exoi  (New England Biolabs)


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    Name:
    Exonuclease I E coli
    Description:
    Exonuclease I E coli 15 000 units
    Catalog Number:
    m0293l
    Price:
    281
    Size:
    15 000 units
    Category:
    Exonucleases
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    New England Biolabs exoi
    Exonuclease I E coli
    Exonuclease I E coli 15 000 units
    https://www.bioz.com/result/exoi/product/New England Biolabs
    Average 95 stars, based on 12260 article reviews
    Price from $9.99 to $1999.99
    exoi - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair"

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw411

    Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.
    Figure Legend Snippet: Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.

    Techniques Used: Agarose Gel Electrophoresis

    2) Product Images from "No Overt Nucleosome Eviction at Deprotected Telomeres ▿No Overt Nucleosome Eviction at Deprotected Telomeres ▿ †"

    Article Title: No Overt Nucleosome Eviction at Deprotected Telomeres ▿No Overt Nucleosome Eviction at Deprotected Telomeres ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01764-07

    A novel assay for the nucleosomal organization near the telomere terminus. (A) Scheme of the last nucleosome assay. Nuclei are treated with MNase, and the isolated DNA fragments are incubated with a biotinylated oligonucleotide representing the C-rich telomeric DNA strand, biotin-(CCCTAA) 6 . Magnetic streptavidin beads are used to pull down DNA fragments containing a telomeric (TTAGGG) n 3′ overhang. The supernatant contains fragments of bulk nucleosomes, while the pulldown contains nucleosomal fragments ending at the telomere terminus. Fragments are fractionated on a 1% agarose gel and subjected to Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. (B) Scheme for the construction of a model telomeric fragment to test the last nucleosome assay. A BglII/KpnI fragment excised from pSXneo.25(T 2 AG 3 ) (dsTel) was annealed and ligated to a single-stranded telomeric oligonucleotide (ssTel) containing the complementary BglII recognition sequence at its 5′ end. CIP, calf intestinal phosphatase. (C) Southern blot detection of the telomeric signal ligation products of dsTel and ssTel (lane 1), dsTel only (lane 2), or ssTel only (lane 3). (D) Southern blot detection of the telomeric signal after annealing the DNA products from lanes 1 and 2 shown in panel C with biotin-(CCCTAA) 6 and separating the supernatant (sup) and pulldown (ppt) following incubation with streptavidin beads. Two percent of the supernatant was loaded next to 50% of the total pulldown. Sizes in base pairs are marked to the left of panels C and D. (E) Detection of the telomeric signal associated with the last nucleosomes in wild-type MEFs. The last nucleosome assay was performed as described above, and the supernatant (2% of total fraction) containing the bulk nucleosomes was loaded in the first eight lanes, while nucleosomal fragments containing the telomeric overhang (50% of total pulldown) were loaded in the last eight lanes. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. (F) Detection of the telomeric signal pulled down by the last nucleosome assay following ExoI treatment of DNA from MNase-digested nuclei. DNA fragments isolated after MNase digestion were mixed and treated with ExoI (300 U in 300 μl), removing the 3′ overhang. ExoI-treated and untreated samples were then subjected to the last nucleosome assay. Two percent of the supernatant (bulk nucleosomes) was loaded next to 50% of the total pulldown (last nucleosomes). Percentage of total TTAGGG signal pulled down was calculated with the formula (2 × pulldown signal)/[(2 × pulldown signal) + (50 × supernatant signal)]. +, present; −, absent.
    Figure Legend Snippet: A novel assay for the nucleosomal organization near the telomere terminus. (A) Scheme of the last nucleosome assay. Nuclei are treated with MNase, and the isolated DNA fragments are incubated with a biotinylated oligonucleotide representing the C-rich telomeric DNA strand, biotin-(CCCTAA) 6 . Magnetic streptavidin beads are used to pull down DNA fragments containing a telomeric (TTAGGG) n 3′ overhang. The supernatant contains fragments of bulk nucleosomes, while the pulldown contains nucleosomal fragments ending at the telomere terminus. Fragments are fractionated on a 1% agarose gel and subjected to Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. (B) Scheme for the construction of a model telomeric fragment to test the last nucleosome assay. A BglII/KpnI fragment excised from pSXneo.25(T 2 AG 3 ) (dsTel) was annealed and ligated to a single-stranded telomeric oligonucleotide (ssTel) containing the complementary BglII recognition sequence at its 5′ end. CIP, calf intestinal phosphatase. (C) Southern blot detection of the telomeric signal ligation products of dsTel and ssTel (lane 1), dsTel only (lane 2), or ssTel only (lane 3). (D) Southern blot detection of the telomeric signal after annealing the DNA products from lanes 1 and 2 shown in panel C with biotin-(CCCTAA) 6 and separating the supernatant (sup) and pulldown (ppt) following incubation with streptavidin beads. Two percent of the supernatant was loaded next to 50% of the total pulldown. Sizes in base pairs are marked to the left of panels C and D. (E) Detection of the telomeric signal associated with the last nucleosomes in wild-type MEFs. The last nucleosome assay was performed as described above, and the supernatant (2% of total fraction) containing the bulk nucleosomes was loaded in the first eight lanes, while nucleosomal fragments containing the telomeric overhang (50% of total pulldown) were loaded in the last eight lanes. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. (F) Detection of the telomeric signal pulled down by the last nucleosome assay following ExoI treatment of DNA from MNase-digested nuclei. DNA fragments isolated after MNase digestion were mixed and treated with ExoI (300 U in 300 μl), removing the 3′ overhang. ExoI-treated and untreated samples were then subjected to the last nucleosome assay. Two percent of the supernatant (bulk nucleosomes) was loaded next to 50% of the total pulldown (last nucleosomes). Percentage of total TTAGGG signal pulled down was calculated with the formula (2 × pulldown signal)/[(2 × pulldown signal) + (50 × supernatant signal)]. +, present; −, absent.

