lambda exonuclease buffer  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Lambda Exonuclease
    Description:
    Lambda Exonuclease 5 000 units
    Catalog Number:
    M0262L
    Price:
    276
    Size:
    5 000 units
    Category:
    Exonucleases
    Score:
    85
    Buy from Supplier


    Structured Review

    New England Biolabs lambda exonuclease buffer
    Lambda Exonuclease
    Lambda Exonuclease 5 000 units
    https://www.bioz.com/result/lambda exonuclease buffer/product/New England Biolabs
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    lambda exonuclease buffer - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis"

    Article Title: Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-103

    In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.
    Figure Legend Snippet: In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Techniques Used: In Situ, DNA Synthesis, Binding Assay, Sequencing, Ligation, Labeling, Hybridization

    Related Articles

    Clone Assay:

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: An SCP2 fragment (bp 28683 to 30305) was cloned into EcoRI-digested pZR131 to yield pZR156. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h.

    Centrifugation:

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol. .. Reaction was stopped by 10 min incubation at 75°C. ssDNA was precipitated with dehydrated alcohol and washed with 70% alcohol.

    Amplification:

    Article Title: Enhanced Functional Potential of Nucleic Acid Aptamer Libraries Patterned to Increase Secondary Structure
    Article Snippet: Output DNA was amplified by PCR (13−15 cycles) with a 5′-phosphorylated reverse primer. .. The reaction was purified using a PCR Purification Kit (Qiagen) and diluted to 1× final concentration of λ-exonuclease buffer (New England Biolabs).

    Article Title: Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection
    Article Snippet: Finally, dsDNA fragments were converted to single-stranded DNA (ssDNA) in 1× λ-exonuclease buffer (NEB) with 10% glycerol, 0.2 μg/μl BSA, and 0.2 U/μl λ-exonuclease (NEB) for 30 min at 37°C. .. The PLP ligation reaction was performed as reported previously ( ).

    Article Title: In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor
    Article Snippet: Paragraph title: In situ rolling-circle amplification of padlock probes. ... The genomic DNA target was made single stranded by 5′-to-3′ exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA).

    Article Title: Rapid high resolution MHC class I genotyping of Chinese rhesus macaques by capillary reference strand mediated conformational analysis
    Article Snippet: The highly polymorphic MHC class I exon 2/exon 3 region was amplified from the cDNA using high-fidelity Phusion™ DNA polymerase (New England BioLabs), phosphorylated forward primer 5P Refstrand (5′-[Phos]GCTACGTGGACGACACGC), and reverse primer 3′Short-RSCA (5′ TTCAGGGCGATGTAATCC). .. Purified products were treated with λ Exonuclease (New England BioLabs) to generate single stranded antisense products for heteroduplex formation.

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: Universal 5′Biotin and 5′Phosphorylated primers were synthesized and HPLC-purified by Integrated DNA Technologies and were complementary to the M13 tails included in the primers used during the first round of amplification. .. Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs).

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: Then dsDNA was amplified under the optimal cycles. .. The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol.

    Synthesized:

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: Universal 5′Biotin and 5′Phosphorylated primers were synthesized and HPLC-purified by Integrated DNA Technologies and were complementary to the M13 tails included in the primers used during the first round of amplification. .. Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs).

    Article Title: An Aptamer-Based Biosensor for the Azole Class of Antifungal Drugs
    Article Snippet: Other oligonucleotides, including amino-functionalized oligonucleotides, were synthesized by IDT (Coralville, IA), Sigma-Aldrich (St. Louis, MO), and Biosearch Technologies (Novato, CA). .. Streptavidin-conjugated magnetic beads and λ exonuclease were purchased from New England Biolabs (Boston, MA).

    Construct:

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: By using a similar strategy , pZR131, containing two functional 381-bp pSLA2 telomeres and the Streptomyces tsr and melC genes of plasmid pIJ702, was constructed. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h.

    Electrophoresis:

    Article Title: Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)--Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria
    Article Snippet: For digestion of the phosphorylated strand, 40 U of lambda exonuclease (New England Biolabs) was mixed with 28 μl of the eluted PCR product in an 80-μl (total volume) mixture containing 1× (final concentration) lambda exonuclease reaction buffer (New England Biolabs). .. For digestion of the phosphorylated strand, 40 U of lambda exonuclease (New England Biolabs) was mixed with 28 μl of the eluted PCR product in an 80-μl (total volume) mixture containing 1× (final concentration) lambda exonuclease reaction buffer (New England Biolabs).

    Plasmid Purification:

    Article Title: Nucleotide Polymorphism-Based Single-Tube Test for Robust Molecular Identification of All Currently Described Brucella Species
    Article Snippet: Briefly, the first step (ligation) was conducted in a 10-μl mixture containing 10 ng purified genomic DNA, 4 U of Pfu DNA ligase (Agilent, Santa Clara, CA), 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl2 , 0.1% Igepal, 0.01 mM ATP, 1 mM dithiothreitol (DTT), and 100 pM of each PLP except the IS 711 probe (50 pM) and the ptsP probe (300 pM). .. The second step (exonuclease treatment) started with the addition of 15 μl of exonuclease mixture consisting of 67 mM glycine-KOH, pH 9.4, 2.5 mM MgCl2 , 50 μg/ml bovine serum albumin (BSA), and 1 U exonuclease λ (New England BioLabs, Ipswich, MA).

    Incubation:

    Article Title: Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology
    Article Snippet: The PCR product was purified on a GFXTM column (GE Healthcare) and incubated with 1 mg of streptavidin-coated magnetic beads (Dynabeads M-270, Invitrogen) for 2 h at 37°C in 400 µl of BW buffer (5 mM Tris–HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl). .. Subsequently, the DNA was washed once with 100 µl of 1× λ exonuclease buffer (NEB).

    Article Title: Complementary strand relocation may play vital roles in RecA-based homology recognition
    Article Snippet: The ssDNA was previously prepared by amplifying a 5-kb fragment using λ-phage as a template where one of the primers was 5′-phosphorylated, and purified using a Macherey–Nagel kit. .. The dsDNA PCR fragment was subsequently incubated with λ-exonuclease enzyme (NEB) at 37°C for 30 min, and the resulting ssDNA was further purified using a Qiagen kit. .. For experiments where free RecA binds to dsDNA, an aliquot of dsDNA in RecA buffer, 1 µM RecA (New England Biolabs), 1 mM ATPγS and the beads were placed in a square micro-cell.

    Article Title: Functional interactions of DNA topoisomerases with a human replication origin
    Article Snippet: Proteins were digested overnight with 200 μg/ml proteinase K and the DNA purification and primer extension were performed as above. .. For mapping the border of the in vitro complex, 50 U λ-exonuclease (New England Biolabs) was added to the preformed complex, incubated at 37°C for 3 h and then stopped with 2% SDS final concentration. .. The DNA was then purified and subjected to primer extension as described above.

    Stripping Membranes:

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: To elute the bound ssDNA, 100 µL of ddH2 O was added to the wells (each well was separated from the strip and sealed with silver paper) and placed in a Thermomixer Compact (Eppendorf, Germany) at temperature of 95°C for 10 min. .. The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol.

    Electrophoretic Mobility Shift Assay:

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assay (EMSA) ... For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Modification:

    Article Title: Complementary strand relocation may play vital roles in RecA-based homology recognition
    Article Snippet: After each modification step was completed, the dsDNA sample was washed three times using Amicon YM-100 filters (Millipore, USA) and 70 mM Tris buffer pH 7.6. .. The dsDNA PCR fragment was subsequently incubated with λ-exonuclease enzyme (NEB) at 37°C for 30 min, and the resulting ssDNA was further purified using a Qiagen kit.

    Transformation Assay:

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: DraI-linearized pZR181, pZR173, and pZR156 DNAs were introduced by transformation into ZX7 and plasmids were isolated. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h.

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. For the ssOR experiment 1 pmole HPLC-purified oligonucletide was used.

    Derivative Assay:

    Article Title: Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase
    Article Snippet: Deoxynucleotide triphosphates (dNTPs) were supplied by Roche (Penzberg, Germany) and were the best quality available (PCR grade). .. Phusion DNA polymerase (derived from a Pyroccccus species DNA polymerase), T4 DNA polymerase (T4-Pol) and lambda (λ) exonuclease were purchased from New England Biolabs (Hitchin, UK). .. NtBpu10I and NbBpu10I nicking enzymes, the restriction endonucleases PstI and DpnI and Thermus aquaticus DNA polymerase (Taq-Pol) were supplied by Fermentas (York, UK). pUC18 was from Stratagene (Agilent, Stockport, UK).

    Hybridization:

    Article Title: In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor
    Article Snippet: The genomic DNA target was made single stranded by 5′-to-3′ exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA). .. The genomic DNA target was made single stranded by 5′-to-3′ exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA).

    High Performance Liquid Chromatography:

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: Universal 5′Biotin and 5′Phosphorylated primers were synthesized and HPLC-purified by Integrated DNA Technologies and were complementary to the M13 tails included in the primers used during the first round of amplification. .. Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs).

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. Native polyacrylamide gel electrophoresis was used for SSCP overnight (14 hours) at 50 V. ssDNA recombination was performed by electroporating 2 pmoles of ssDNA generated from the in vitro Redα digestion.

    Ligation:

    Article Title: Nucleotide Polymorphism-Based Single-Tube Test for Robust Molecular Identification of All Currently Described Brucella Species
    Article Snippet: Briefly, the first step (ligation) was conducted in a 10-μl mixture containing 10 ng purified genomic DNA, 4 U of Pfu DNA ligase (Agilent, Santa Clara, CA), 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl2 , 0.1% Igepal, 0.01 mM ATP, 1 mM dithiothreitol (DTT), and 100 pM of each PLP except the IS 711 probe (50 pM) and the ptsP probe (300 pM). .. The second step (exonuclease treatment) started with the addition of 15 μl of exonuclease mixture consisting of 67 mM glycine-KOH, pH 9.4, 2.5 mM MgCl2 , 50 μg/ml bovine serum albumin (BSA), and 1 U exonuclease λ (New England BioLabs, Ipswich, MA).

    Article Title: Complementary strand relocation may play vital roles in RecA-based homology recognition
    Article Snippet: The dsDNA PCR fragment was subsequently incubated with λ-exonuclease enzyme (NEB) at 37°C for 30 min, and the resulting ssDNA was further purified using a Qiagen kit. .. The dsDNA PCR fragment was subsequently incubated with λ-exonuclease enzyme (NEB) at 37°C for 30 min, and the resulting ssDNA was further purified using a Qiagen kit.

    Generated:

    Article Title: Rapid high resolution MHC class I genotyping of Chinese rhesus macaques by capillary reference strand mediated conformational analysis
    Article Snippet: Amplicons were generated and purified under the conditions described above for custom sizing standard. .. Purified products were treated with λ Exonuclease (New England BioLabs) to generate single stranded antisense products for heteroduplex formation.

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany).

    other:

    Article Title: Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis
    Article Snippet: Exonuclease digestion was performed in a buffer containing 1× lambda exonuclease buffer (NEB), 0.2 μg/μl BSA (NEB) and 1u/μl lambda exonuclease (NEB) for 1 min at 37°C.

    Sequencing:

    Article Title: Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)--Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria
    Article Snippet: For digestion of the phosphorylated strand, 40 U of lambda exonuclease (New England Biolabs) was mixed with 28 μl of the eluted PCR product in an 80-μl (total volume) mixture containing 1× (final concentration) lambda exonuclease reaction buffer (New England Biolabs). .. The samples were separated by electrophoresis in a 0.625× MDE gel (FMC Bioproducts) with 1× TBE buffer.

