apai cut λ dna  (New England Biolabs)


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    Name:
    Lambda DNA
    Description:
    Lambda DNA 1 250 ug
    Catalog Number:
    N3011L
    Price:
    276
    Category:
    Genomic DNA
    Size:
    1 250 ug
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    Structured Review

    New England Biolabs apai cut λ dna
    Lambda DNA
    Lambda DNA 1 250 ug
    https://www.bioz.com/result/apai cut λ dna/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    apai cut λ dna - by Bioz Stars, 2021-05
    99/100 stars

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    1) Product Images from "Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase"

    Article Title: Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17443-7

    Direct visualization of RPA-facilitated processive fork unwinding by individual CMG molecules. a  A 10-kb fragment of λ DNA was ligated to a short fork DNA substrate at one end and to a digoxigenin-modified DNA fragment on the opposite end. The 5′ tail of the forked end contained a biotin to attach the 10-kb substrate to biotin-functionalized glass through biotin-streptavidin binding. The digoxigenin-modified end was coupled to anti-digoxigenin-coated microsphere to stretch DNA molecules by buffer flow, and to subsequently attach this end to the surface (details are described in the Methods section). The DNA substrate was labeled with Cy3 at the 3′ dT 40  ssDNA tail near the fork junction. LD655-labeled CMG was bound to the dT 40  ssDNA in the presence of ATPγS.  b  A sample stretched 10-kb linear DNA stained with a fluorescent dsDNA intercalator Sytox Orange. Average length of stretched DNA molecules were determined by measuring the end-to-end distance of 717 individual DNA molecules ( n  = 2 independent experiments).  c  After binding CMG on the surface-immobilized DNA, EGFP-RPA, and ATP was introduced to initiate unwinding. While CMG unwinds DNA at the fork, EGFP-RPA binds both strands of unwound DNA.  d  Kymograph showing a representative 10-kb DNA being unwound entirely by a single CMG complex. EGFP-RPA (left panel), LD655-labeled CMG (center panel), and 3′ Cy3 (right panel) are imaged during unwinding under near-TIRF conditions. Images were acquired in the absence of buffer flow.  e  Histogram of CMG-catalyzed DNA-unwinding rates on stretched DNA (74 individual DNA molecules analyzed from two independent experiments). Source data are provided as a Source Data file.
    Figure Legend Snippet: Direct visualization of RPA-facilitated processive fork unwinding by individual CMG molecules. a A 10-kb fragment of λ DNA was ligated to a short fork DNA substrate at one end and to a digoxigenin-modified DNA fragment on the opposite end. The 5′ tail of the forked end contained a biotin to attach the 10-kb substrate to biotin-functionalized glass through biotin-streptavidin binding. The digoxigenin-modified end was coupled to anti-digoxigenin-coated microsphere to stretch DNA molecules by buffer flow, and to subsequently attach this end to the surface (details are described in the Methods section). The DNA substrate was labeled with Cy3 at the 3′ dT 40 ssDNA tail near the fork junction. LD655-labeled CMG was bound to the dT 40 ssDNA in the presence of ATPγS. b A sample stretched 10-kb linear DNA stained with a fluorescent dsDNA intercalator Sytox Orange. Average length of stretched DNA molecules were determined by measuring the end-to-end distance of 717 individual DNA molecules ( n  = 2 independent experiments). c After binding CMG on the surface-immobilized DNA, EGFP-RPA, and ATP was introduced to initiate unwinding. While CMG unwinds DNA at the fork, EGFP-RPA binds both strands of unwound DNA. d Kymograph showing a representative 10-kb DNA being unwound entirely by a single CMG complex. EGFP-RPA (left panel), LD655-labeled CMG (center panel), and 3′ Cy3 (right panel) are imaged during unwinding under near-TIRF conditions. Images were acquired in the absence of buffer flow. e Histogram of CMG-catalyzed DNA-unwinding rates on stretched DNA (74 individual DNA molecules analyzed from two independent experiments). Source data are provided as a Source Data file.