    Techniques Used: Isolation, Incubation, Agarose Gel Electrophoresis, Southern Blot, Hybridization, Sequencing, Ligation

    Related Articles

    Amplification:

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    Article Snippet: .. Specific target amplification reactions were performed with the following thermocycling conditions: one cycle at 95 °C for 10 min, and 14 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was treated with exonuclease I (New England Biolabs) and diluted 10-fold. .. High throughput real time PCR was then conducted on the Fluidigm BioMark HD system using SsoFast EvaGreen Supermix with Low ROX (Bio-Rad) and Fluidigm 48.48 Dynamic Array integrated fluidic circuits.

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
    Article Snippet: Initial fragment amplification was carried out using primers kdr CL-F (5′-AAA TGT CTC GCC CAA ATC AG-3′) and kdr CL-R (5′-GCA CCT GCA AAA CAA TGT CA-3′) described previously [ ]. .. Cycling condition were as follows: 95 °C for 5min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s, followed by a final extension step at 72 °C for 10 min. PCR products were cleaned up using Exonuclease I (Exo I) and Shrimp Alkaline Phosphate (Exo-SAP protocol) according to the protocol of New England Biolabs (NEB, MA, USA).

    Article Title: Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks
    Article Snippet: The first-strand cDNA, synthesized using the qScript cDNA Synthesis kit, was first amplified for specific target amplification (STA). .. Unincorporated primers were then cleaned up using Exonuclease I (New England Biolabs).

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    Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function *
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    Article Title: Mitochondrial Introgression, Color Pattern Variation, and Severe Demographic Bottlenecks in Three Species of Malagasy Poison Frogs, Genus Mantella
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    Stable Transfection:

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
    Article Snippet: HepG2, HepaRG, Hepa1-6, Hepa56D, and HeLa cells stably expressing hNTCP were cultivated as described previously ( , ). .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Synthesized:

    Article Title: Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks
    Article Snippet: The first-strand cDNA, synthesized using the qScript cDNA Synthesis kit, was first amplified for specific target amplification (STA). .. Unincorporated primers were then cleaned up using Exonuclease I (New England Biolabs).

    Quantitative RT-PCR:

    Article Title: Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *
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    Article Snippet: Paragraph title: High-throughput quantitative RT-PCR analysis ... Unincorporated primers were then cleaned up using Exonuclease I (New England Biolabs).

    Real-time Polymerase Chain Reaction:

    Article Title: Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *
    Article Snippet: Specific target amplification reactions were performed with the following thermocycling conditions: one cycle at 95 °C for 10 min, and 14 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was treated with exonuclease I (New England Biolabs) and diluted 10-fold. .. High throughput real time PCR was then conducted on the Fluidigm BioMark HD system using SsoFast EvaGreen Supermix with Low ROX (Bio-Rad) and Fluidigm 48.48 Dynamic Array integrated fluidic circuits.

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
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    Incubation:

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    Article Title: Hybridization of glaucous gull (Larus hyperboreus) and herring gull (Larus argentatus) in Iceland: mitochondrial and microsatellite data
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    Article Title: Rapid Identification of Pseudallescheria and Scedosporium Strains by Using Rolling Circle Amplification
    Article Snippet: Unbound padlock probes and ITS templates were removed by exonucleolysis in 20-μl volumes containing 10 U exonuclease I and 10 U exonuclease III (New England BioLabs, Leusden, The Netherlands) for 30 min at 37°C. .. Lytic activity was stopped by incubation at 94°C for 30 min.

    Article Title: Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland ▿
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    Activity Assay:

    Article Title: Rapid Identification of Pseudallescheria and Scedosporium Strains by Using Rolling Circle Amplification
    Article Snippet: Unbound padlock probes and ITS templates were removed by exonucleolysis in 20-μl volumes containing 10 U exonuclease I and 10 U exonuclease III (New England BioLabs, Leusden, The Netherlands) for 30 min at 37°C. .. Lytic activity was stopped by incubation at 94°C for 30 min.