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: Oligonucleotides were named according to the length of double-stranded (D) and Single-stranded (S) telomeric sequence in the probe. .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs). .. In a similar way, we produced biotinylated baits targeting a somatic indel in patient NB-pt02.

    Sonication:

    Article Title: In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor
    Article Snippet: The genomic DNA target was made single stranded by 5′-to-3′ exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA). .. The genomic DNA target was detected by hybridization to a circularizable, linear, oligonucleotide padlock probe (Fig. ).

    Affinity Purification:

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: The successful creation of partial ds probe was assayed by mobility shift compared to ssDNA reverse strand on 6% EMSA gel. .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction. .. The reaction was carried out at 25 °C for 15 min and samples were loaded onto 6% polyacrylamide (29:1 acrylamide:bis-acrylamide) gel and run in 1x TBE (89 mM Tris-borate, 2 mM EDTA, pH 8.0), 150 V at 4 °C.

    Binding Assay:

    Article Title: Enhanced Functional Potential of Nucleic Acid Aptamer Libraries Patterned to Increase Secondary Structure
    Article Snippet: The bound DNA was eluted twice using 30-min incubations with 200 μL of binding buffer containing 1 μM streptavidin. .. The reaction was purified using a PCR Purification Kit (Qiagen) and diluted to 1× final concentration of λ-exonuclease buffer (New England Biolabs).

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: The successful creation of partial ds probe was assayed by mobility shift compared to ssDNA reverse strand on 6% EMSA gel. .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction. .. The reaction was carried out at 25 °C for 15 min and samples were loaded onto 6% polyacrylamide (29:1 acrylamide:bis-acrylamide) gel and run in 1x TBE (89 mM Tris-borate, 2 mM EDTA, pH 8.0), 150 V at 4 °C.

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol. .. Reaction was stopped by 10 min incubation at 75°C. ssDNA was precipitated with dehydrated alcohol and washed with 70% alcohol.

    In Vivo:

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. For the ssOR experiment 1 pmole HPLC-purified oligonucletide was used.

    Magnetic Beads:

    Article Title: Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology
    Article Snippet: The PCR product was purified on a GFXTM column (GE Healthcare) and incubated with 1 mg of streptavidin-coated magnetic beads (Dynabeads M-270, Invitrogen) for 2 h at 37°C in 400 µl of BW buffer (5 mM Tris–HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl). .. Subsequently, the DNA was washed once with 100 µl of 1× λ exonuclease buffer (NEB).

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: The new batch of PCR amplicons was purified with 1.5× volumes of Agencourt AMPure XP magnetic beads, eluted in a final volume of 25 µl and quantified in a Qubit fluorometer. .. Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs).

    Article Title: An Aptamer-Based Biosensor for the Azole Class of Antifungal Drugs
    Article Snippet: Carboxyl Dynabeads (14305D), the Oligreen single-stranded DNA (ssDNA) assay kit, and BODIPY fluorescent dye were purchased from Life Technologies, Inc. (Carlsbad, CA). .. Streptavidin-conjugated magnetic beads and λ exonuclease were purchased from New England Biolabs (Boston, MA). .. Azole drugs were purchased from Santa Cruz Biotechnologies (Dallas, TX).

    Mutagenesis:

    Article Title: Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase
    Article Snippet: Phusion DNA polymerase (derived from a Pyroccccus species DNA polymerase), T4 DNA polymerase (T4-Pol) and lambda (λ) exonuclease were purchased from New England Biolabs (Hitchin, UK). .. Phusion DNA polymerase (derived from a Pyroccccus species DNA polymerase), T4 DNA polymerase (T4-Pol) and lambda (λ) exonuclease were purchased from New England Biolabs (Hitchin, UK).

    Isolation:

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: DraI-linearized pZR181, pZR173, and pZR156 DNAs were introduced by transformation into ZX7 and plasmids were isolated. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h.

    Polymerase Chain Reaction:

    Article Title: Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology
    Article Snippet: The PCR product was purified on a GFXTM column (GE Healthcare) and incubated with 1 mg of streptavidin-coated magnetic beads (Dynabeads M-270, Invitrogen) for 2 h at 37°C in 400 µl of BW buffer (5 mM Tris–HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl). .. Subsequently, the DNA was washed once with 100 µl of 1× λ exonuclease buffer (NEB).

    Article Title: Enhanced Functional Potential of Nucleic Acid Aptamer Libraries Patterned to Increase Secondary Structure
    Article Snippet: PCR product yields were monitored by agarose gel electrophoresis. .. The reaction was purified using a PCR Purification Kit (Qiagen) and diluted to 1× final concentration of λ-exonuclease buffer (New England Biolabs). .. Five units of λ-exonuclease were added, and the solution was incubated for 30 min at 37 °C.

    Article Title: Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)--Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria
    Article Snippet: Samples were eluted with 30 μl of Tris-HCl (pH 8.0). .. For digestion of the phosphorylated strand, 40 U of lambda exonuclease (New England Biolabs) was mixed with 28 μl of the eluted PCR product in an 80-μl (total volume) mixture containing 1× (final concentration) lambda exonuclease reaction buffer (New England Biolabs). .. The reaction mixtures were incubated at 37°C for 2 h, purified with Qiaquick columns (Qiagen), and eluted as previously described.

    Article Title:
    Article Snippet: The PCR products were then gel purified using the Qiagen QIAquick PCR purification kit as described in the manufacturer's protocol. .. After purification, the PCR product was treated with λ-exonuclease (New England Biolabs) at 37 °C for 2 h to remove the non-biotinylated strand. .. 10 μg of the purified products were hybridized on the GeneChip® Human Genome U133 +2 Array (Affymetrix) using the Affymetrix hybridization buffer and then stained with streptavidin-phycoerythrin (Molecular Probes).

    Article Title: Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase
    Article Snippet: Deoxynucleotide triphosphates (dNTPs) were supplied by Roche (Penzberg, Germany) and were the best quality available (PCR grade). .. Phusion DNA polymerase (derived from a Pyroccccus species DNA polymerase), T4 DNA polymerase (T4-Pol) and lambda (λ) exonuclease were purchased from New England Biolabs (Hitchin, UK).

    Article Title: Complementary strand relocation may play vital roles in RecA-based homology recognition
    Article Snippet: The ssDNA was previously prepared by amplifying a 5-kb fragment using λ-phage as a template where one of the primers was 5′-phosphorylated, and purified using a Macherey–Nagel kit. .. The dsDNA PCR fragment was subsequently incubated with λ-exonuclease enzyme (NEB) at 37°C for 30 min, and the resulting ssDNA was further purified using a Qiagen kit. .. For experiments where free RecA binds to dsDNA, an aliquot of dsDNA in RecA buffer, 1 µM RecA (New England Biolabs), 1 mM ATPγS and the beads were placed in a square micro-cell.

    Article Title: Rapid high resolution MHC class I genotyping of Chinese rhesus macaques by capillary reference strand mediated conformational analysis
    Article Snippet: Paragraph title: cDNA synthesis and PCR of Chinese rhesus macaque MHC class I sequences ... Purified products were treated with λ Exonuclease (New England BioLabs) to generate single stranded antisense products for heteroduplex formation.

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: The new batch of PCR amplicons was purified with 1.5× volumes of Agencourt AMPure XP magnetic beads, eluted in a final volume of 25 µl and quantified in a Qubit fluorometer. .. Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs). .. The reaction was incubated during 30 min at 37 °C and the enzyme was heat-inactivated at 75 °C during 10 min. Single-stranded biotinylated baits were purified with 1.5x volumes of AMPure XP beads and finally eluted in 25 µl of 10 mM Tris-HCl, 0.1 mM EDTA, pH = 8.0 buffer.

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: A PCR fragment (primers 5′-TCTAGAGTTGCTCATGCCGCCACC-3′ and 5′-AAGCTTGTGTGGACCATGCTGCCT-3′) containing the rlrA and rorA genes (required for linear replication) of linear plasmid pSLA2 was cloned into XbaI-treated pZR173 to yield pZR181. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h.

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: One hundred microliters of the PCR mixture contained 50 µL Premix Taq solution, 4 µL template, 1 µL of 10 µM fluorescein labeled forward primer F-fam, 1 µL of 10 µM phosphate labeled reverse primer R-pho, and 44 µL of ddH2 O. PCRs conditions were as follows: preincubation at 94°C for 5 min; optical number of cycles consisting of denaturation at 94°C for 0.5 min, annealing at 56°C for 0.5 min and extension at 72°C for 0.5 min; and final extension at 72°C for 10 min. .. The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol. .. Reaction was stopped by 10 min incubation at 75°C. ssDNA was precipitated with dehydrated alcohol and washed with 70% alcohol.

    Labeling:

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: One hundred microliters of the PCR mixture contained 50 µL Premix Taq solution, 4 µL template, 1 µL of 10 µM fluorescein labeled forward primer F-fam, 1 µL of 10 µM phosphate labeled reverse primer R-pho, and 44 µL of ddH2 O. PCRs conditions were as follows: preincubation at 94°C for 5 min; optical number of cycles consisting of denaturation at 94°C for 0.5 min, annealing at 56°C for 0.5 min and extension at 72°C for 0.5 min; and final extension at 72°C for 10 min. .. The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol.

    Purification:

    Article Title: Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology
    Article Snippet: The PCR product was purified on a GFXTM column (GE Healthcare) and incubated with 1 mg of streptavidin-coated magnetic beads (Dynabeads M-270, Invitrogen) for 2 h at 37°C in 400 µl of BW buffer (5 mM Tris–HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl). .. Subsequently, the DNA was washed once with 100 µl of 1× λ exonuclease buffer (NEB).

    Article Title: Enhanced Functional Potential of Nucleic Acid Aptamer Libraries Patterned to Increase Secondary Structure
    Article Snippet: PCR product yields were monitored by agarose gel electrophoresis. .. The reaction was purified using a PCR Purification Kit (Qiagen) and diluted to 1× final concentration of λ-exonuclease buffer (New England Biolabs). .. Five units of λ-exonuclease were added, and the solution was incubated for 30 min at 37 °C.

    Article Title: Nucleotide Polymorphism-Based Single-Tube Test for Robust Molecular Identification of All Currently Described Brucella Species
    Article Snippet: Briefly, the first step (ligation) was conducted in a 10-μl mixture containing 10 ng purified genomic DNA, 4 U of Pfu DNA ligase (Agilent, Santa Clara, CA), 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl2 , 0.1% Igepal, 0.01 mM ATP, 1 mM dithiothreitol (DTT), and 100 pM of each PLP except the IS 711 probe (50 pM) and the ptsP probe (300 pM). .. The second step (exonuclease treatment) started with the addition of 15 μl of exonuclease mixture consisting of 67 mM glycine-KOH, pH 9.4, 2.5 mM MgCl2 , 50 μg/ml bovine serum albumin (BSA), and 1 U exonuclease λ (New England BioLabs, Ipswich, MA).

    Article Title: Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)--Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria
    Article Snippet: PCR products were purified with Qiaquick columns by using a protocol recommended by the manufacturer (Qiagen, Hilden, Germany). .. For digestion of the phosphorylated strand, 40 U of lambda exonuclease (New England Biolabs) was mixed with 28 μl of the eluted PCR product in an 80-μl (total volume) mixture containing 1× (final concentration) lambda exonuclease reaction buffer (New England Biolabs).

    Article Title:
    Article Snippet: The PCR products were then gel purified using the Qiagen QIAquick PCR purification kit as described in the manufacturer's protocol. .. After purification, the PCR product was treated with λ-exonuclease (New England Biolabs) at 37 °C for 2 h to remove the non-biotinylated strand. .. 10 μg of the purified products were hybridized on the GeneChip® Human Genome U133 +2 Array (Affymetrix) using the Affymetrix hybridization buffer and then stained with streptavidin-phycoerythrin (Molecular Probes).