    Techniques Used: Recombinase Polymerase Amplification, Modification, Binding Assay, Labeling, Staining

    Related Articles

    Purification:

    Article Title: Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase
    Article Snippet: Forked 10-kb linear DNA used in single-molecule assays (Fig. ) was generated by treating λ DNA with ApaI (NEB). .. 10-kb fragment from ApaI-cut λ DNA was purified from a 0.5% agarose gel using Monarch gel extraction kit (NEB). .. We generated the fork end containing a 5′ biotin and a Cy3 on the 3′ dT40 tail by annealing Oligo-Bio-4 and Oligo-Cy3-1, which leaves a 12-nt ssDNA complementary to one end of the 10 kb λ-ApaI fragment.

    Agarose Gel Electrophoresis:

    Article Title: Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase
    Article Snippet: Forked 10-kb linear DNA used in single-molecule assays (Fig. ) was generated by treating λ DNA with ApaI (NEB). .. 10-kb fragment from ApaI-cut λ DNA was purified from a 0.5% agarose gel using Monarch gel extraction kit (NEB). .. We generated the fork end containing a 5′ biotin and a Cy3 on the 3′ dT40 tail by annealing Oligo-Bio-4 and Oligo-Cy3-1, which leaves a 12-nt ssDNA complementary to one end of the 10 kb λ-ApaI fragment.

    Gel Extraction:

    Article Title: Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase
    Article Snippet: Forked 10-kb linear DNA used in single-molecule assays (Fig. ) was generated by treating λ DNA with ApaI (NEB). .. 10-kb fragment from ApaI-cut λ DNA was purified from a 0.5% agarose gel using Monarch gel extraction kit (NEB). .. We generated the fork end containing a 5′ biotin and a Cy3 on the 3′ dT40 tail by annealing Oligo-Bio-4 and Oligo-Cy3-1, which leaves a 12-nt ssDNA complementary to one end of the 10 kb λ-ApaI fragment.

    Marker:

    Article Title: Simple and Cost-Effective Restriction Endonuclease Analysis of Human Adenoviruses
    Article Snippet: After electrophoresis, the DNA in the gel was stained for 30 minutes in GelRed (Biotium, Hayward, CA) solution made in TAE buffer. .. Lambda DNA-HindIII digest marker (New England Biolabs) photographs were taken under UV light. .. For electrophoresis, a prototype strain was always added as a control.

    Ligation:

    Article Title: Watching Individual Proteins Acting on Single Molecules of DNA
    Article Snippet: .. Bacteriophage λ DNA (New England Biolabs, Ipswich, MA) is biotinylated by ligation to a 3′-biotinylated 12-mer oligonucleotide (5′-GGGCGGCG ACCT-3′ or 5′-AGGTCGCCGCCC-3′, Operon Technologies, Huntsville, AL) that is complementary to one of the cohesive ends of λ DNA ( ). .. In all the subsequent protocols, the pipetting of solutions containing λ DNA should be performed with cut pipette tips to minimize shearing of the DNA.

    Staining:

    Article Title: Massively-Parallel Ultra-High-Aspect-Ratio Nanochannels as Mesoporous Membranes
    Article Snippet: .. The λ-DNA Hind digest (New England Biolabs) was stained with the intercalating fluorescence dye YOYO-1 (Invitrogen) at a dye-to-base pair ratio of ~1:10 in 5× TBE buffer. ..

    Fluorescence:

    Article Title: Massively-Parallel Ultra-High-Aspect-Ratio Nanochannels as Mesoporous Membranes
    Article Snippet: .. The λ-DNA Hind digest (New England Biolabs) was stained with the intercalating fluorescence dye YOYO-1 (Invitrogen) at a dye-to-base pair ratio of ~1:10 in 5× TBE buffer. ..

    Chromatin Immunoprecipitation:

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing
    Article Snippet: Following antibodies were used in ChIP assays: anti-H3K4me1 (ab8895, Abcam) and anti-H3K27me3 (07–449, Millipore). .. Library generation for genome-scale MAB-seq/BS-seq 1 ug unenriched genomic DNA (whole-genome) or 200–500 ng of ChIP DNA (H3K4me1- or H3K27me3-enriched) was first spiked-in with unmethylated lambda DNA (1:400), which was then repaired and ligated to methylated (5mC) custom adapters (forward 5’-ACACTCTTTCCCTACACGACGCTCTTCC GATC*T-3’; reverse 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’; the asterisk denotes phosphorothioate bond) with the NEBNext Ultra DNA Library Prep kit from Illumina (NEB). .. Adaptor-ligated DNA was then purified with 1.2x AMPure XP beads (Beckman Coulter).

