lambda exonuclease buffer  (New England Biolabs)


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    Name:
    Lambda Exonuclease
    Description:
    Lambda Exonuclease 5 000 units
    Catalog Number:
    m0262l
    Price:
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    Size:
    5 000 units
    Category:
    Exonucleases
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    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 4 article reviews
    Price from $9.99 to $1999.99
    lambda exonuclease buffer - by Bioz Stars, 2020-05
    99/100 stars

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    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

    Binding Assay:

    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. ..

    In Vitro:

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Ligation:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Ethanol Precipitation:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Purification:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Incubation:

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    other:

    Article Title: Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks *
    Article Snippet: Subsequent treatments with topoisomerase IV (John Innes Enterprises), topoisomerase I, T7 endonuclease I, or λ-exonuclease (all New England Biolabs) used the manufacturers' recommended conditions.

    Nuclease Assay:

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Polymerase Chain Reaction:

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences
    Article Snippet: .. The digested PCR product was treated with 0.1 units lambda exonuclease (New England Biolabs) at 37°C for 15 min in the same restriction enzyme buffer. .. The probes were phosphorylated with 5 units of T4 polynucleotide kinase (NEB) in 50 mM Tris·HCl, pH7.9; 10 mM MgCl2 . dHPLC analysis was used to monitor the efficiency of lambda exonuclease digestion.

    Affinity Purification:

    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. ..

    Recombinant:

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

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    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 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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, Ethanol Precipitation

    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.

    Journal: BMC Molecular Biology

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

    doi: 10.1186/1471-2199-8-103

    Figure Lengend 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.

    Article Snippet: Lambda exonuclease 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.

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

    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: Journal of Virology

    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

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Journal: Nature Communications

    Article Title: Simplified ChIP-exo assays

    doi: 10.1038/s41467-018-05265-7

    Figure Lengend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Article Snippet: The λ exonuclease digestion (100 µl) containing: 20 U λ exonuclease (NEB), 1 × λ exonuclease reaction buffer (NEB), 0.1% Triton-X 100, and 5% DMSO was incubated for 30 min at 37 °C; then washed with 10 mM Tris-HCl, pH 8.0 at 4 °C.

    Techniques: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced