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    New England Biolabs large klenow fragment new england biolabs
    Large Klenow Fragment New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher klenow dna polymerase
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    New England Biolabs exo klenow dna polymerase
    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic <t>DNA</t> and treated with 0.02 U μl −1 T4pol at <t>25°C</t> for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
    Exo Klenow Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna polymerase i
    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic <t>DNA</t> and treated with 0.02 U μl −1 T4pol at <t>25°C</t> for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
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    New England Biolabs klenow dna polymerase exo
    Effects of blocking oligonucleotides on FLEA-PCR-amplified libraries. A genomic <t>DNA</t> HeLa cell clone was subjected to FLEA-PCR using <t>T7</t> DNA polymerase with and without addition of internal sequences blocking oligonucleotide. A plasmid library for each
    Klenow Dna Polymerase Exo, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bst polymerase
    Effects of blocking oligonucleotides on FLEA-PCR-amplified libraries. A genomic <t>DNA</t> HeLa cell clone was subjected to FLEA-PCR using <t>T7</t> DNA polymerase with and without addition of internal sequences blocking oligonucleotide. A plasmid library for each
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    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Journal: Nucleic Acids Research

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    doi: 10.1093/nar/gkx1249

    Figure Lengend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Article Snippet: Assembly reactions with other exonucleases were cycled as follows: T5 exonuclease (T5exo; NEB, cat. no. M0363): 50°C for 30 min, then held at 4°C; T7 exonuclease (T7exo; NEB, cat. no. M0263): 25°C for 20 min, 50°C for 30 min, then held at 4°C; DNA polymerase I Klenow fragment (Kle; NEB, cat. no. M0210), T7 DNA polymerase (T7pol; NEB, cat. no. M0274) and λ exonuclease (λexo; NEB, cat. no. M0262): 25°C for 20 min, 75°C for 20 min, 50°C for 30 min, then held at 4°C; Exonuclease III (ExoIII; NEB, cat. no. M0206): 37°C for 20 min, 75°C for 20 min, 50°C for 30 min, then held at 4°C; Phusion DNA polymerase (Phu; NEB, cat. no. M0530): 37°C for 20 min, 50°C for 30 min, then held at 4°C.

    Techniques: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

    The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Journal: PLoS ONE

    Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    doi: 10.1371/journal.pone.0020303

    Figure Lengend Snippet: The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Article Snippet: Materials The exo I and Klenow DNA polymerase were purchased from New England Biolab (Ipswich, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Incubation, BAC Assay

    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Purification, Polymerase Chain Reaction, Construct

    Effects of blocking oligonucleotides on FLEA-PCR-amplified libraries. A genomic DNA HeLa cell clone was subjected to FLEA-PCR using T7 DNA polymerase with and without addition of internal sequences blocking oligonucleotide. A plasmid library for each

    Journal:

    Article Title: Flanking-sequence exponential anchored-polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant-host-junction sequences

    doi: 10.1080/14653240802192636

    Figure Lengend Snippet: Effects of blocking oligonucleotides on FLEA-PCR-amplified libraries. A genomic DNA HeLa cell clone was subjected to FLEA-PCR using T7 DNA polymerase with and without addition of internal sequences blocking oligonucleotide. A plasmid library for each

    Article Snippet: Washed beads were resuspended in 20 μL 1 × DNA polymerase buffer (either Klenow or T7 DNA polymerase buffer, both from New England Biolabs, Beverly, MA, USA), 500 rMol dNTP, 5 μm anchoring primer and 5 μm internal blocking primer (for use with T7 DNA polymerase).

    Techniques: Blocking Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Journal: Nucleic Acids Research

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    doi: 10.1093/nar/gkn575

    Figure Lengend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Article Snippet: Primer extension with Klenow fragment of DNA polymerase Primer extension experiments using large fragment (Klenow) of DNA polymerase I (New England Biolabs) were performed at 25°C using the HIV-1 tat reverse primer (5′-AATACTATGGTCCACACAACTATTGCT-3′) that was unmodified or contained a single OXP modification.

    Techniques: Polyacrylamide Gel Electrophoresis, Modification, Incubation