    Expressing:

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
    Article Snippet: HepG2, HepaRG, Hepa1-6, Hepa56D, and HeLa cells stably expressing hNTCP were cultivated as described previously ( , ). .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Modification:

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
    Article Snippet: HepG2hNTCP , Hepa1-6hNTCP , Hepa56DhNTCP , and HeLahNTCP cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM nonessential amino acids. .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Article Title: Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana
    Article Snippet: DNA isolation, PCR amplification and sequencing Genomic DNA was extracted from a pool of leaves from three plants per accession using a modified CTAB procedure [ ]. .. The fragments of 30 worldwide distributed accessions (An-2, Bl-1, Bsch-2, Bur-0, C24, Can-0, Cha-0, Col-0, Ct-1, Cvi-0, Edi-0, El-0, Er-0, Est-1, Gre-0, Ler-1, Mt-0, Nok-2, Oy-0, Ra-0, Rsch-0, Sap-0, Sha(kdara), Stw-0, Te-0, Tsu-1, Van-0, Wil, Ws-3, Yo-0) were amplified with the proof-reading polymerase Phusion (Finnzymes) and purified enzymatically by using Exonuclease I and Antarctic Phosphatase (New England Biolabs).

    Gel Purification:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: .. After gel purification and reverse transcription as described above using the RT primer 5′-CAAGCAGAAGACGGCATACGAGC-3′, first-strand cDNA was treated with Exonuclease I (New England Biolabs Inc.) as described to remove excess RT primers. ..

    Cell Culture:

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
    Article Snippet: HepG2hNTCP , Hepa1-6hNTCP , Hepa56DhNTCP , and HeLahNTCP cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM nonessential amino acids. .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    other:

    Article Title: Construction and electrophoretic migration of single-stranded DNA knots and catenanes
    Article Snippet: T4 DNA ligase and exonuclease I, exonuclease III and T4 polynucleotide kinase were supplied by New England Biolabs.

    DNA Sequencing:

    Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function *
    Article Snippet: To remove primers and unincorporated dNTPs, exonuclease I (New England Biolabs) and calf intestine alkaline phosphatase (Roche Applied Science) were used. .. The BigDye XTerminator purification kit was used as purification method for DNA sequencing with the purpose of removing unincorporated BigDye terminators.

    Article Title: Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing
    Article Snippet: .. Prior to DNA sequencing, enzymatic cleanup of each PCR was performed using a combination of Exonuclease I (Exo; New England BioLabs, Ipswich, MA) and shrimp alkaline phosphatase (SAP; USB, Affymetrix, Cleveland, OH) as described previously ( ). .. For the sequencing of specific PCR products, 1 μl of the ExoSAP-treated DNA was mixed with 6 μl of 5× sequencing buffer and 2 μl of sequencing reaction mix from a BigDye Terminator cycle sequencing kit, version 1.1 (Applied Biosystems, Foster City, CA), 2 μl of amplicon-specific primer (2 μmol/μl), and 9 μl of PCR-grade water for a final volume of 20 μl.

    Sequencing:

    Article Title: Visual short-term memory deficits associated with GBA mutation and Parkinson’s disease
    Article Snippet: Briefly, DNA was treated with an ExoSAP reaction as follows: 1 × SAP buffer, shrimp alkaline phosphatase (500 U; SAP, Promega), Exonuclease I (2 U; NEB). .. The sequencing reaction was performed according to BigDye® Terminator v3.1 Cycle Sequencing protocol (Applied Biosystems).

    Article Title: Hybridization of glaucous gull (Larus hyperboreus) and herring gull (Larus argentatus) in Iceland: mitochondrial and microsatellite data
    Article Snippet: .. Prior to sequencing, excess primers and nucleotides were enzymatically removed from PCR amplification products using a mixture of exonuclease I and Antarctic phosphatase (New England BioLabs). .. Cycle sequencing was carried out using the BigDye Terminator v. 1.1 Cycle Sequencing Kit on ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

    Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function *
    Article Snippet: Paragraph title: Direct Sequencing of PCR-amplified LRP4 DNA ... To remove primers and unincorporated dNTPs, exonuclease I (New England Biolabs) and calf intestine alkaline phosphatase (Roche Applied Science) were used.

    Article Title: Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿
    Article Snippet: .. Plasmid DNA purified with NucleoBond BAC 100 cartridges (Macherey-Nagel, Düren, Germany) or the 3,760-bp espP PCR product (Table ) purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (GE Healthcare, München, Germany) was used as a template for bidirectional sequencing with an automated ABI Prism 3130 Avant Genetic Analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), ABI Prism BigDye Terminator Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied Biosystems), and customized primers. .. Sequences were analyzed using MEGA software, version 3.1 , and aligned with ClustalW , and homology was determined using the EMBL-GenBank database ( ).

    Article Title: Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing
    Article Snippet: Prior to DNA sequencing, enzymatic cleanup of each PCR was performed using a combination of Exonuclease I (Exo; New England BioLabs, Ipswich, MA) and shrimp alkaline phosphatase (SAP; USB, Affymetrix, Cleveland, OH) as described previously ( ). .. For the sequencing of specific PCR products, 1 μl of the ExoSAP-treated DNA was mixed with 6 μl of 5× sequencing buffer and 2 μl of sequencing reaction mix from a BigDye Terminator cycle sequencing kit, version 1.1 (Applied Biosystems, Foster City, CA), 2 μl of amplicon-specific primer (2 μmol/μl), and 9 μl of PCR-grade water for a final volume of 20 μl.