    Lambda DNA Preparation:

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing
    Article Snippet: Following antibodies were used in ChIP assays: anti-H3K4me1 (ab8895, Abcam) and anti-H3K27me3 (07–449, Millipore). .. Library generation for genome-scale MAB-seq/BS-seq 1 ug unenriched genomic DNA (whole-genome) or 200–500 ng of ChIP DNA (H3K4me1- or H3K27me3-enriched) was first spiked-in with unmethylated lambda DNA (1:400), which was then repaired and ligated to methylated (5mC) custom adapters (forward 5’-ACACTCTTTCCCTACACGACGCTCTTCC GATC*T-3’; reverse 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’; the asterisk denotes phosphorothioate bond) with the NEBNext Ultra DNA Library Prep kit from Illumina (NEB). .. Adaptor-ligated DNA was then purified with 1.2x AMPure XP beads (Beckman Coulter).

    Methylation:

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing
    Article Snippet: Following antibodies were used in ChIP assays: anti-H3K4me1 (ab8895, Abcam) and anti-H3K27me3 (07–449, Millipore). .. Library generation for genome-scale MAB-seq/BS-seq 1 ug unenriched genomic DNA (whole-genome) or 200–500 ng of ChIP DNA (H3K4me1- or H3K27me3-enriched) was first spiked-in with unmethylated lambda DNA (1:400), which was then repaired and ligated to methylated (5mC) custom adapters (forward 5’-ACACTCTTTCCCTACACGACGCTCTTCC GATC*T-3’; reverse 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’; the asterisk denotes phosphorothioate bond) with the NEBNext Ultra DNA Library Prep kit from Illumina (NEB). .. Adaptor-ligated DNA was then purified with 1.2x AMPure XP beads (Beckman Coulter).

    In Vitro:

    Article Title: Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging
    Article Snippet: We labeled the constitutive heterochromatin marker Histone H3K9me3 with the primary rabbit polyclonal antibody (ab8898, Abcam, Cambridge, England) and a secondary donkey anti-rabbit antibody (sc-362291, Santa Cruz Biotechnology, Dallas, TX). .. Preparation of DNA in vitro Solutions For the DNA in vitro solution experiments, double-stranded λ-DNA was obtained from New England Biolabs (Ipswich, MA). ..

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    New England Biolabs apai cut λ dna
    Direct visualization of RPA-facilitated processive fork unwinding by individual CMG molecules. a  A 10-kb fragment of λ DNA was ligated to a short fork DNA substrate at one end and to a digoxigenin-modified DNA fragment on the opposite end. The 5′ tail of the forked end contained a biotin to attach the 10-kb substrate to biotin-functionalized glass through biotin-streptavidin binding. The digoxigenin-modified end was coupled to anti-digoxigenin-coated microsphere to stretch DNA molecules by buffer flow, and to subsequently attach this end to the surface (details are described in the Methods section). The DNA substrate was labeled with Cy3 at the 3′ dT 40  ssDNA tail near the fork junction. LD655-labeled CMG was bound to the dT 40  ssDNA in the presence of ATPγS.  b  A sample stretched 10-kb linear DNA stained with a fluorescent dsDNA intercalator Sytox Orange. Average length of stretched DNA molecules were determined by measuring the end-to-end distance of 717 individual DNA molecules ( n  = 2 independent experiments).  c  After binding CMG on the surface-immobilized DNA, EGFP-RPA, and ATP was introduced to initiate unwinding. While CMG unwinds DNA at the fork, EGFP-RPA binds both strands of unwound DNA.  d  Kymograph showing a representative 10-kb DNA being unwound entirely by a single CMG complex. EGFP-RPA (left panel), LD655-labeled CMG (center panel), and 3′ Cy3 (right panel) are imaged during unwinding under near-TIRF conditions. Images were acquired in the absence of buffer flow.  e  Histogram of CMG-catalyzed DNA-unwinding rates on stretched DNA (74 individual DNA molecules analyzed from two independent experiments). Source data are provided as a Source Data file.
    Apai Cut λ Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apai cut λ dna/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    apai cut λ dna - by Bioz Stars, 2021-05
    99/100 stars
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    Direct visualization of RPA-facilitated processive fork unwinding by individual CMG molecules. a  A 10-kb fragment of λ DNA was ligated to a short fork DNA substrate at one end and to a digoxigenin-modified DNA fragment on the opposite end. The 5′ tail of the forked end contained a biotin to attach the 10-kb substrate to biotin-functionalized glass through biotin-streptavidin binding. The digoxigenin-modified end was coupled to anti-digoxigenin-coated microsphere to stretch DNA molecules by buffer flow, and to subsequently attach this end to the surface (details are described in the Methods section). The DNA substrate was labeled with Cy3 at the 3′ dT 40  ssDNA tail near the fork junction. LD655-labeled CMG was bound to the dT 40  ssDNA in the presence of ATPγS.  b  A sample stretched 10-kb linear DNA stained with a fluorescent dsDNA intercalator Sytox Orange. Average length of stretched DNA molecules were determined by measuring the end-to-end distance of 717 individual DNA molecules ( n  = 2 independent experiments).  c  After binding CMG on the surface-immobilized DNA, EGFP-RPA, and ATP was introduced to initiate unwinding. While CMG unwinds DNA at the fork, EGFP-RPA binds both strands of unwound DNA.  d  Kymograph showing a representative 10-kb DNA being unwound entirely by a single CMG complex. EGFP-RPA (left panel), LD655-labeled CMG (center panel), and 3′ Cy3 (right panel) are imaged during unwinding under near-TIRF conditions. Images were acquired in the absence of buffer flow.  e  Histogram of CMG-catalyzed DNA-unwinding rates on stretched DNA (74 individual DNA molecules analyzed from two independent experiments). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase

    doi: 10.1038/s41467-020-17443-7

    Figure Lengend Snippet: Direct visualization of RPA-facilitated processive fork unwinding by individual CMG molecules. a A 10-kb fragment of λ DNA was ligated to a short fork DNA substrate at one end and to a digoxigenin-modified DNA fragment on the opposite end. The 5′ tail of the forked end contained a biotin to attach the 10-kb substrate to biotin-functionalized glass through biotin-streptavidin binding. The digoxigenin-modified end was coupled to anti-digoxigenin-coated microsphere to stretch DNA molecules by buffer flow, and to subsequently attach this end to the surface (details are described in the Methods section). The DNA substrate was labeled with Cy3 at the 3′ dT 40 ssDNA tail near the fork junction. LD655-labeled CMG was bound to the dT 40 ssDNA in the presence of ATPγS. b A sample stretched 10-kb linear DNA stained with a fluorescent dsDNA intercalator Sytox Orange. Average length of stretched DNA molecules were determined by measuring the end-to-end distance of 717 individual DNA molecules ( n  = 2 independent experiments). c After binding CMG on the surface-immobilized DNA, EGFP-RPA, and ATP was introduced to initiate unwinding. While CMG unwinds DNA at the fork, EGFP-RPA binds both strands of unwound DNA. d Kymograph showing a representative 10-kb DNA being unwound entirely by a single CMG complex. EGFP-RPA (left panel), LD655-labeled CMG (center panel), and 3′ Cy3 (right panel) are imaged during unwinding under near-TIRF conditions. Images were acquired in the absence of buffer flow. e Histogram of CMG-catalyzed DNA-unwinding rates on stretched DNA (74 individual DNA molecules analyzed from two independent experiments). Source data are provided as a Source Data file.

    Article Snippet: 10-kb fragment from ApaI-cut λ DNA was purified from a 0.5% agarose gel using Monarch gel extraction kit (NEB).

    Techniques: Recombinase Polymerase Amplification, Modification, Binding Assay, Labeling, Staining