    Article Title: Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase
    Article Snippet: Phusion DNA polymerase (derived from a Pyroccccus species DNA polymerase), T4 DNA polymerase (T4-Pol) and lambda (λ) exonuclease were purchased from New England Biolabs (Hitchin, UK). .. Phusion DNA polymerase (derived from a Pyroccccus species DNA polymerase), T4 DNA polymerase (T4-Pol) and lambda (λ) exonuclease were purchased from New England Biolabs (Hitchin, UK).

    Article Title: Complementary strand relocation may play vital roles in RecA-based homology recognition
    Article Snippet: The ssDNA was previously prepared by amplifying a 5-kb fragment using λ-phage as a template where one of the primers was 5′-phosphorylated, and purified using a Macherey–Nagel kit. .. The dsDNA PCR fragment was subsequently incubated with λ-exonuclease enzyme (NEB) at 37°C for 30 min, and the resulting ssDNA was further purified using a Qiagen kit. .. For experiments where free RecA binds to dsDNA, an aliquot of dsDNA in RecA buffer, 1 µM RecA (New England Biolabs), 1 mM ATPγS and the beads were placed in a square micro-cell.

    Article Title: Rapid high resolution MHC class I genotyping of Chinese rhesus macaques by capillary reference strand mediated conformational analysis
    Article Snippet: Amplicons were generated and purified under the conditions described above for custom sizing standard. .. Purified products were treated with λ Exonuclease (New England BioLabs) to generate single stranded antisense products for heteroduplex formation. .. Fluorescently labeled reference strands were generated from MHC class I clones containing Mamu-B * 07 , Mamu-A1 * 7402 , or Mafa-B * 430101 ( ).

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: The new batch of PCR amplicons was purified with 1.5× volumes of Agencourt AMPure XP magnetic beads, eluted in a final volume of 25 µl and quantified in a Qubit fluorometer. .. Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs).

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: was performed to isolate ssDNA from asymmetrically phosphorylated dsDNA. .. 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. 2.5 volumes of denaturating buffer (95% formamide, 10 mM NaOH, 0.05% xylene cyanol, 0.05% bromophenol blue) was added to 1 volume of digestion product before boiling for 5 minutes at 95°C and placing on ice.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Rapid high resolution MHC class I genotyping of Chinese rhesus macaques by capillary reference strand mediated conformational analysis
    Article Snippet: Synthesis of complementary DNA (cDNA) was performed using the Superscript™ III First-Strand Synthesis System for RT-PCR (Invitrogen). .. Purified products were treated with λ Exonuclease (New England BioLabs) to generate single stranded antisense products for heteroduplex formation.

    shRNA:

    Article Title:
    Article Snippet: Paragraph title: shRNA-based Screening ... After purification, the PCR product was treated with λ-exonuclease (New England Biolabs) at 37 °C for 2 h to remove the non-biotinylated strand.

    Microscopy:

    Article Title: In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor
    Article Snippet: Cytospin culture material on the microscope slides was uniformly treated as follows. .. The genomic DNA target was made single stranded by 5′-to-3′ exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany).

    Concentration Assay:

    Article Title: Enhanced Functional Potential of Nucleic Acid Aptamer Libraries Patterned to Increase Secondary Structure
    Article Snippet: PCR product yields were monitored by agarose gel electrophoresis. .. The reaction was purified using a PCR Purification Kit (Qiagen) and diluted to 1× final concentration of λ-exonuclease buffer (New England Biolabs). .. Five units of λ-exonuclease were added, and the solution was incubated for 30 min at 37 °C.

    Article Title: Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)--Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria
    Article Snippet: Samples were eluted with 30 μl of Tris-HCl (pH 8.0). .. For digestion of the phosphorylated strand, 40 U of lambda exonuclease (New England Biolabs) was mixed with 28 μl of the eluted PCR product in an 80-μl (total volume) mixture containing 1× (final concentration) lambda exonuclease reaction buffer (New England Biolabs). .. The reaction mixtures were incubated at 37°C for 2 h, purified with Qiaquick columns (Qiagen), and eluted as previously described.

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction. .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: The new batch of PCR amplicons was purified with 1.5× volumes of Agencourt AMPure XP magnetic beads, eluted in a final volume of 25 µl and quantified in a Qubit fluorometer. .. Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs). .. The reaction was incubated during 30 min at 37 °C and the enzyme was heat-inactivated at 75 °C during 10 min. Single-stranded biotinylated baits were purified with 1.5x volumes of AMPure XP beads and finally eluted in 25 µl of 10 mM Tris-HCl, 0.1 mM EDTA, pH = 8.0 buffer.

    Article Title: Functional interactions of DNA topoisomerases with a human replication origin
    Article Snippet: Proteins were digested overnight with 200 μg/ml proteinase K and the DNA purification and primer extension were performed as above. .. For mapping the border of the in vitro complex, 50 U λ-exonuclease (New England Biolabs) was added to the preformed complex, incubated at 37°C for 3 h and then stopped with 2% SDS final concentration. .. The DNA was then purified and subjected to primer extension as described above.

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol. .. After centrifugation, the pellet was dissolved in binding buffer and used as the enriched library for the next selection round.

    Chromatin Immunoprecipitation:

    Article Title:
    Article Snippet: After purification, the PCR product was treated with λ-exonuclease (New England Biolabs) at 37 °C for 2 h to remove the non-biotinylated strand. .. 10 μg of the purified products were hybridized on the GeneChip® Human Genome U133 +2 Array (Affymetrix) using the Affymetrix hybridization buffer and then stained with streptavidin-phycoerythrin (Molecular Probes).

    Plasmid Preparation:

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: A PCR fragment (primers 5′-TCTAGAGTTGCTCATGCCGCCACC-3′ and 5′-AAGCTTGTGTGGACCATGCTGCCT-3′) containing the rlrA and rorA genes (required for linear replication) of linear plasmid pSLA2 was cloned into XbaI-treated pZR173 to yield pZR181. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h.

    Software:

    Article Title:
    Article Snippet: After purification, the PCR product was treated with λ-exonuclease (New England Biolabs) at 37 °C for 2 h to remove the non-biotinylated strand. .. 10 μg of the purified products were hybridized on the GeneChip® Human Genome U133 +2 Array (Affymetrix) using the Affymetrix hybridization buffer and then stained with streptavidin-phycoerythrin (Molecular Probes).

    Functional Assay:

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: By using a similar strategy , pZR131, containing two functional 381-bp pSLA2 telomeres and the Streptomyces tsr and melC genes of plasmid pIJ702, was constructed. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h.

    Selection:

    Article Title: Enhanced Functional Potential of Nucleic Acid Aptamer Libraries Patterned to Increase Secondary Structure
    Article Snippet: The reaction was purified using a PCR Purification Kit (Qiagen) and diluted to 1× final concentration of λ-exonuclease buffer (New England Biolabs). .. Five units of λ-exonuclease were added, and the solution was incubated for 30 min at 37 °C.

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: During every round of selection, a pilot PCR was first performed to determine the optimal cycle number of amplification as described by Lou et al. . .. The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol.

    Agarose Gel Electrophoresis:

    Article Title: Enhanced Functional Potential of Nucleic Acid Aptamer Libraries Patterned to Increase Secondary Structure
    Article Snippet: PCR product yields were monitored by agarose gel electrophoresis. .. The reaction was purified using a PCR Purification Kit (Qiagen) and diluted to 1× final concentration of λ-exonuclease buffer (New England Biolabs).

    Article Title: Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
    Article Snippet: DraI-linearized pZR181, pZR173, and pZR156 DNAs were introduced by transformation into ZX7 and plasmids were isolated. .. Aliquots of DNA were either untreated (in TE buffer) or treated with E. coli exonuclease III (Fermentas, Inc.) and bacteriophage λ exonuclease (New England Biolabs) and electrophoresed in a 0.6% agarose gel at 3 V/cm for 6 h. .. Plasmid pFQ8 contained full-length pFP11 and vector pHZ132.

    Article Title: Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin
    Article Snippet: The dsDNA PCR product was digested by λ exonuclease (NEB, UK) for 2 h at 37°C to generate ssDNA, according to the manufacturer's protocol. .. The concentration of ssDNA was evaluated by electrophoretic analysis .

    In Vitro:

    Article Title: Functional interactions of DNA topoisomerases with a human replication origin
    Article Snippet: Proteins were digested overnight with 200 μg/ml proteinase K and the DNA purification and primer extension were performed as above. .. For mapping the border of the in vitro complex, 50 U λ-exonuclease (New England Biolabs) was added to the preformed complex, incubated at 37°C for 3 h and then stopped with 2% SDS final concentration. .. The DNA was then purified and subjected to primer extension as described above.

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany).

    Produced:

    Article Title: Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology
    Article Snippet: DNA with a 3′ ss overhang was made as follows: using the URA3 gene from Saccharomyces cerevisiae as template DNA, an 810-bp polymerase chain reaction (PCR) fragment was produced using primer URA3 which was 5′ phosphorylated and primer BIO 5′ which was 5′ biotinylated. .. Subsequently, the DNA was washed once with 100 µl of 1× λ exonuclease buffer (NEB).

    Article Title: Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits
    Article Snippet: Aliquots of PCR products at ~15 ng/µl were now treated with 25 U of lambda exonuclease (New England Biolabs) in a final volume of 50 µl containing 1x final concentration of lambda exonuclease reaction buffer (New England Biolabs). .. The reaction was incubated during 30 min at 37 °C and the enzyme was heat-inactivated at 75 °C during 10 min. Single-stranded biotinylated baits were purified with 1.5x volumes of AMPure XP beads and finally eluted in 25 µl of 10 mM Tris-HCl, 0.1 mM EDTA, pH = 8.0 buffer.

    Mobility Shift:

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: The successful creation of partial ds probe was assayed by mobility shift compared to ssDNA reverse strand on 6% EMSA gel. .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    In Situ:

    Article Title: Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
    Article Snippet: We adapted a previously described protocol for in situ detection of mitochondrial DNA to work with the padlock gap probes ( ). .. Genomic and mitochondrial DNA was restriction digested using 0.5 U/μl of Msc I (New England Biolabs) in NEB buffer 4 supplemented with 0.2 μg/μl BSA (New England Biolabs) at 37°C for 30 min. Exonucleolysis was carried out in the same step by adding 0.4 U/μl of the 5′-3′ T7 exonuclease (New England Biolabs) or the 5′-3′ λ exonuclease (New England Biolabs) to the reaction mix.

    Article Title: In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor
    Article Snippet: Paragraph title: In situ rolling-circle amplification of padlock probes. ... The genomic DNA target was made single stranded by 5′-to-3′ exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA).

    Gel Extraction:

    Article Title: Single-stranded heteroduplex intermediates in ? Red homologous recombination
    Article Snippet: was performed to isolate ssDNA from asymmetrically phosphorylated dsDNA. .. 100 pmol of dsDNA were digested with 1 unit of λ exonuclease (New England Biolabs) for 1 hour at 37°C, followed by purification using gel extraction kit (QIAGEN, Germany). .. 2.5 volumes of denaturating buffer (95% formamide, 10 mM NaOH, 0.05% xylene cyanol, 0.05% bromophenol blue) was added to 1 volume of digestion product before boiling for 5 minutes at 95°C and placing on ice.

    Fluorescence In Situ Hybridization:

    Article Title: In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor
    Article Snippet: The genomic DNA target was made single stranded by 5′-to-3′ exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA). .. The genomic DNA target was detected by hybridization to a circularizable, linear, oligonucleotide padlock probe (Fig. ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs λ exonuclease buffer
    Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of <t>λ-exonuclease</t> (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .
    λ Exonuclease Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/λ exonuclease buffer/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    λ exonuclease buffer - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Binding Assay, Liquid Chromatography

    Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: In Vivo, Binding Assay, Mutagenesis

    ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Liquid Chromatography, Ethanol Precipitation

    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Amplification, Functional Assay, Ligation, Purification, Polymerase Chain Reaction, Sequencing, Genomic Sequencing, Generated, In Vitro, Footprinting, In Vivo

    Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

    Techniques: In Vivo, Chromatin Immunoprecipitation, Variant Assay, Binding Assay

    Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.

    Journal:

    Article Title: Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

    doi: 10.1128/JVI.00166-17

    Figure Lengend Snippet: Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.

    Article Snippet: Finally, dsDNA fragments were converted to single-stranded DNA (ssDNA) in 1× λ-exonuclease buffer (NEB) with 10% glycerol, 0.2 μg/μl BSA, and 0.2 U/μl λ-exonuclease (NEB) for 30 min at 37°C.

    Techniques: In Situ, Infection, Plasmid Purification

    HAdV-5 DNA and mRNA detection by rolling-circle amplification (RCA). (A) Simplified schematic overview of concurrent viral DNA and mRNA detection. Viral mRNA is converted to cDNA by reverse transcription, followed by mRNA degradation by RNase H. HAdV-5 DNA endonucleolytic cleavage with MscI and λ-exonuclease digestion expose ssDNA target sequence for padlock probe (PLP) binding. Target annealed PLPs are circularized with DNA ligase and amplified by RCA. The generated rolling-circle product (RCP) is visualized upon binding of fluorophore-conjugated oligonucleotides under a fluorescence microscope. Arrows represent 3′ extremities, and flat ends represent 5′ extremities. (B) PLP target sites on HAdV-5 DNA and mRNAs. The E1A mRNA and MLTU mRNA PLP target sequences (uppercase letters) are presented on the corresponding HAdV-5 genome regions. 5′ and 3′ arms of the PLP target sequences are indicated in green and blue, respectively. The MscI restriction site adjacent to the HAdV-5 genomic DNA padlock probe sequence target is in yellow. RT primer binding sites adjacent to E1A and MLTU target sites (italic). HAdV-5 genome numbering is based on NCBI reference sequence .

    Journal:

    Article Title: Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

    doi: 10.1128/JVI.00166-17

    Figure Lengend Snippet: HAdV-5 DNA and mRNA detection by rolling-circle amplification (RCA). (A) Simplified schematic overview of concurrent viral DNA and mRNA detection. Viral mRNA is converted to cDNA by reverse transcription, followed by mRNA degradation by RNase H. HAdV-5 DNA endonucleolytic cleavage with MscI and λ-exonuclease digestion expose ssDNA target sequence for padlock probe (PLP) binding. Target annealed PLPs are circularized with DNA ligase and amplified by RCA. The generated rolling-circle product (RCP) is visualized upon binding of fluorophore-conjugated oligonucleotides under a fluorescence microscope. Arrows represent 3′ extremities, and flat ends represent 5′ extremities. (B) PLP target sites on HAdV-5 DNA and mRNAs. The E1A mRNA and MLTU mRNA PLP target sequences (uppercase letters) are presented on the corresponding HAdV-5 genome regions. 5′ and 3′ arms of the PLP target sequences are indicated in green and blue, respectively. The MscI restriction site adjacent to the HAdV-5 genomic DNA padlock probe sequence target is in yellow. RT primer binding sites adjacent to E1A and MLTU target sites (italic). HAdV-5 genome numbering is based on NCBI reference sequence .

    Article Snippet: Finally, dsDNA fragments were converted to single-stranded DNA (ssDNA) in 1× λ-exonuclease buffer (NEB) with 10% glycerol, 0.2 μg/μl BSA, and 0.2 U/μl λ-exonuclease (NEB) for 30 min at 37°C.

    Techniques: Amplification, Sequencing, Plasmid Purification, Binding Assay, Generated, Fluorescence, Microscopy

    Overproduction of Din7 enhances 5′−3′ dsDNA exonuclease activity in mitochondria. ( A ) Principle of the 5′−3′ and 3′−5′ exonuclease activity assay. The 5′- and 3′- 32 P-labels were preferentially removed by 5′−3′ and 3′−5′ exonuclease activity, and they are resistant to 3′−5′ and 5′−3′ exonucleases, respectively. ( B ) Schematic diagram of the construction of the Din7 deletion mutant (Din7Δ). Immunoblot analysis of overproduced C-terminally 6× His-tagged Din7 or Din7Δ in mitochondrial extracts (bottom). Mitochondria were isolated from cells bearing WT/pYES2/CT, WT/pYES2/CT-DIN7 and WT/pYES2/CT-din7Δ (overproducing Din7Δ with a C-terminal 6× His tag). The signals of overproduced Din7 and Din7Δ were detected using a monoclonal anti-6× His-tag antibody. As a control, the levels of porin (a constitutively expressed protein, used here as a control) were determined by immunoblot analysis using a monoclonal anti-porin antibody to adjust the protein concentrations of mitochondrial extracts from all three strains to the same level. ( C  and  E ) Analyses of 5′−3′ or 3′−5′ exonuclease activity in mitochondrial extracts. The 5′- or 3′- 32 P-labeled pUC119/HincII dsDNA fragments were incubated at 37°C for 15 min with increasing concentrations of mitochondrial extracts. After removing the proteins by treating the extracts with proteinase K, the DNA molecules were separated on a 1% agarose gel. ( D  and  F ) Quantitative representation of the 5′−3′ and 3′−5′ exonuclease activities in mitochondrial extracts, detected in (C) and (E). Signals relative to untreated DNA are plotted. ds, double-stranded; ss, single-stranded. Open circles, incubated with mitochondrial extracts from wild-type cells (cells without Din7 overproduction); closed circles, incubated with mitochondrial extracts from Din7-overproducing cells; closed triangles, incubated with mitochondrial extracts from Din7Δ-overproducing cells. Each bar represents the results of at least two independent experiments. ( G ) The generation of single-stranded DNA by Din7. HincII-linearized pUC119 DNA (104 µM) was treated at 37°C for 5 min with increasing amounts of mitochondrial extracts derived from cells of WT/pYES2/CT, WT/pYES2/CT-DIN7 or WT/pYES2/CT-din7Δ in the same buffer used for detection of λ exonuclease activity. Then, each reaction solution (10 µl) was separated into duplicate aliquots. Proteinase K was added to one aliquot to stop the reaction. The second aliquot from each set was treated at 37°C for 10 min with 2.25 U mung bean nuclease followed by addition of proteinase K to stop the reaction. Mung bean nuclease digests only the single-stranded DNA, leaving the double-stranded DNA intact. Samples were then electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lane 1, 1-kb plus ladder used as a size marker; lane 2, HincII-linearized pUC119 DNA; lane 3, HincII-linearized pUC119 DNA treated in a reaction mixture without mitochondrial extracts; lanes 4–7, HincII-linearized pUC119 DNA treated with increasing amounts of mitochondrial extracts; lane 8, HincII-linearized pUC119 DNA treated only with mung bean nuclease; lanes 9–12, samples treated as in lanes 4–7, treated additionally with mung bean nuclease. The samples treated with mitochondrial extracts overexpressing  DIN7  contain both single-stranded DNA fragments with discrete sizes and double-stranded DNA because discrete DNA signals smaller than those of the double-stranded DNA in the middle were removed by mung bean nuclease treatments.

    Journal: Nucleic Acids Research

    Article Title: Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast

    doi: 10.1093/nar/gkt273

    Figure Lengend Snippet: Overproduction of Din7 enhances 5′−3′ dsDNA exonuclease activity in mitochondria. ( A ) Principle of the 5′−3′ and 3′−5′ exonuclease activity assay. The 5′- and 3′- 32 P-labels were preferentially removed by 5′−3′ and 3′−5′ exonuclease activity, and they are resistant to 3′−5′ and 5′−3′ exonucleases, respectively. ( B ) Schematic diagram of the construction of the Din7 deletion mutant (Din7Δ). Immunoblot analysis of overproduced C-terminally 6× His-tagged Din7 or Din7Δ in mitochondrial extracts (bottom). Mitochondria were isolated from cells bearing WT/pYES2/CT, WT/pYES2/CT-DIN7 and WT/pYES2/CT-din7Δ (overproducing Din7Δ with a C-terminal 6× His tag). The signals of overproduced Din7 and Din7Δ were detected using a monoclonal anti-6× His-tag antibody. As a control, the levels of porin (a constitutively expressed protein, used here as a control) were determined by immunoblot analysis using a monoclonal anti-porin antibody to adjust the protein concentrations of mitochondrial extracts from all three strains to the same level. ( C and E ) Analyses of 5′−3′ or 3′−5′ exonuclease activity in mitochondrial extracts. The 5′- or 3′- 32 P-labeled pUC119/HincII dsDNA fragments were incubated at 37°C for 15 min with increasing concentrations of mitochondrial extracts. After removing the proteins by treating the extracts with proteinase K, the DNA molecules were separated on a 1% agarose gel. ( D and F ) Quantitative representation of the 5′−3′ and 3′−5′ exonuclease activities in mitochondrial extracts, detected in (C) and (E). Signals relative to untreated DNA are plotted. ds, double-stranded; ss, single-stranded. Open circles, incubated with mitochondrial extracts from wild-type cells (cells without Din7 overproduction); closed circles, incubated with mitochondrial extracts from Din7-overproducing cells; closed triangles, incubated with mitochondrial extracts from Din7Δ-overproducing cells. Each bar represents the results of at least two independent experiments. ( G ) The generation of single-stranded DNA by Din7. HincII-linearized pUC119 DNA (104 µM) was treated at 37°C for 5 min with increasing amounts of mitochondrial extracts derived from cells of WT/pYES2/CT, WT/pYES2/CT-DIN7 or WT/pYES2/CT-din7Δ in the same buffer used for detection of λ exonuclease activity. Then, each reaction solution (10 µl) was separated into duplicate aliquots. Proteinase K was added to one aliquot to stop the reaction. The second aliquot from each set was treated at 37°C for 10 min with 2.25 U mung bean nuclease followed by addition of proteinase K to stop the reaction. Mung bean nuclease digests only the single-stranded DNA, leaving the double-stranded DNA intact. Samples were then electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lane 1, 1-kb plus ladder used as a size marker; lane 2, HincII-linearized pUC119 DNA; lane 3, HincII-linearized pUC119 DNA treated in a reaction mixture without mitochondrial extracts; lanes 4–7, HincII-linearized pUC119 DNA treated with increasing amounts of mitochondrial extracts; lane 8, HincII-linearized pUC119 DNA treated only with mung bean nuclease; lanes 9–12, samples treated as in lanes 4–7, treated additionally with mung bean nuclease. The samples treated with mitochondrial extracts overexpressing DIN7 contain both single-stranded DNA fragments with discrete sizes and double-stranded DNA because discrete DNA signals smaller than those of the double-stranded DNA in the middle were removed by mung bean nuclease treatments.

    Article Snippet: The 5′-labeled pUC119/HincII dsDNA fragments (75 nM) and the 3′-labeled pUC119/BamHI dsDNA fragments (87 nM) were incubated at 37°C for 15 min with various amounts (0, 4, 20, 32 and 50 µg/ml in protein) of mitochondrial extracts in a 10-µl reaction solution containing 1× λ exonuclease buffer (New England BioLabs Inc., MA, USA): 67 mM glycine–KOH (pH 9.4), 2.5 mM MgCl2 and 50 µg/ml of bovine serum albumin.

    Techniques: Activity Assay, Mutagenesis, Isolation, Labeling, Incubation, Agarose Gel Electrophoresis, Derivative Assay, Staining, Marker