    Article Title: Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland ▿
    Article Snippet: For this purpose, primers for the amplification and sequencing of 13 targets per strain (Table ) were divided into four amplification groups (AGs), taking into account the PCR product length and amplification efficiency for all targets. .. To enzymatically purify the samples from residual deoxynucleotides and excess primers, 8.0 μl of the AG1, AG2, or AG3 PCR product and 4.0 μl of the AG4 PCR product was transferred into new reaction tubes, followed by the addition of 1.0 μl rAPid Alkaline Phosphatase (1 U/μl; Roche Diagnostics), 0.2 μl of the corresponding buffer, and 0.05 μl exonuclease I ( Exo I; 20 U/μl; New England Biolabs, Ipswich, MA).

    Article Title: Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana
    Article Snippet: Paragraph title: DNA isolation, PCR amplification and sequencing ... The fragments of 30 worldwide distributed accessions (An-2, Bl-1, Bsch-2, Bur-0, C24, Can-0, Cha-0, Col-0, Ct-1, Cvi-0, Edi-0, El-0, Er-0, Est-1, Gre-0, Ler-1, Mt-0, Nok-2, Oy-0, Ra-0, Rsch-0, Sap-0, Sha(kdara), Stw-0, Te-0, Tsu-1, Van-0, Wil, Ws-3, Yo-0) were amplified with the proof-reading polymerase Phusion (Finnzymes) and purified enzymatically by using Exonuclease I and Antarctic Phosphatase (New England Biolabs).

    Cellular Antioxidant Activity Assay:

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
    Article Snippet: Initial fragment amplification was carried out using primers kdr CL-F (5′-AAA TGT CTC GCC CAA ATC AG-3′) and kdr CL-R (5′-GCA CCT GCA AAA CAA TGT CA-3′) described previously [ ]. .. Cycling condition were as follows: 95 °C for 5min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s, followed by a final extension step at 72 °C for 10 min. PCR products were cleaned up using Exonuclease I (Exo I) and Shrimp Alkaline Phosphate (Exo-SAP protocol) according to the protocol of New England Biolabs (NEB, MA, USA).

    DNA Extraction:

    Article Title: Mitochondrial Introgression, Color Pattern Variation, and Severe Demographic Bottlenecks in Three Species of Malagasy Poison Frogs, Genus Mantella
    Article Snippet: Paragraph title: 2.1. Tissue Sampling, DNA Extraction, and Amplification ... The successfully amplified double-stranded PCR products, treated with Exonuclease I (New England Biolabs, Ipswich MA, USA) and Shrimp Alkaline Phosphatase (Promega) to inactivate remaining primers and dNTPs, were directly used for the cycle-sequencing reaction using dye-labeled dideoxy terminators (Applied Biosystems, Foster City, CA, USA) with the amplification primers on an ABI 3130xl automated DNA sequencer.

    Article Title: Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana
    Article Snippet: Paragraph title: DNA isolation, PCR amplification and sequencing ... The fragments of 30 worldwide distributed accessions (An-2, Bl-1, Bsch-2, Bur-0, C24, Can-0, Cha-0, Col-0, Ct-1, Cvi-0, Edi-0, El-0, Er-0, Est-1, Gre-0, Ler-1, Mt-0, Nok-2, Oy-0, Ra-0, Rsch-0, Sap-0, Sha(kdara), Stw-0, Te-0, Tsu-1, Van-0, Wil, Ws-3, Yo-0) were amplified with the proof-reading polymerase Phusion (Finnzymes) and purified enzymatically by using Exonuclease I and Antarctic Phosphatase (New England Biolabs).

    Mutagenesis:

    Article Title: Visual short-term memory deficits associated with GBA mutation and Parkinson’s disease
    Article Snippet: For the L444P mutation primers used were: 5’-GGAGGACCCAATTGGGTGCGT-3’ and 5’-ACGCTGTCTTCAGCCCACTTC-3’. .. Briefly, DNA was treated with an ExoSAP reaction as follows: 1 × SAP buffer, shrimp alkaline phosphatase (500 U; SAP, Promega), Exonuclease I (2 U; NEB).

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
    Article Snippet: Paragraph title: Investigation of the role of the 1014F knockdown resistance mutation in pyrethroid/DDT resistance ... Cycling condition were as follows: 95 °C for 5min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s, followed by a final extension step at 72 °C for 10 min. PCR products were cleaned up using Exonuclease I (Exo I) and Shrimp Alkaline Phosphate (Exo-SAP protocol) according to the protocol of New England Biolabs (NEB, MA, USA).

    Isolation:

    Article Title: Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *
    Article Snippet: Paragraph title: RNA Isolation and Fluidigm BioMark RT-qPCR Analysis ... Specific target amplification reactions were performed with the following thermocycling conditions: one cycle at 95 °C for 10 min, and 14 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was treated with exonuclease I (New England Biolabs) and diluted 10-fold.

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
    Article Snippet: PHH were isolated from liver specimens obtained after partial hepatectomy and following written informed consent of the patients (approved by the ethics commission of Hannover Medical School/Ethik-Kommission der MHH, no. 252-2008). .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Polymerase Chain Reaction:

    Article Title: Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *
    Article Snippet: Specific target amplification reactions were performed with the following thermocycling conditions: one cycle at 95 °C for 10 min, and 14 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was treated with exonuclease I (New England Biolabs) and diluted 10-fold. .. PCR was performed using the thermal protocol GE Fast 96 × 96 PCR + Melt v2.pcl.

