buffer (20 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 0.5 mM EDTA, 5% glycerol, and 4 μM S-adenosylmethionine) at 37°C for 1 h. The plasmid or PCR products was recovered by phenol extraction and ethanol precipitation and digested with UPV230 protein.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The inhibitory activity of UPV229 against the endonuclease activity of UPV230 was assessed by incubating purified UPV229 recombinant protein and 1 μg of closed circular pT7Blue plasmid or 250 ng of PCR products in a total volume of 20 μl containing methylation buffer (20 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 0.5 mM EDTA, 5% glycerol, and 4 μM S-adenosylmethionine) at 37°C for 1 h. The plasmid or PCR products was recovered by phenol extraction and ethanol precipitation and digested with UPV230 protein.

    Agarose Gel Electrophoresis:

    Article Title: A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment
    Article Snippet: .. After verification of the PCR products on agarose gel, 3 μL was used to digest with 7.5 U of Nla III (New England Biolabs) in a final volume of 10 μL and fragments were separated on a 4% agarose gel. ..

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The reaction products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The reaction products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Proximity Ligation Assay:

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei
    Article Snippet: Next, chromatin is digested with 500 units of Apo I or Nla III (New England Biolabs; 33°C, 30–45 min) without shaking. .. Following resuspension in 1 ml of ice‐cold PB, spatially proximal chromatin ends are ligated together in intact nuclei (an idea based on the original “proximity ligation” assay by Cullen et al , ) and supported by recent findings that, even under cross‐linked conditions, ligations predominantly occur within the “chromatin cage” of intact nuclei; Gavrilov et al , ) by adding 100 units of T4 DNA ligase (5 U/μl stock; Invitrogen) and 10 μl BSA (10 mg/ml stock; Sigma‐Aldrich), and incubating at 16°C for 6–12 h without shaking.

    Ethanol Precipitation:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The inhibitory activity of UPV229 against the endonuclease activity of UPV230 was assessed by incubating purified UPV229 recombinant protein and 1 μg of closed circular pT7Blue plasmid or 250 ng of PCR products in a total volume of 20 μl containing methylation buffer (20 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 0.5 mM EDTA, 5% glycerol, and 4 μM S-adenosylmethionine) at 37°C for 1 h. The plasmid or PCR products was recovered by phenol extraction and ethanol precipitation and digested with UPV230 protein.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan). .. The inhibitory activity of UPV229 against the endonuclease activity of UPV230 was assessed by incubating purified UPV229 recombinant protein and 1 μg of closed circular pT7Blue plasmid or 250 ng of PCR products in a total volume of 20 μl containing methylation buffer (20 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 0.5 mM EDTA, 5% glycerol, and 4 μM S-adenosylmethionine) at 37°C for 1 h. The plasmid or PCR products was recovered by phenol extraction and ethanol precipitation and digested with UPV230 protein.

    Laser Capture Microdissection:

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning
    Article Snippet: Nuclei were digested with 700 units of Nla III (NEB, #R0125) or Mse I (NEB, #R0525) restriction enzyme at 37°C overnight, followed by ligation under dilute conditions at 16°C overnight. .. LAM-HTGTS libraries were then prepared and analyzed as described in “HTGTS-Rep-Seq to determine VH utilization frequencies” section (see also ) and data was aligned to /mm9 hybrid genome as described in with an additional modification in which Chr12 coordinates from 114671120 to 114734564 in the /mm9 hybrid genome were replaced with CCCCT to incorporate the DH FL16.1 to JH 4 rearrangement for aligning data obtained from the DH FL16.1JH 4 rearranged v-Abl pro-B lines.

    Concentration Assay:

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination
    Article Snippet: .. For examination of high molecular weight telomeric DNA, isolated genomic DNA was digested with Alu I, Hpa II and Nla III (NEB Beverly, MA), at enzyme concentration of 1 U/mg for 2 h, and then supplemented with an equal amount of each enzyme for an additional 2 h. The telomere restriction fragments were then separated by size exclusion chromatography and the eluted fractions monitored for DNA concentration and telomeric DNA abundance. ..

    CTG Assay:

    Article Title: A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment
    Article Snippet: The 154-bp PCR product with the polymorphic restriction site at position 2005 was amplified by PCR using forward primer 5'-GTC AAG CTC ATC CTG GTG TG-3' and reverse primer 5'-CCA TGT TGG TCT CCT TCC AG-3'. .. After verification of the PCR products on agarose gel, 3 μL was used to digest with 7.5 U of Nla III (New England Biolabs) in a final volume of 10 μL and fragments were separated on a 4% agarose gel.

    High Throughput Screening Assay:

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum
    Article Snippet: Paragraph title: Expression tag library construction and high-throughput tag sequencing by Illumina sequencing technology ... Double-stranded cDNA was digested with Nla III (NEB, Ipswich, MA, USA).

    Staining:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The reaction products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: The reaction products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining. .. Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

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    New England Biolabs restriction enzyme nlaiii
    Size fractionation of digested <t>DNA</t> by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with <t>NlaIII</t> . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.
    Restriction Enzyme Nlaiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.

    Journal: BMC Genomics

    Article Title: Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

    doi: 10.1186/1471-2164-13-72

    Figure Lengend Snippet: Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.

    Article Snippet: Standard method Approximately 500 ng of DNA was digested with the restriction enzyme NlaIII (NEB, Ipswich, Massachusetts, cat. no. R0125) for 1 to 6 hour at 37°C.

    Techniques: Fractionation, In Silico, Magnetic Beads

    (A) Developmental imprinting pattern of Kcnq1 . Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with Nla III on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.

    Journal: Epigenetics & Chromatin

    Article Title: Enhancer-driven chromatin interactions during development promote escape from silencing by a long non-coding RNA

    doi: 10.1186/1756-8935-4-21

    Figure Lengend Snippet: (A) Developmental imprinting pattern of Kcnq1 . Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with Nla III on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.

    Article Snippet: Following the PCR, a restriction digest was performed with the Nla III (New England Biolabs, Ipswich, MA no. R0125) for 1 h at 37°C.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Amplification, Positive Control, Negative Control