    Article Title: Visual short-term memory deficits associated with GBA mutation and Parkinson’s disease
    Article Snippet: The resulting PCR products were digested with XhoI (NEB) for N370S and NciI (NEB) for L444P and resolved by agarose gel electrophoresis. .. Briefly, DNA was treated with an ExoSAP reaction as follows: 1 × SAP buffer, shrimp alkaline phosphatase (500 U; SAP, Promega), Exonuclease I (2 U; NEB).

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
    Article Snippet: .. Cycling condition were as follows: 95 °C for 5min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s, followed by a final extension step at 72 °C for 10 min. PCR products were cleaned up using Exonuclease I (Exo I) and Shrimp Alkaline Phosphate (Exo-SAP protocol) according to the protocol of New England Biolabs (NEB, MA, USA). ..

    Article Title: Hybridization of glaucous gull (Larus hyperboreus) and herring gull (Larus argentatus) in Iceland: mitochondrial and microsatellite data
    Article Snippet: .. Prior to sequencing, excess primers and nucleotides were enzymatically removed from PCR amplification products using a mixture of exonuclease I and Antarctic phosphatase (New England BioLabs). .. Cycle sequencing was carried out using the BigDye Terminator v. 1.1 Cycle Sequencing Kit on ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

    Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function *
    Article Snippet: Paragraph title: Direct Sequencing of PCR-amplified LRP4 DNA ... To remove primers and unincorporated dNTPs, exonuclease I (New England Biolabs) and calf intestine alkaline phosphatase (Roche Applied Science) were used.

    Article Title: Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿
    Article Snippet: .. Plasmid DNA purified with NucleoBond BAC 100 cartridges (Macherey-Nagel, Düren, Germany) or the 3,760-bp espP PCR product (Table ) purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (GE Healthcare, München, Germany) was used as a template for bidirectional sequencing with an automated ABI Prism 3130 Avant Genetic Analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), ABI Prism BigDye Terminator Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied Biosystems), and customized primers. .. Sequences were analyzed using MEGA software, version 3.1 , and aligned with ClustalW , and homology was determined using the EMBL-GenBank database ( ).

    Article Title: Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing
    Article Snippet: .. Prior to DNA sequencing, enzymatic cleanup of each PCR was performed using a combination of Exonuclease I (Exo; New England BioLabs, Ipswich, MA) and shrimp alkaline phosphatase (SAP; USB, Affymetrix, Cleveland, OH) as described previously ( ). .. For the sequencing of specific PCR products, 1 μl of the ExoSAP-treated DNA was mixed with 6 μl of 5× sequencing buffer and 2 μl of sequencing reaction mix from a BigDye Terminator cycle sequencing kit, version 1.1 (Applied Biosystems, Foster City, CA), 2 μl of amplicon-specific primer (2 μmol/μl), and 9 μl of PCR-grade water for a final volume of 20 μl.

    Article Title: Mitochondrial Introgression, Color Pattern Variation, and Severe Demographic Bottlenecks in Three Species of Malagasy Poison Frogs, Genus Mantella
    Article Snippet: .. The successfully amplified double-stranded PCR products, treated with Exonuclease I (New England Biolabs, Ipswich MA, USA) and Shrimp Alkaline Phosphatase (Promega) to inactivate remaining primers and dNTPs, were directly used for the cycle-sequencing reaction using dye-labeled dideoxy terminators (Applied Biosystems, Foster City, CA, USA) with the amplification primers on an ABI 3130xl automated DNA sequencer. ..

    Article Title: Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland ▿
    Article Snippet: .. To enzymatically purify the samples from residual deoxynucleotides and excess primers, 8.0 μl of the AG1, AG2, or AG3 PCR product and 4.0 μl of the AG4 PCR product was transferred into new reaction tubes, followed by the addition of 1.0 μl rAPid Alkaline Phosphatase (1 U/μl; Roche Diagnostics), 0.2 μl of the corresponding buffer, and 0.05 μl exonuclease I ( Exo I; 20 U/μl; New England Biolabs, Ipswich, MA). .. The samples were incubated in the 9800 Fast Thermal Cycler (Applied Biosystems) for 30 min at 37°C and then for 20 min at 80°C to inactivate the enzymes.

    Article Title: Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana
    Article Snippet: Paragraph title: DNA isolation, PCR amplification and sequencing ... The fragments of 30 worldwide distributed accessions (An-2, Bl-1, Bsch-2, Bur-0, C24, Can-0, Cha-0, Col-0, Ct-1, Cvi-0, Edi-0, El-0, Er-0, Est-1, Gre-0, Ler-1, Mt-0, Nok-2, Oy-0, Ra-0, Rsch-0, Sap-0, Sha(kdara), Stw-0, Te-0, Tsu-1, Van-0, Wil, Ws-3, Yo-0) were amplified with the proof-reading polymerase Phusion (Finnzymes) and purified enzymatically by using Exonuclease I and Antarctic Phosphatase (New England Biolabs).

    Purification:

    Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function *
    Article Snippet: To remove primers and unincorporated dNTPs, exonuclease I (New England Biolabs) and calf intestine alkaline phosphatase (Roche Applied Science) were used. .. Sequencing was carried out directly on purified fragments with the ABI 310 Genetic Analyzer (Applied Biosystems), using an ABI Prism BigDye terminator cycle sequencing ready reaction kit, version 1.1 (Applied Biosystems).

    Article Title: Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿
    Article Snippet: .. Plasmid DNA purified with NucleoBond BAC 100 cartridges (Macherey-Nagel, Düren, Germany) or the 3,760-bp espP PCR product (Table ) purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (GE Healthcare, München, Germany) was used as a template for bidirectional sequencing with an automated ABI Prism 3130 Avant Genetic Analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), ABI Prism BigDye Terminator Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied Biosystems), and customized primers. .. Sequences were analyzed using MEGA software, version 3.1 , and aligned with ClustalW , and homology was determined using the EMBL-GenBank database ( ).

    Article Title: Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland ▿
    Article Snippet: Paragraph title: Multiplex PCR amplification and purification. ... To enzymatically purify the samples from residual deoxynucleotides and excess primers, 8.0 μl of the AG1, AG2, or AG3 PCR product and 4.0 μl of the AG4 PCR product was transferred into new reaction tubes, followed by the addition of 1.0 μl rAPid Alkaline Phosphatase (1 U/μl; Roche Diagnostics), 0.2 μl of the corresponding buffer, and 0.05 μl exonuclease I ( Exo I; 20 U/μl; New England Biolabs, Ipswich, MA).

    Article Title: Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana
    Article Snippet: .. The fragments of 30 worldwide distributed accessions (An-2, Bl-1, Bsch-2, Bur-0, C24, Can-0, Cha-0, Col-0, Ct-1, Cvi-0, Edi-0, El-0, Er-0, Est-1, Gre-0, Ler-1, Mt-0, Nok-2, Oy-0, Ra-0, Rsch-0, Sap-0, Sha(kdara), Stw-0, Te-0, Tsu-1, Van-0, Wil, Ws-3, Yo-0) were amplified with the proof-reading polymerase Phusion (Finnzymes) and purified enzymatically by using Exonuclease I and Antarctic Phosphatase (New England Biolabs). .. The templates were directly used for sequencing on an ABI 3130xl automated sequencer (Applied Biosystems), using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

    High Throughput Screening Assay:

    Article Title: Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *
    Article Snippet: Specific target amplification reactions were performed with the following thermocycling conditions: one cycle at 95 °C for 10 min, and 14 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was treated with exonuclease I (New England Biolabs) and diluted 10-fold. .. High throughput real time PCR was then conducted on the Fluidigm BioMark HD system using SsoFast EvaGreen Supermix with Low ROX (Bio-Rad) and Fluidigm 48.48 Dynamic Array integrated fluidic circuits.

    Article Title: Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks
    Article Snippet: Paragraph title: High-throughput quantitative RT-PCR analysis ... Unincorporated primers were then cleaned up using Exonuclease I (New England Biolabs).

    Activated Clotting Time Assay:

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
    Article Snippet: Probes were labelled with two specific fluorophores, FAM and HEX: FAM to detect the resistant allele [(5′-ACG ACA AAA TTT C-3′ for 1014F kdr ), (5′-ACG ACT GAA TTT C-3′ for 1014S kdr )] and HEX (5′-CTT ACG ACT AAA TTT C-3′) to detect the susceptible allele. .. Cycling condition were as follows: 95 °C for 5min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s, followed by a final extension step at 72 °C for 10 min. PCR products were cleaned up using Exonuclease I (Exo I) and Shrimp Alkaline Phosphate (Exo-SAP protocol) according to the protocol of New England Biolabs (NEB, MA, USA).

    Chromatin Immunoprecipitation:

    Article Title: Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks
    Article Snippet: Unincorporated primers were then cleaned up using Exonuclease I (New England Biolabs). .. High-throughput qPCRs were performed on the Biomark HD (Fluidigm) in a microfluidic multiplex 48.48 dynamic array chip according to the Fluidigm Advanced Development Protocol with EvaGreen.

    Plasmid Preparation:

    Article Title: Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿
    Article Snippet: .. Plasmid DNA purified with NucleoBond BAC 100 cartridges (Macherey-Nagel, Düren, Germany) or the 3,760-bp espP PCR product (Table ) purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (GE Healthcare, München, Germany) was used as a template for bidirectional sequencing with an automated ABI Prism 3130 Avant Genetic Analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), ABI Prism BigDye Terminator Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied Biosystems), and customized primers. .. Sequences were analyzed using MEGA software, version 3.1 , and aligned with ClustalW , and homology was determined using the EMBL-GenBank database ( ).

    Software:

    Article Title: Hybridization of glaucous gull (Larus hyperboreus) and herring gull (Larus argentatus) in Iceland: mitochondrial and microsatellite data
    Article Snippet: Prior to sequencing, excess primers and nucleotides were enzymatically removed from PCR amplification products using a mixture of exonuclease I and Antarctic phosphatase (New England BioLabs). .. PCR was performed in a 15 μl reaction containing the same concentrations as listed above; the products were sent for genotyping to GATCBiotech AG, Germany and scored with the Gene Marker v. 5.1 software package (SoftGenetics LLC 2004).

    Article Title: Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿
    Article Snippet: Plasmid DNA purified with NucleoBond BAC 100 cartridges (Macherey-Nagel, Düren, Germany) or the 3,760-bp espP PCR product (Table ) purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (GE Healthcare, München, Germany) was used as a template for bidirectional sequencing with an automated ABI Prism 3130 Avant Genetic Analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), ABI Prism BigDye Terminator Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied Biosystems), and customized primers. .. Sequences were analyzed using MEGA software, version 3.1 , and aligned with ClustalW , and homology was determined using the EMBL-GenBank database ( ).

    Multiplex Assay:

    Article Title: Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei *
    Article Snippet: Reference , never edited, pre-edited, and edited ( , ) transcript cDNAs were then preamplified in multiplex specific target amplification reactions using TaqMan PreAmp Master Mix (Life Technologies). .. Specific target amplification reactions were performed with the following thermocycling conditions: one cycle at 95 °C for 10 min, and 14 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was treated with exonuclease I (New England Biolabs) and diluted 10-fold.

    Article Title: Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks
    Article Snippet: Unincorporated primers were then cleaned up using Exonuclease I (New England Biolabs). .. High-throughput qPCRs were performed on the Biomark HD (Fluidigm) in a microfluidic multiplex 48.48 dynamic array chip according to the Fluidigm Advanced Development Protocol with EvaGreen.

    Article Title: Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland ▿
    Article Snippet: Paragraph title: Multiplex PCR amplification and purification. ... To enzymatically purify the samples from residual deoxynucleotides and excess primers, 8.0 μl of the AG1, AG2, or AG3 PCR product and 4.0 μl of the AG4 PCR product was transferred into new reaction tubes, followed by the addition of 1.0 μl rAPid Alkaline Phosphatase (1 U/μl; Roche Diagnostics), 0.2 μl of the corresponding buffer, and 0.05 μl exonuclease I ( Exo I; 20 U/μl; New England Biolabs, Ipswich, MA).

    Selection:

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
    Article Snippet: The assay was performed on an Agilent MX3005 real-time PCR machine with cycling conditions of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. To assess the genetic diversity and detect potential signatures of selection acting on the voltage-gated sodium channel, a portion of this gene spanning exon 20 was sequenced in 15 An. coluzzii and 14 An. arabiensis females. .. Cycling condition were as follows: 95 °C for 5min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s, followed by a final extension step at 72 °C for 10 min. PCR products were cleaned up using Exonuclease I (Exo I) and Shrimp Alkaline Phosphate (Exo-SAP protocol) according to the protocol of New England Biolabs (NEB, MA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Visual short-term memory deficits associated with GBA mutation and Parkinson’s disease
    Article Snippet: The resulting PCR products were digested with XhoI (NEB) for N370S and NciI (NEB) for L444P and resolved by agarose gel electrophoresis. .. Briefly, DNA was treated with an ExoSAP reaction as follows: 1 × SAP buffer, shrimp alkaline phosphatase (500 U; SAP, Promega), Exonuclease I (2 U; NEB).

    Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function *
    Article Snippet: Amplification of the fragments was verified by agarose gel electrophoresis, simultaneously running a Generuler 100bp Plus DNA Ladder (Fermentas). .. To remove primers and unincorporated dNTPs, exonuclease I (New England Biolabs) and calf intestine alkaline phosphatase (Roche Applied Science) were used.

    Article Title: Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland ▿
    Article Snippet: Each PCR was run in a 9800 Fast Thermal Cycler (Applied Biosystems, Foster City, CA) under the following universal conditions: 3 min of denaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C and a final extension step at 72°C for 7 min. Multiplex PCR products (3.0 μl from each AG) were analyzed on a 1.5% agarose gel stained with 0.3 μg/ml ethidium bromide. .. To enzymatically purify the samples from residual deoxynucleotides and excess primers, 8.0 μl of the AG1, AG2, or AG3 PCR product and 4.0 μl of the AG4 PCR product was transferred into new reaction tubes, followed by the addition of 1.0 μl rAPid Alkaline Phosphatase (1 U/μl; Roche Diagnostics), 0.2 μl of the corresponding buffer, and 0.05 μl exonuclease I ( Exo I; 20 U/μl; New England Biolabs, Ipswich, MA).

    Sampling:

    Article Title: Mitochondrial Introgression, Color Pattern Variation, and Severe Demographic Bottlenecks in Three Species of Malagasy Poison Frogs, Genus Mantella
    Article Snippet: Paragraph title: 2.1. Tissue Sampling, DNA Extraction, and Amplification ... The successfully amplified double-stranded PCR products, treated with Exonuclease I (New England Biolabs, Ipswich MA, USA) and Shrimp Alkaline Phosphatase (Promega) to inactivate remaining primers and dNTPs, were directly used for the cycle-sequencing reaction using dye-labeled dideoxy terminators (Applied Biosystems, Foster City, CA, USA) with the amplification primers on an ABI 3130xl automated DNA sequencer.

    Concentration Assay:

    Article Title: Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks
    Article Snippet: Briefly, a 12-cycle preamplification reaction was performed for each sample in 5 µL by pooling all primer pairs (final concentration, 50 nM), 1.25 µL cDNA, and 2.5 µL 2× PreAmp Master Mix (Applied Biosystems) following the manufacturer’s protocol. .. Unincorporated primers were then cleaned up using Exonuclease I (New England Biolabs).

    CTG Assay:

    Article Title: A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon
    Article Snippet: The primers kdr _F (5′-CAT TTT TCT TGG CCA CTG TAG TGA T-3′) and kdr _R (5′-CGA TCT TGG TCC ATG TTA ATT TGC A-3′) were used without modififcation. .. Cycling condition were as follows: 95 °C for 5min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s, followed by a final extension step at 72 °C for 10 min. PCR products were cleaned up using Exonuclease I (Exo I) and Shrimp Alkaline Phosphate (Exo-SAP protocol) according to the protocol of New England Biolabs (NEB, MA, USA).

    BAC Assay:

    Article Title: Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿
    Article Snippet: .. Plasmid DNA purified with NucleoBond BAC 100 cartridges (Macherey-Nagel, Düren, Germany) or the 3,760-bp espP PCR product (Table ) purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (GE Healthcare, München, Germany) was used as a template for bidirectional sequencing with an automated ABI Prism 3130 Avant Genetic Analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), ABI Prism BigDye Terminator Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied Biosystems), and customized primers. .. Sequences were analyzed using MEGA software, version 3.1 , and aligned with ClustalW , and homology was determined using the EMBL-GenBank database ( ).

    Marker:

    Article Title: Hybridization of glaucous gull (Larus hyperboreus) and herring gull (Larus argentatus) in Iceland: mitochondrial and microsatellite data
    Article Snippet: Prior to sequencing, excess primers and nucleotides were enzymatically removed from PCR amplification products using a mixture of exonuclease I and Antarctic phosphatase (New England BioLabs). .. PCR was performed in a 15 μl reaction containing the same concentrations as listed above; the products were sent for genotyping to GATCBiotech AG, Germany and scored with the Gene Marker v. 5.1 software package (SoftGenetics LLC 2004).

    Staining:

    Article Title: Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland ▿
    Article Snippet: Each PCR was run in a 9800 Fast Thermal Cycler (Applied Biosystems, Foster City, CA) under the following universal conditions: 3 min of denaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C and a final extension step at 72°C for 7 min. Multiplex PCR products (3.0 μl from each AG) were analyzed on a 1.5% agarose gel stained with 0.3 μg/ml ethidium bromide. .. To enzymatically purify the samples from residual deoxynucleotides and excess primers, 8.0 μl of the AG1, AG2, or AG3 PCR product and 4.0 μl of the AG4 PCR product was transferred into new reaction tubes, followed by the addition of 1.0 μl rAPid Alkaline Phosphatase (1 U/μl; Roche Diagnostics), 0.2 μl of the corresponding buffer, and 0.05 μl exonuclease I ( Exo I; 20 U/μl; New England Biolabs, Ipswich, MA).

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    New England Biolabs exonuclease i
    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following <t>Exonuclease</t> I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    Exonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Journal: PLoS ONE

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    doi: 10.1371/journal.pone.0016925

    Figure Lengend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Article Snippet: In cases where the COLIGO was still contaminated by > 5% of the linear oligonucleotide after elution (as determined by gel staining), an Exonuclease I (NEB) digest was done.

    Techniques: Polyacrylamide Gel Electrophoresis

    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Journal: Nucleic Acids Research

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

    doi: 10.1093/nar/gkr424

    Figure Lengend Snippet: Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Article Snippet: After a 5-min incubation at 37°C, a 20 µl extension mix containing 66.8 mM Tris–HCl, 16.8 mM ammonium sulfate, 1 mM dithiothreitol, 33 mM magnesium chloride, 62.5 U/ml T4 DNA Polymerase [or 62.5 U/ml Klenow fragment exo(−), 125 U/ml Klenow fragment, 125 U/ml DNA Polymerase I (Fermentas), 250 U/ml Exonuclease I (New England Biolabs)] was added.

    Techniques: Activity Assay, Hybridization, Generated

    Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Journal: International Journal of Molecular Sciences

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    doi: 10.3390/ijms17091457

    Figure Lengend Snippet: Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Article Snippet: After that, non-hybridized single-stranded DNA, including the probe, was digested with 1 U exonuclease I (New England BioLabs, Inc., M0293, Beijing, China) in the same tube according to the instruction protocol for 30 min at 37 °C.

    Techniques: Hybridization, Nucleic Acid Electrophoresis, Electrophoresis, Blocking Assay, Incubation, Labeling

    Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Journal: International Journal of Molecular Sciences

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    doi: 10.3390/ijms17091457

    Figure Lengend Snippet: Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Article Snippet: After that, non-hybridized single-stranded DNA, including the probe, was digested with 1 U exonuclease I (New England BioLabs, Inc., M0293, Beijing, China) in the same tube according to the instruction protocol for 30 min at 37 °C.

    Techniques: Northern Blot, Hybridization

    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation