taq dna polymerase  (New England Biolabs)


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    Name:
    Taq DNA Polymerase with Standard Taq Buffer
    Description:
    Taq DNA Polymerase with Standard Taq Buffer 20 000 units
    Catalog Number:
    M0273E
    Price:
    1200
    Size:
    20 000 units
    Category:
    Thermostable DNA Polymerases
    Score:
    85
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    New England Biolabs taq dna polymerase
    Taq DNA Polymerase with Standard Taq Buffer
    Taq DNA Polymerase with Standard Taq Buffer 20 000 units
    https://www.bioz.com/result/taq dna polymerase/product/New England Biolabs
    Average 99 stars, based on 927 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization"

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp150

    DNA polymerization by PCR using a caged primer (17 nt) containing the first caged thymidine (blue square) 10 nt from the 5′ end. PCR generates a caged template which results in a stop of Taq and Pfu polymerase due to the presence of a single-caged thymidine.
    Figure Legend Snippet: DNA polymerization by PCR using a caged primer (17 nt) containing the first caged thymidine (blue square) 10 nt from the 5′ end. PCR generates a caged template which results in a stop of Taq and Pfu polymerase due to the presence of a single-caged thymidine.

    Techniques Used: Polymerase Chain Reaction

    Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.
    Figure Legend Snippet: Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.

    Techniques Used: Polymerase Chain Reaction

    2) Product Images from "CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis"

    Article Title: CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv878

    Schematics of Cas9n/gRNA target sequence specific fluorescent labeling for whole genome DNA mapping. The Cas9n fluorescent nick-labeling system uses a guide RNA (gRNA) to direct the Cas9 nuclease to a targeted site. The gRNA is composed of a trans-activating crRNA (tracrRNA) and a crRNA that contains a 20 nucleotide sequence that is complementary to the site of interest. A mutation in the RuvC-like domain nuclease alters the Cas9 enzyme to make only a single cut three nucleotides upstream of a protospacer adjacent motif (PAM) of the 3′–5′ strand of the target DNA. In the nick labeling method, after Cas9n D10A generates a nick, fluorophores are directly incorporated to the nick sites using Taq DNA Polymerase. These fluorophores can be detected using fluorescence microscopy.
    Figure Legend Snippet: Schematics of Cas9n/gRNA target sequence specific fluorescent labeling for whole genome DNA mapping. The Cas9n fluorescent nick-labeling system uses a guide RNA (gRNA) to direct the Cas9 nuclease to a targeted site. The gRNA is composed of a trans-activating crRNA (tracrRNA) and a crRNA that contains a 20 nucleotide sequence that is complementary to the site of interest. A mutation in the RuvC-like domain nuclease alters the Cas9 enzyme to make only a single cut three nucleotides upstream of a protospacer adjacent motif (PAM) of the 3′–5′ strand of the target DNA. In the nick labeling method, after Cas9n D10A generates a nick, fluorophores are directly incorporated to the nick sites using Taq DNA Polymerase. These fluorophores can be detected using fluorescence microscopy.

    Techniques Used: Sequencing, Labeling, Mutagenesis, Fluorescence, Microscopy

    3) Product Images from "Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing"

    Article Title: Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096727

    Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.
    Figure Legend Snippet: Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.

    Techniques Used: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Gas Chromatography

    4) Product Images from "One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning"

    Article Title: One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm583

    Plasmid re-circularization in pJBN250 transformed cells. Mutagenized PCR products from pAK009, (a pUNI10- URA3 derivative), were transformed into BW23474 cells harboring pJBN250. Plasmid DNA from nine Kn R transformants was analyzed before ( A ) or after ( B ) HindIII digestion, predicted to produce fragments of 1481, 1300 and 757 bp from correct recombinants (a map of the predicted recombinant is depicted above the gel panel). DNA was separated on 0.8% agarose gels and visualized by ethidium bromide staining. ( C ) Equal amounts of linearized pAK009 DNA (5 ng) were used as template for PCR reactions with or without (mock) Taq polymerase. The gel inset shows aliquots of the reaction products. Equivalent volumes of these reactions were transformed into BW23474 (BW) or BW23474/pJBN250 (BW/N250) strains, and dilutions were plated to calculate the total number of Kn R colonies. In the bottom three rows of the table, mutagenized libraries were constructed using pooled PCR reactions from pAK009, pJJ005 and pAS003 (pUNI10 derivatives containing the indicated S. cerevisiae genes) as described in Methods section. In these cases, the concentration of the pooled PCR reaction products was quantified, and used to calculate recombination efficiency by dividing the total number of Kn R colonies by the amount of PCR-amplified DNA used in the transformations. In all cases, transformation efficiency for circular plasmids was evaluated by determining the number of Ap R transformants obtained from a known amount of pBR322.
    Figure Legend Snippet: Plasmid re-circularization in pJBN250 transformed cells. Mutagenized PCR products from pAK009, (a pUNI10- URA3 derivative), were transformed into BW23474 cells harboring pJBN250. Plasmid DNA from nine Kn R transformants was analyzed before ( A ) or after ( B ) HindIII digestion, predicted to produce fragments of 1481, 1300 and 757 bp from correct recombinants (a map of the predicted recombinant is depicted above the gel panel). DNA was separated on 0.8% agarose gels and visualized by ethidium bromide staining. ( C ) Equal amounts of linearized pAK009 DNA (5 ng) were used as template for PCR reactions with or without (mock) Taq polymerase. The gel inset shows aliquots of the reaction products. Equivalent volumes of these reactions were transformed into BW23474 (BW) or BW23474/pJBN250 (BW/N250) strains, and dilutions were plated to calculate the total number of Kn R colonies. In the bottom three rows of the table, mutagenized libraries were constructed using pooled PCR reactions from pAK009, pJJ005 and pAS003 (pUNI10 derivatives containing the indicated S. cerevisiae genes) as described in Methods section. In these cases, the concentration of the pooled PCR reaction products was quantified, and used to calculate recombination efficiency by dividing the total number of Kn R colonies by the amount of PCR-amplified DNA used in the transformations. In all cases, transformation efficiency for circular plasmids was evaluated by determining the number of Ap R transformants obtained from a known amount of pBR322.

    Techniques Used: Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Recombinant, Staining, Construct, Concentration Assay, Amplification

    5) Product Images from "Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method"

    Article Title: Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method

    Journal:

    doi: 10.1080/21655979.2017.1308986

    Principle and optimization of sDATEL assembly method. (A) Schematic diagram of sDATEL. The length of overhang was set as 30 nt while the primers for amplifying DNA fragments were designed with 40 nt. After denaturation and annealing, the displaced overhangs at the fork structures were cleaved by Taq DNA polymerase and the nicks were joined by Taq DNA ligase; (B) Schematic diagram of DATEL; (C), (D) and (E) were the effects on the assembly efficiency that caused by pH, NAD+ and Mg2+ , respectively.
    Figure Legend Snippet: Principle and optimization of sDATEL assembly method. (A) Schematic diagram of sDATEL. The length of overhang was set as 30 nt while the primers for amplifying DNA fragments were designed with 40 nt. After denaturation and annealing, the displaced overhangs at the fork structures were cleaved by Taq DNA polymerase and the nicks were joined by Taq DNA ligase; (B) Schematic diagram of DATEL; (C), (D) and (E) were the effects on the assembly efficiency that caused by pH, NAD+ and Mg2+ , respectively.

    Techniques Used:

    6) Product Images from "Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing"

    Article Title: Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096727

    Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.
    Figure Legend Snippet: Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.

    Techniques Used: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Gas Chromatography

    7) Product Images from "Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V"

    Article Title: Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-81

    Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.
    Figure Legend Snippet: Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Produced, Plasmid Preparation

    8) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.
    Figure Legend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Techniques Used: Labeling, Gas Chromatography, Incubation, Negative Control

    Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.
    Figure Legend Snippet: Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Techniques Used: Gas Chromatography, Polymerase Chain Reaction, Sequencing, Generated, Produced, Purification, Incubation

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.
    Figure Legend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Techniques Used: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.
    Figure Legend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Techniques Used: Gas Chromatography, Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation, Activated Clotting Time Assay

    9) Product Images from "RNase H1 directs origin-specific initiation of DNA replication in human mitochondria"

    Article Title: RNase H1 directs origin-specific initiation of DNA replication in human mitochondria

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007781

    DNA replication initiation defects in RNase H1 deficient cells. A. Schematic representation of the control region of mtDNA with the positions of primer extension primers 1 and 2 indicated. B. Primer extension of mtDNA from control and RNase H1 patient cells with primer 1 (corresponding to mtDNA positions 8-29, see panel A). Primer 1 was annealed to mtDNA from control (WT) and RNase H1 patient (mut RH1) cells and extended over the OriH, CSB and LSP regions with Taq DNA polymerase (see method details). Control cell DNA (WT) in lanes 1-2 and RNase H1 patient cell DNA in lanes 3-4. Untreated DNA in lanes 1 and 3, and RNase H2 treated DNA in lanes 2 and 4. The NEB LMW ladder is indicated in black, mtDNA positions in green, mapped 5′-ends in blue and mapped RNA to DNA transition points in red. G, C, A and T sequencing ladders are found on the left-hand side. A schematic representation of the control region with OriH, CSBI-III and LSP is shown on the right-hand side. C. Primer extension of mtDNA from control and RNase H1 patient cells with primer 2 (corresponding to mtDNA positions 16,231-16,251, see panel A). Sample loading and colored indications as in panel B. D. 5′-end sequencing (5′-End-seq) of control cell mtDNA. E. Hydrolytic end sequencing (HydEn-seq) of control cell mtDNA to map 5′-ends with attached ribonucleotides. F. 5′-end sequencing (5′-End-seq) of RNase H1 patient cell mtDNA. G. Hydrolytic end sequencing (HydEn-seq) of RNase H1 patient cell mtDNA to map 5′-ends with attached ribonucleotides.
    Figure Legend Snippet: DNA replication initiation defects in RNase H1 deficient cells. A. Schematic representation of the control region of mtDNA with the positions of primer extension primers 1 and 2 indicated. B. Primer extension of mtDNA from control and RNase H1 patient cells with primer 1 (corresponding to mtDNA positions 8-29, see panel A). Primer 1 was annealed to mtDNA from control (WT) and RNase H1 patient (mut RH1) cells and extended over the OriH, CSB and LSP regions with Taq DNA polymerase (see method details). Control cell DNA (WT) in lanes 1-2 and RNase H1 patient cell DNA in lanes 3-4. Untreated DNA in lanes 1 and 3, and RNase H2 treated DNA in lanes 2 and 4. The NEB LMW ladder is indicated in black, mtDNA positions in green, mapped 5′-ends in blue and mapped RNA to DNA transition points in red. G, C, A and T sequencing ladders are found on the left-hand side. A schematic representation of the control region with OriH, CSBI-III and LSP is shown on the right-hand side. C. Primer extension of mtDNA from control and RNase H1 patient cells with primer 2 (corresponding to mtDNA positions 16,231-16,251, see panel A). Sample loading and colored indications as in panel B. D. 5′-end sequencing (5′-End-seq) of control cell mtDNA. E. Hydrolytic end sequencing (HydEn-seq) of control cell mtDNA to map 5′-ends with attached ribonucleotides. F. 5′-end sequencing (5′-End-seq) of RNase H1 patient cell mtDNA. G. Hydrolytic end sequencing (HydEn-seq) of RNase H1 patient cell mtDNA to map 5′-ends with attached ribonucleotides.

    Techniques Used: Sequencing

    10) Product Images from "High-Throughput, Sensitive Quantification of Repopulating Hematopoietic Stem Cell Clones"

    Article Title: High-Throughput, Sensitive Quantification of Repopulating Hematopoietic Stem Cell Clones

    Journal:

    doi: 10.1128/JVI.01355-10

    Sequence frequency distribution for different lengths of VIS DNAs. The length of VIS DNA was calculated based on the distance of the nearest Taq α I site from the junction added to 50 bp of vector DNA. Individual VIS were distributed primarily from
    Figure Legend Snippet: Sequence frequency distribution for different lengths of VIS DNAs. The length of VIS DNA was calculated based on the distance of the nearest Taq α I site from the junction added to 50 bp of vector DNA. Individual VIS were distributed primarily from

    Techniques Used: Sequencing, Plasmid Preparation

    11) Product Images from "Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs"

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj512

    PCR amplification of UV-damaged DNA. ( A ) Human genomic DNA (K562) was damaged by exposure to UVC at a flux rate of 0.197 J/cm 2 /min. Two nanograms of damaged genomic DNA were amplified with primers specific for the Major and Precise Alu subfamilies ( 21 ). The PCR contained, inter alia, 2.5 U Taq Gold DNA Polymerase, 20 pmol each of the forward and reverse primers and, if applicable, 100 nM Dpo4. The forward primer was labeled at its 5′ end with the reactive fluorescent dye 6-FAM. Amplified fragments were separated by capillary electrophoresis and detected by laser-induced fluorescence of the incorporated dye-labeled primer. The electropherograms depict Alu element amplification products of the UVC damaged genomic substrate with Taq Gold DNA polymerase alone ( 1 ) or with a cocktail of Taq DNA polymerase and 100 nM Dpo4 ( 2 ). ( B ) Experimental details are the same as (A) except that the DNA was exposed to UVC at the same flux rate for 30 min and PCR was performed at 85°C, as noted in Materials and Methods. Results show the Alu element products obtained with AmpliTaq DNA polymerase alone ( 1 ) or with a cocktail of Taq DNA polymerase and 100 nM Ste ( 2 ) or Ain ( 3 ).
    Figure Legend Snippet: PCR amplification of UV-damaged DNA. ( A ) Human genomic DNA (K562) was damaged by exposure to UVC at a flux rate of 0.197 J/cm 2 /min. Two nanograms of damaged genomic DNA were amplified with primers specific for the Major and Precise Alu subfamilies ( 21 ). The PCR contained, inter alia, 2.5 U Taq Gold DNA Polymerase, 20 pmol each of the forward and reverse primers and, if applicable, 100 nM Dpo4. The forward primer was labeled at its 5′ end with the reactive fluorescent dye 6-FAM. Amplified fragments were separated by capillary electrophoresis and detected by laser-induced fluorescence of the incorporated dye-labeled primer. The electropherograms depict Alu element amplification products of the UVC damaged genomic substrate with Taq Gold DNA polymerase alone ( 1 ) or with a cocktail of Taq DNA polymerase and 100 nM Dpo4 ( 2 ). ( B ) Experimental details are the same as (A) except that the DNA was exposed to UVC at the same flux rate for 30 min and PCR was performed at 85°C, as noted in Materials and Methods. Results show the Alu element products obtained with AmpliTaq DNA polymerase alone ( 1 ) or with a cocktail of Taq DNA polymerase and 100 nM Ste ( 2 ) or Ain ( 3 ).

    Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Electrophoresis, Fluorescence

    12) Product Images from "Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization"

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp150

    DNA polymerization by PCR using a caged primer (17 nt) containing the first caged thymidine (blue square) 10 nt from the 5′ end. PCR generates a caged template which results in a stop of Taq and Pfu polymerase due to the presence of a single-caged thymidine.
    Figure Legend Snippet: DNA polymerization by PCR using a caged primer (17 nt) containing the first caged thymidine (blue square) 10 nt from the 5′ end. PCR generates a caged template which results in a stop of Taq and Pfu polymerase due to the presence of a single-caged thymidine.

    Techniques Used: Polymerase Chain Reaction

    Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.
    Figure Legend Snippet: Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.

    Techniques Used: Polymerase Chain Reaction

    13) Product Images from "Examining Sources of Error in PCR by Single-Molecule Sequencing"

    Article Title: Examining Sources of Error in PCR by Single-Molecule Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169774

    Fidelity measurements and mutational spectrum of DNA polymerases. (A) Base substitution error rates of various DNA polymerases relative to Taq polymerase. (B) Proportion of each type of base substitution error as a percentage of the total errors for each polymerase.
    Figure Legend Snippet: Fidelity measurements and mutational spectrum of DNA polymerases. (A) Base substitution error rates of various DNA polymerases relative to Taq polymerase. (B) Proportion of each type of base substitution error as a percentage of the total errors for each polymerase.

    Techniques Used:

    14) Product Images from "Generating a synthetic genome by whole genome assembly: ?X174 bacteriophage from synthetic oligonucleotides"

    Article Title: Generating a synthetic genome by whole genome assembly: ?X174 bacteriophage from synthetic oligonucleotides

    Journal:

    doi: 10.1073/pnas.2237126100

    PCA of full-length synφX molecules. The first stage of PCA (50-μl reaction volume) was carried out for 35 cycles with 0.2, 0.5, or 1 μlofthe Taq ligation product. PCA products were analyzed on a 0.8% E-gel. ( A ) Lanes 1 and 6, 1-kb ladder; lane 2, 0.5 μlof Taq ligation product; lane 3, 2 μl of the 0.2-μl PCA; lane 4, 2 μl of the 0.5-μl PCA; lane 5, 2 μl of the 1-μl PCA. The second stage of PCA was for an additional 35 cycles in five new 50-μl reactions. For reaction 1, the 0.2-μl first-stage reaction was continued without change for another 35 cycles with the addition of 0.5 μl of fresh HF polymerase mixture. For reactions 2 and 3, 10 and 20 μl of the 0.5-μl first-stage PCA product was used. For reactions 4 and 5, 5 and 10 μl of the 1-μl first-stage PCA product was used. Analysis was on 0.8% E-gels. ( B ) Formamide-denatured DNA. ( C ) Native DNA. ( B and C ) Lanes 1 and 7, 1-kb ladder; lane 2, 2 μl of reaction 1; lanes 3 and 4, 2 μl of reactions 2 and 3; lanes 5 and 6, 2 μl of reactions 4 and 5. ( D ) PCR amplification of the products of the second set of PCA products as shown in B and C .( E ) Taq ligase assembly of 259 oligonucleotides. A 0.5-μl sample of the ligation products was analyzed on a 2% E-gel (Invitrogen) in duplex form (lane N). One microliter of the ligation products was mixed with 20 μl of formamide, heated to 95°C for 2 min, and then analyzed (lane D). Denatured standards run approximately the same as native standards, based on other experiments (data not shown).
    Figure Legend Snippet: PCA of full-length synφX molecules. The first stage of PCA (50-μl reaction volume) was carried out for 35 cycles with 0.2, 0.5, or 1 μlofthe Taq ligation product. PCA products were analyzed on a 0.8% E-gel. ( A ) Lanes 1 and 6, 1-kb ladder; lane 2, 0.5 μlof Taq ligation product; lane 3, 2 μl of the 0.2-μl PCA; lane 4, 2 μl of the 0.5-μl PCA; lane 5, 2 μl of the 1-μl PCA. The second stage of PCA was for an additional 35 cycles in five new 50-μl reactions. For reaction 1, the 0.2-μl first-stage reaction was continued without change for another 35 cycles with the addition of 0.5 μl of fresh HF polymerase mixture. For reactions 2 and 3, 10 and 20 μl of the 0.5-μl first-stage PCA product was used. For reactions 4 and 5, 5 and 10 μl of the 1-μl first-stage PCA product was used. Analysis was on 0.8% E-gels. ( B ) Formamide-denatured DNA. ( C ) Native DNA. ( B and C ) Lanes 1 and 7, 1-kb ladder; lane 2, 2 μl of reaction 1; lanes 3 and 4, 2 μl of reactions 2 and 3; lanes 5 and 6, 2 μl of reactions 4 and 5. ( D ) PCR amplification of the products of the second set of PCA products as shown in B and C .( E ) Taq ligase assembly of 259 oligonucleotides. A 0.5-μl sample of the ligation products was analyzed on a 2% E-gel (Invitrogen) in duplex form (lane N). One microliter of the ligation products was mixed with 20 μl of formamide, heated to 95°C for 2 min, and then analyzed (lane D). Denatured standards run approximately the same as native standards, based on other experiments (data not shown).

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification

    15) Product Images from "Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction"

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145682

    Difference in DNA polymerase behaviors. The tetracycline resistance gene assembly product was PCR amplified by Taq DNA polymerase (Lane 1) and Pfx DNA polymerase (Lane 2). Pfx amplification caused polymerization and produced some high molecular weight products, thus the Taq polymerase was chosen for subsequent studies. The last lane contained the 100-bp DNA ladder.
    Figure Legend Snippet: Difference in DNA polymerase behaviors. The tetracycline resistance gene assembly product was PCR amplified by Taq DNA polymerase (Lane 1) and Pfx DNA polymerase (Lane 2). Pfx amplification caused polymerization and produced some high molecular weight products, thus the Taq polymerase was chosen for subsequent studies. The last lane contained the 100-bp DNA ladder.

    Techniques Used: Polymerase Chain Reaction, Amplification, Produced, Molecular Weight

    16) Product Images from "Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides"

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008430

    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.
    Figure Legend Snippet: PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Techniques Used:

    Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.
    Figure Legend Snippet: Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Techniques Used: Produced

    17) Product Images from "Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells"

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034778

    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.
    Figure Legend Snippet: Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Techniques Used: Isolation, Polymerase Chain Reaction, Expressing, TRAP Assay, Activity Assay

    18) Product Images from "Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells"

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034778

    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.
    Figure Legend Snippet: Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Techniques Used: Isolation, Polymerase Chain Reaction, Expressing, TRAP Assay, Activity Assay

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    GWAS:

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    Amplification:

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    Synthesized:

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    TA Cloning:

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    Incubation:

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    RNA Binding Assay:

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    Crystallization Assay:

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    Countercurrent Chromatography:

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    Electroporation:

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    Activation Assay:

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    Buffer Exchange:

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    Northern Blot:

    Article Title: Genetic variants in the enhancer region of the thymidylate synthase gene in the Chilean population
    Article Snippet: The SJH and CLC are located in the northern and eastern parts of Santiago, respectively. .. Amplification reactions were performed in a 25-µl volume containing 0.2 µg genomic DNA, 0.2 m m dNTPs , 0.5 µ m of each primer, 1.0 units of Taq polymerase (New England BioLabs, Ipswich, MA, USA) and 2.5 µl of 10 × Taq PCR buffer.

    Hemagglutination Assay:

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: The sense primer annealed to the GAL4 DNA-binding domain and HA tag and extended to the CDH1 start codon. .. EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs).

    DNA Sequencing:

    Article Title: Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes
    Article Snippet: PCR was performed in triplicate 50-μl reactions containing 1× ThermoPol PCR buffer (New England Biolabs, Mississauga, Ontario, Canada), 0.5 μM concentrations of each primer, 0.5 mM deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (New Englands Biolabs), and 50 ng of DNA. .. Triplicate reactions were combined and cloned with the TOPO TA cloning kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's protocol.

    Sequencing:

    Article Title: Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus
    Article Snippet: PCR amplification was performed with 50-μl volumes containing 1× PCR buffer containing 1.5 mM MgCl2 (New England Biolabs, Herts, United Kingdom), 100 mM deoxynucleoside triphosphates, 0.1 μM of each primer (MWG Biotech AG, Ebersberg, Germany), 2.5 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom), 100 ng template DNA, and PCR-grade water (Invitrogen, CA). .. PCR amplification was performed with 50-μl volumes containing 1× PCR buffer containing 1.5 mM MgCl2 (New England Biolabs, Herts, United Kingdom), 100 mM deoxynucleoside triphosphates, 0.1 μM of each primer (MWG Biotech AG, Ebersberg, Germany), 2.5 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom), 100 ng template DNA, and PCR-grade water (Invitrogen, CA).

    Article Title: Discovery of Bacterial sRNAs by High-Throughput Sequencing
    Article Snippet: Taq DNA Polymerase (with 10× Buffer) (NEB, Ipswich, MA). .. Taq DNA Polymerase (with 10× Buffer) (NEB, Ipswich, MA).

    Article Title: Identification of Quantitative Trait Loci and Candidate Genes for Maize Starch Granule Size through Association Mapping
    Article Snippet: Paragraph title: Sequencing and candidate gene association analysis ... PCR reaction mixes (20 µl) contained 1 µl of NEB (New England BioLabs Inc., Ipswich, MA, USA) Taq DNA Polymerase, 4 µl of 5× NEB PCR Buffer, 0.5 µl of dNTP mixture, 0.5 µl each of the two primers, and 1 µl of template DNA.

    Article Title: Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes
    Article Snippet: PCR was performed in triplicate 50-μl reactions containing 1× ThermoPol PCR buffer (New England Biolabs, Mississauga, Ontario, Canada), 0.5 μM concentrations of each primer, 0.5 mM deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (New Englands Biolabs), and 50 ng of DNA. .. Triplicate reactions were combined and cloned with the TOPO TA cloning kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's protocol.

    Binding Assay:

    Article Title: Automated selection of aptamers against cholangiocarcinoma cells on an integrated microfluidic platform
    Article Snippet: With the aim of reducing the non-specific binding of the aptamers, one additional round of negative selection with MMNK-1 cell lines was performed with the 6th SELEX round PCR product for the SNU-478 and HuCCT-1 cell lines. .. The PCR chamber was preloaded with 0.5 μ l of the forward primer (5′-GGCAGGAAGACAAACA-3′, 0.5 μ M), 0.5 μ l of the reverse primer (5′-ACAGCACCACAGACCA-3′, 0.5 μ M), 0.5 μ l of deoxynucleotide triphosphates (0.2 mM), 2 μ l of ssDNA (0.1 μ M) or the previous amplified product from the last round of Cell-SELEX, 0.125 μ l of Taq DNA polymerase (New England Biolabs, USA), and double-distilled water (ddH2 O) to a final volume of 25 μ l. The PCR was initiated at 95 °C for 10 min followed by 30 cycles of 95 °C for 30 s, 63 °C for 15 s, and 72 °C for 30 s. A final extension step at 72 °C for 10 min was also incorporated into the protocol.

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA . .. The templates for the rhlAΔ1 (primers rhlA SDM delta RB1 F and rhlA SDM WT RB2R), rhlAΔ2 (primers rhlA SDM WT RB1 F and rhlA SDM delta RB2R), and rhlAΔ1Δ2 (primers rhlA SDM delta RB1 F and rhlA SDM delta RB2R) oligonucleotides were created using site-directed mutagenesis of the pGEMT- rhlA prom vector.

    DNA Extraction:

    Article Title: Genetic variations among three major ethnic groups in Nigeria using RAPD
    Article Snippet: Materials: Genomic DNA isolation kits were purchased from Norgen Biotek Corporation, (Thorold, Canada). .. Agarose from Cleaver Scientific Limited, Quickload 100bp DNA ladder from New England Biolabs Inc., PCR Master-mix with standard buffer (Quick-Ld2X) containing Taq polymerase was also from New England Biolabs Inc. and the oligonucleotide primers were from Inqaba, South Africa.

    Article Title: A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production
    Article Snippet: Paragraph title: DNA Isolation and Manipulation ... Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were purchased from NEB.

    Article Title: The Alternative Sigma Factor ?H Is Required for Toxin Gene Expression by Bacillus anthracis
    Article Snippet: Paragraph title: DNA isolation and manipulation. ... Restriction enzymes and T4 DNA ligase were purchased from Promega (Madison, WI) and Fisher Scientific, and Taq DNA polymerase was purchased from New England Biolabs (Beverly, MA).

    Mutagenesis:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA . .. DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA .

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: Paragraph title: EP-PCR mutagenesis of CDH1- Δ 200 and yeast two-hybrid screening ... EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs).

    Article Title: Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants
    Article Snippet: Nucleotide analogue mutagenesis was carried out in the presence of 2 and 20 μM 8-oxo-2′-deoxyguanosine (8-oxo-dGTP) and 6-(2-deoxy-β- d -ribofuranosyl)-3,4-dihydro-8H-pyrimido-(4,5-c)(1,2)oxazin-7-one (dPTP) ( ). .. Using the plasmid pZE21:: tyrA WT as template, 10, 20, and 30 amplification cycles with the primers mentioned above were performed using Taq DNA polymerase (New England Biolabs).

    Article Title: Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer
    Article Snippet: Finally, we screened the individual plate by the same PCR technique to identify the mutant of interest. .. PCRs were performed using Taq polymerase (New England Biolabs, Massachusetts) with the following protocol: denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 68°C for 30 s, and amplification at 72°C for 3 min.

    Isolation:

    Article Title: Automated selection of aptamers against cholangiocarcinoma cells on an integrated microfluidic platform
    Article Snippet: The product of this final round was then used in a negative selection step with White blood cells (WBCs) isolated from whole blood (described in more detail below). .. The PCR chamber was preloaded with 0.5 μ l of the forward primer (5′-GGCAGGAAGACAAACA-3′, 0.5 μ M), 0.5 μ l of the reverse primer (5′-ACAGCACCACAGACCA-3′, 0.5 μ M), 0.5 μ l of deoxynucleotide triphosphates (0.2 mM), 2 μ l of ssDNA (0.1 μ M) or the previous amplified product from the last round of Cell-SELEX, 0.125 μ l of Taq DNA polymerase (New England Biolabs, USA), and double-distilled water (ddH2 O) to a final volume of 25 μ l. The PCR was initiated at 95 °C for 10 min followed by 30 cycles of 95 °C for 30 s, 63 °C for 15 s, and 72 °C for 30 s. A final extension step at 72 °C for 10 min was also incorporated into the protocol.

    Article Title: A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production
    Article Snippet: E. coli transformation, plasmid isolation, and recombinant techniques were performed using standard methods ( ). .. Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were purchased from NEB.

    Article Title: Genetic variants in the enhancer region of the thymidylate synthase gene in the Chilean population
    Article Snippet: To determine the TSER variants, DNA was isolated by routine methods and amplified by polymerase chain reaction (PCR) [ ]. .. Amplification reactions were performed in a 25-µl volume containing 0.2 µg genomic DNA, 0.2 m m dNTPs , 0.5 µ m of each primer, 1.0 units of Taq polymerase (New England BioLabs, Ipswich, MA, USA) and 2.5 µl of 10 × Taq PCR buffer.

    Article Title: The Alternative Sigma Factor ?H Is Required for Toxin Gene Expression by Bacillus anthracis
    Article Snippet: Extraction of chromosomal DNA from B. anthracis cultures was carried out using a Mo Bio genomic isolation kit (Mo Bio Laboratories, Solana Beach, CA). .. Restriction enzymes and T4 DNA ligase were purchased from Promega (Madison, WI) and Fisher Scientific, and Taq DNA polymerase was purchased from New England Biolabs (Beverly, MA).

    Size-exclusion Chromatography:

    Article Title: Targeted gene inactivation in zebrafish using engineered zinc finger nucleases
    Article Snippet: Colonies from the desired selection plates were washed off the plates and their plasmid DNA was recovered as a pool as previously described . .. Each pool of individual fingers was amplified from the plasmid DNA by PCR: 50 μl reaction with 1 units NEB DNA Taq polymerase using 50 ng plasmid DNA template and 1 mM each primer ( ); denature for 1 min at 95°C, 28 cycles consisting of 94°C for 20 sec, 55°C for 20 sec and 72°C for 20 sec, followed by a final extension at 72°C for 6 min. .. The PCR products were run on a 1.5% agarose TAE gel, and the appropriate bands were excised and purified using a Qiagen PCR purification kit.

    Electrophoretic Mobility Shift Assay:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: Electrophoretic mobility shift assays (EMSAs) were performed using purified AlgR proteins, as described previously ( ). .. DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA .

    Purification:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: Electrophoretic mobility shift assays (EMSAs) were performed using purified AlgR proteins, as described previously ( ). .. DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA .

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs). .. After 16 cycles of amplification, 1 ml of product was purified using a Qiagen PCR purification column (Qiagen, Inc.) and co-transformed into YJB1212 with ∼2.5 μg of gapped CDH1- Δ 200-URA3 -pAS2 vector.

    Article Title: Eco-Evolutionary Processes Generating Diversity Among Bottlenose Dolphin, Tursiops truncatus, Populations off Baja California, Mexico
    Article Snippet: The PCRs were performed in 25µL volumes consisting of 10 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2 , 0.25 mM each dNTP, 0.12 µM each primer: L15812 (TRO): 5′ CCT CCC TAA GAC TCA AGG AAG 3′ and (D): 5′ CCT GAA GTA AGA ACC AGA TG 3′ (Rosel et al. ), 1.25 U of Taq DNA polymerase (NEB, UK), and approximately 50 ng of genomic DNA. .. The PCRs were performed in 25µL volumes consisting of 10 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2 , 0.25 mM each dNTP, 0.12 µM each primer: L15812 (TRO): 5′ CCT CCC TAA GAC TCA AGG AAG 3′ and (D): 5′ CCT GAA GTA AGA ACC AGA TG 3′ (Rosel et al. ), 1.25 U of Taq DNA polymerase (NEB, UK), and approximately 50 ng of genomic DNA.

    Article Title: Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes
    Article Snippet: Bacterial 16S rRNA genes were selectively amplified from the purified DNA by PCR using the forward primer 27f and the reverse primer 1492R ( ). .. PCR was performed in triplicate 50-μl reactions containing 1× ThermoPol PCR buffer (New England Biolabs, Mississauga, Ontario, Canada), 0.5 μM concentrations of each primer, 0.5 mM deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (New Englands Biolabs), and 50 ng of DNA.

    Polymerase Chain Reaction:

    Article Title: Automated selection of aptamers against cholangiocarcinoma cells on an integrated microfluidic platform
    Article Snippet: The gauge pressure and frequency were then regulated such that an aliquot of the supernatant was transferred to the PCR chamber. .. The PCR chamber was preloaded with 0.5 μ l of the forward primer (5′-GGCAGGAAGACAAACA-3′, 0.5 μ M), 0.5 μ l of the reverse primer (5′-ACAGCACCACAGACCA-3′, 0.5 μ M), 0.5 μ l of deoxynucleotide triphosphates (0.2 mM), 2 μ l of ssDNA (0.1 μ M) or the previous amplified product from the last round of Cell-SELEX, 0.125 μ l of Taq DNA polymerase (New England Biolabs, USA), and double-distilled water (ddH2 O) to a final volume of 25 μ l. The PCR was initiated at 95 °C for 10 min followed by 30 cycles of 95 °C for 30 s, 63 °C for 15 s, and 72 °C for 30 s. A final extension step at 72 °C for 10 min was also incorporated into the protocol. .. The amplified products were then transferred to the positive selection chamber, and the gauge pressure and frequency were fine-tuned such that an aliquot of the amplified product was used for the next round of SELEX, with the remainder collected via the waste collection chamber.

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: Electrophoretic mobility shift assays (EMSAs) were performed using purified AlgR proteins, as described previously ( ). .. DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA . .. Oligonucleotides were 5′-end labeled with [γ-32 P]ATP (6,000 Ci/mmol) (Perkin-Elmer) with polynucleotide kinase (New England BioLabs).

    Article Title: Post-crystallization improvement of RNA crystal diffraction quality
    Article Snippet: Oligonucleotides for PCR amplification. .. Taq DNA polymerase, 5,000 U/mL (New England Biolabs).

    Article Title: Genetic variations among three major ethnic groups in Nigeria using RAPD
    Article Snippet: Materials: Genomic DNA isolation kits were purchased from Norgen Biotek Corporation, (Thorold, Canada). .. Agarose from Cleaver Scientific Limited, Quickload 100bp DNA ladder from New England Biolabs Inc., PCR Master-mix with standard buffer (Quick-Ld2X) containing Taq polymerase was also from New England Biolabs Inc. and the oligonucleotide primers were from Inqaba, South Africa. .. All other chemicals were of molecular biology grade.

    Article Title: Promoter Activation by CII, a Potent Transcriptional Activator from Bacteriophage 186
    Article Snippet: CII145 was amplified from pZS45-CII145 by high fidelity PCR using primers 289 and RSP (see for sequences). .. The resulting PCR product was used as template for error-prone PCR with Taq DNA polymerase (New England Biolabs) and MgCl2 supplemented to a total concentration of 6 m m . .. The product of error-prone PCR was subcloned back into pZS45-CII145 via BamHI and XhoI restriction sites and transformed into screening strain IM18 by electroporation.

    Article Title: Direct Selection of RNA Beacon Aptamers
    Article Snippet: Reactions contained 0.125 μM DM001, 1 μM DM003, 1 μM DM004 , 200 μM dNTPs, 1X buffer (provided with enzyme) and 12.5 U/mL Taq DNA polymerase (New England Biolabs). .. Reactions contained 0.125 μM DM001, 1 μM DM003, 1 μM DM004 , 200 μM dNTPs, 1X buffer (provided with enzyme) and 12.5 U/mL Taq DNA polymerase (New England Biolabs).

    Article Title: Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus
    Article Snippet: These genes were chosen based on sequence analysis that revealed that these targets were highly conserved. .. PCR amplification was performed with 50-μl volumes containing 1× PCR buffer containing 1.5 mM MgCl2 (New England Biolabs, Herts, United Kingdom), 100 mM deoxynucleoside triphosphates, 0.1 μM of each primer (MWG Biotech AG, Ebersberg, Germany), 2.5 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom), 100 ng template DNA, and PCR-grade water (Invitrogen, CA). .. Thermal PCR conditions for O:1 were 95°C for 1 min, followed by 35 cycles of 94°C for 1 min, 56°C for 1 min (annealing), and 72°C for 1 min with a final extension of 72°C for 5 min, while O:2 PCR was annealed at 51°C for 1 min. Amplified products were analyzed by electrophoresis through a 1% (wt/vol) agarose gel at 80 V for 60 min in 1× TBE.

    Article Title: Targeted gene inactivation in zebrafish using engineered zinc finger nucleases
    Article Snippet: Colonies from the desired selection plates were washed off the plates and their plasmid DNA was recovered as a pool as previously described . .. Each pool of individual fingers was amplified from the plasmid DNA by PCR: 50 μl reaction with 1 units NEB DNA Taq polymerase using 50 ng plasmid DNA template and 1 mM each primer ( ); denature for 1 min at 95°C, 28 cycles consisting of 94°C for 20 sec, 55°C for 20 sec and 72°C for 20 sec, followed by a final extension at 72°C for 6 min. .. The PCR products were run on a 1.5% agarose TAE gel, and the appropriate bands were excised and purified using a Qiagen PCR purification kit.

    Article Title: Specific Roles of the iroBCDEN Genes in Virulence of an Avian Pathogenic Escherichia coli O78 Strain and in Production of Salmochelins
    Article Snippet: Two hundred ninety-eight APEC and 32 commensal fecal isolates were screened by PCR for the presence of the iroB and iroN genes. .. The PCRs were achieved using Taq DNA polymerase (New England Biolabs), at an annealing temperature of 58°C and an extension time of 1 min at 72°C for 25 cycles.

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: The antisense primer annealed to URA3 and extended to the last amino acid of CDH1 . .. EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs). .. After 16 cycles of amplification, 1 ml of product was purified using a Qiagen PCR purification column (Qiagen, Inc.) and co-transformed into YJB1212 with ∼2.5 μg of gapped CDH1- Δ 200-URA3 -pAS2 vector.

    Article Title: Discovery of Bacterial sRNAs by High-Throughput Sequencing
    Article Snippet: Paragraph title: 2.6. PCR ... Taq DNA Polymerase (with 10× Buffer) (NEB, Ipswich, MA).

    Article Title: Identification of Quantitative Trait Loci and Candidate Genes for Maize Starch Granule Size through Association Mapping
    Article Snippet: DNA was extracted from seedlings of 26 maize lines with the largest starch granule sizes and 21 maize lines with the smallest starch granule sizes . .. PCR reaction mixes (20 µl) contained 1 µl of NEB (New England BioLabs Inc., Ipswich, MA, USA) Taq DNA Polymerase, 4 µl of 5× NEB PCR Buffer, 0.5 µl of dNTP mixture, 0.5 µl each of the two primers, and 1 µl of template DNA. .. The PCR reaction was carried out in a Bio-Rad Thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA)with an initial denaturation at 94 °C for 3 min followed by 34 cycles of denaturation for 10 s at 94 °C, annealing for 1 min at 64 °C and extension for 1 min at 68 °C, with a final extension for 10 min at 68 °C.

    Article Title: Eco-Evolutionary Processes Generating Diversity Among Bottlenose Dolphin, Tursiops truncatus, Populations off Baja California, Mexico
    Article Snippet: The PCRs were performed in 25µL volumes consisting of 10 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2 , 0.25 mM each dNTP, 0.12 µM each primer: L15812 (TRO): 5′ CCT CCC TAA GAC TCA AGG AAG 3′ and (D): 5′ CCT GAA GTA AGA ACC AGA TG 3′ (Rosel et al. ), 1.25 U of Taq DNA polymerase (NEB, UK), and approximately 50 ng of genomic DNA. .. The PCRs were performed in 25µL volumes consisting of 10 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2 , 0.25 mM each dNTP, 0.12 µM each primer: L15812 (TRO): 5′ CCT CCC TAA GAC TCA AGG AAG 3′ and (D): 5′ CCT GAA GTA AGA ACC AGA TG 3′ (Rosel et al. ), 1.25 U of Taq DNA polymerase (NEB, UK), and approximately 50 ng of genomic DNA.

    Article Title: Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants
    Article Snippet: Paragraph title: Error-prone PCR and selection of feedback-inhibition-resistant tyrA mutants. ... Using the plasmid pZE21:: tyrA WT as template, 10, 20, and 30 amplification cycles with the primers mentioned above were performed using Taq DNA polymerase (New England Biolabs).

    Article Title: Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer
    Article Snippet: Finally, we screened the individual plate by the same PCR technique to identify the mutant of interest. .. PCRs were performed using Taq polymerase (New England Biolabs, Massachusetts) with the following protocol: denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 68°C for 30 s, and amplification at 72°C for 3 min.

    Article Title: Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes
    Article Snippet: Bacterial 16S rRNA genes were selectively amplified from the purified DNA by PCR using the forward primer 27f and the reverse primer 1492R ( ). .. PCR was performed in triplicate 50-μl reactions containing 1× ThermoPol PCR buffer (New England Biolabs, Mississauga, Ontario, Canada), 0.5 μM concentrations of each primer, 0.5 mM deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (New Englands Biolabs), and 50 ng of DNA. .. The conditions used for PCR amplification were as follows: initial denaturation at 94°C for 5 min and then 25 cycles of (denaturation 94°C for 30 s, primer annealing at 52°C for 30 s, and chain extension for 1 min at 72°C), followed by a final extension at 72°C for 10 min. A PTC-200 thermocycler (MJ Research, Inc., Waltham, MA) was used for PCR.

    Article Title: Genetic variants in the enhancer region of the thymidylate synthase gene in the Chilean population
    Article Snippet: Two oligonucleotide primers were synthesized specifically to amplify the polymorphic region of TSER (sense primer 5′-GTGGCTCCTGCGT TTCCCCC-3′ and antisense primer 5′-GCTCCGAGC CGG CCACAGGCATGGCGCGG-3′) [ ]. .. Amplification reactions were performed in a 25-µl volume containing 0.2 µg genomic DNA, 0.2 m m dNTPs , 0.5 µ m of each primer, 1.0 units of Taq polymerase (New England BioLabs, Ipswich, MA, USA) and 2.5 µl of 10 × Taq PCR buffer. .. Cycling conditions were: one cycle at 94 °C for 5 min with hot start; 30 cycles of 40 s at 94 °C, 1 min at 62 °C and 40 s at 72 °C; and one elongation cycle for 5 min at 72 °C.

    Selection:

    Article Title: Automated selection of aptamers against cholangiocarcinoma cells on an integrated microfluidic platform
    Article Snippet: After each round of Cell-SELEX, the magnetic field was activated such that the negative control cell-magnetic bead complexes could be trapped in the negative selection micropump/micromixer chamber. .. The PCR chamber was preloaded with 0.5 μ l of the forward primer (5′-GGCAGGAAGACAAACA-3′, 0.5 μ M), 0.5 μ l of the reverse primer (5′-ACAGCACCACAGACCA-3′, 0.5 μ M), 0.5 μ l of deoxynucleotide triphosphates (0.2 mM), 2 μ l of ssDNA (0.1 μ M) or the previous amplified product from the last round of Cell-SELEX, 0.125 μ l of Taq DNA polymerase (New England Biolabs, USA), and double-distilled water (ddH2 O) to a final volume of 25 μ l. The PCR was initiated at 95 °C for 10 min followed by 30 cycles of 95 °C for 30 s, 63 °C for 15 s, and 72 °C for 30 s. A final extension step at 72 °C for 10 min was also incorporated into the protocol.

    Article Title: Promoter Activation by CII, a Potent Transcriptional Activator from Bacteriophage 186
    Article Snippet: The resulting PCR product was used as template for error-prone PCR with Taq DNA polymerase (New England Biolabs) and MgCl2 supplemented to a total concentration of 6 m m . .. The product of error-prone PCR was subcloned back into pZS45-CII145 via BamHI and XhoI restriction sites and transformed into screening strain IM18 by electroporation.

    Article Title: Targeted gene inactivation in zebrafish using engineered zinc finger nucleases
    Article Snippet: Colonies from the desired selection plates were washed off the plates and their plasmid DNA was recovered as a pool as previously described . .. Each pool of individual fingers was amplified from the plasmid DNA by PCR: 50 μl reaction with 1 units NEB DNA Taq polymerase using 50 ng plasmid DNA template and 1 mM each primer ( ); denature for 1 min at 95°C, 28 cycles consisting of 94°C for 20 sec, 55°C for 20 sec and 72°C for 20 sec, followed by a final extension at 72°C for 6 min.

    Article Title: Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants
    Article Snippet: Paragraph title: Error-prone PCR and selection of feedback-inhibition-resistant tyrA mutants. ... Using the plasmid pZE21:: tyrA WT as template, 10, 20, and 30 amplification cycles with the primers mentioned above were performed using Taq DNA polymerase (New England Biolabs).

    Microscopy:

    Article Title: Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
    Article Snippet: Nested PCR was conducted in cases of disagreement between study microscopy and the real-time PCR results. .. Reactions were performed in 20 μL total volume containing 1X buffer, 2.5 mM MgCl2 , 200 mM dNTPs, 200 nM primers, and 1.25 units of Taq Polymerase (New England Biolabs, Ipswich, MA).

    Gel Extraction:

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs). .. After 16 cycles of amplification, 1 ml of product was purified using a Qiagen PCR purification column (Qiagen, Inc.) and co-transformed into YJB1212 with ∼2.5 μg of gapped CDH1- Δ 200-URA3 -pAS2 vector.

    Nested PCR:

    Article Title: Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
    Article Snippet: Paragraph title: Nested PCR ... Reactions were performed in 20 μL total volume containing 1X buffer, 2.5 mM MgCl2 , 200 mM dNTPs, 200 nM primers, and 1.25 units of Taq Polymerase (New England Biolabs, Ipswich, MA).

    Two Hybrid Screening:

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: Paragraph title: EP-PCR mutagenesis of CDH1- Δ 200 and yeast two-hybrid screening ... EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs).

    Activated Clotting Time Assay:

    Article Title: Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus
    Article Snippet: The O:1 serotype-specific PCR used the forward primer ESO:1F (5′-CAC GTT CGC CCT GCA AAA AT-3′) and a reverse primer, ESO:1R (5′-GCA AGC GGC CAG ACT GGA TA-3′), designed from sequence information within wehC to generate a 341-bp amplicon. .. PCR amplification was performed with 50-μl volumes containing 1× PCR buffer containing 1.5 mM MgCl2 (New England Biolabs, Herts, United Kingdom), 100 mM deoxynucleoside triphosphates, 0.1 μM of each primer (MWG Biotech AG, Ebersberg, Germany), 2.5 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom), 100 ng template DNA, and PCR-grade water (Invitrogen, CA).

    Plasmid Preparation:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA . .. DNA fragments were prepared by PCR amplification of PAO1 chromosomal DNA with Taq polymerase (New England BioLabs) and the oligonucleotides listed in for fimU and rhlA .

    Article Title: Targeted gene inactivation in zebrafish using engineered zinc finger nucleases
    Article Snippet: Colonies from the desired selection plates were washed off the plates and their plasmid DNA was recovered as a pool as previously described . .. Each pool of individual fingers was amplified from the plasmid DNA by PCR: 50 μl reaction with 1 units NEB DNA Taq polymerase using 50 ng plasmid DNA template and 1 mM each primer ( ); denature for 1 min at 95°C, 28 cycles consisting of 94°C for 20 sec, 55°C for 20 sec and 72°C for 20 sec, followed by a final extension at 72°C for 6 min. .. The PCR products were run on a 1.5% agarose TAE gel, and the appropriate bands were excised and purified using a Qiagen PCR purification kit.

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs). .. After 16 cycles of amplification, 1 ml of product was purified using a Qiagen PCR purification column (Qiagen, Inc.) and co-transformed into YJB1212 with ∼2.5 μg of gapped CDH1- Δ 200-URA3 -pAS2 vector.

    Article Title: A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production
    Article Snippet: Non-methylated plasmid DNA for electroporation into B. anthracis ( ; ) was obtained from E. coli GM2163 cells. .. Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were purchased from NEB.

    Article Title: Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants
    Article Snippet: Nucleotide analogue mutagenesis was carried out in the presence of 2 and 20 μM 8-oxo-2′-deoxyguanosine (8-oxo-dGTP) and 6-(2-deoxy-β- d -ribofuranosyl)-3,4-dihydro-8H-pyrimido-(4,5-c)(1,2)oxazin-7-one (dPTP) ( ). .. Using the plasmid pZE21:: tyrA WT as template, 10, 20, and 30 amplification cycles with the primers mentioned above were performed using Taq DNA polymerase (New England Biolabs). .. The 1.1-kbp PCR products were gel purified, and the mutated tyrA genes were amplified in a second PCR under regular conditions.

    Article Title: The Alternative Sigma Factor ?H Is Required for Toxin Gene Expression by Bacillus anthracis
    Article Snippet: B. anthracis was electroporated with unmethylated plasmid DNA from E. coli GM2163 as described elsewhere ( ). .. Restriction enzymes and T4 DNA ligase were purchased from Promega (Madison, WI) and Fisher Scientific, and Taq DNA polymerase was purchased from New England Biolabs (Beverly, MA).

    Software:

    Article Title: Eco-Evolutionary Processes Generating Diversity Among Bottlenose Dolphin, Tursiops truncatus, Populations off Baja California, Mexico
    Article Snippet: The PCRs were performed in 25µL volumes consisting of 10 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2 , 0.25 mM each dNTP, 0.12 µM each primer: L15812 (TRO): 5′ CCT CCC TAA GAC TCA AGG AAG 3′ and (D): 5′ CCT GAA GTA AGA ACC AGA TG 3′ (Rosel et al. ), 1.25 U of Taq DNA polymerase (NEB, UK), and approximately 50 ng of genomic DNA. .. PCR products were purified using purification spin columns (QIAGEN, UK) and then sequenced in an automatic sequencer (ABI 3730 Gene Analyzer, Applied Biosystems).

    Negative Control:

    Article Title: Automated selection of aptamers against cholangiocarcinoma cells on an integrated microfluidic platform
    Article Snippet: After each round of Cell-SELEX, the magnetic field was activated such that the negative control cell-magnetic bead complexes could be trapped in the negative selection micropump/micromixer chamber. .. The PCR chamber was preloaded with 0.5 μ l of the forward primer (5′-GGCAGGAAGACAAACA-3′, 0.5 μ M), 0.5 μ l of the reverse primer (5′-ACAGCACCACAGACCA-3′, 0.5 μ M), 0.5 μ l of deoxynucleotide triphosphates (0.2 mM), 2 μ l of ssDNA (0.1 μ M) or the previous amplified product from the last round of Cell-SELEX, 0.125 μ l of Taq DNA polymerase (New England Biolabs, USA), and double-distilled water (ddH2 O) to a final volume of 25 μ l. The PCR was initiated at 95 °C for 10 min followed by 30 cycles of 95 °C for 30 s, 63 °C for 15 s, and 72 °C for 30 s. A final extension step at 72 °C for 10 min was also incorporated into the protocol.

    Denaturing Gradient Gel Electrophoresis:

    Article Title: Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes
    Article Snippet: PCR was performed in triplicate 50-μl reactions containing 1× ThermoPol PCR buffer (New England Biolabs, Mississauga, Ontario, Canada), 0.5 μM concentrations of each primer, 0.5 mM deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (New Englands Biolabs), and 50 ng of DNA. .. Positive clones (42 clones) were sequenced by the University Health Network Research DNA Sequencing Facility (Toronto, Ontario, Canada) with the primer 27f, and then the sequence closest match was identified with the blastn utility of GenBank.

    Agarose Gel Electrophoresis:

    Article Title: Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
    Article Snippet: EP-PCR conditions were as follows: 2 μg/ml CDH1- Δ 200-URA3 -pAS2, 0.2 μM of each primer, 1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP, 6 mM MgCl2 , 50 μM MnCl2 , 1 × PCR buffer (−MgCl2 ) (Invitrogen) and 0.5 U/μl Taq polymerase (New England Biolabs). .. After 16 cycles of amplification, 1 ml of product was purified using a Qiagen PCR purification column (Qiagen, Inc.) and co-transformed into YJB1212 with ∼2.5 μg of gapped CDH1- Δ 200-URA3 -pAS2 vector.

    In Vitro:

    Article Title: Direct Selection of RNA Beacon Aptamers
    Article Snippet: The original RNA pool was produced by in vitro transcription. .. Reactions contained 0.125 μM DM001, 1 μM DM003, 1 μM DM004 , 200 μM dNTPs, 1X buffer (provided with enzyme) and 12.5 U/mL Taq DNA polymerase (New England Biolabs).

    Transformation Assay:

    Article Title: A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production
    Article Snippet: E. coli transformation, plasmid isolation, and recombinant techniques were performed using standard methods ( ). .. Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were purchased from NEB.

    Article Title: Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants
    Article Snippet: Using the plasmid pZE21:: tyrA WT as template, 10, 20, and 30 amplification cycles with the primers mentioned above were performed using Taq DNA polymerase (New England Biolabs). .. The 1.1-kbp PCR products were gel purified, and the mutated tyrA genes were amplified in a second PCR under regular conditions.

    Article Title: The Alternative Sigma Factor ?H Is Required for Toxin Gene Expression by Bacillus anthracis
    Article Snippet: Preparation of plasmid DNA from E. coli , transformation of E. coli , and recombinant DNA techniques were performed using standard procedures ( ). .. Restriction enzymes and T4 DNA ligase were purchased from Promega (Madison, WI) and Fisher Scientific, and Taq DNA polymerase was purchased from New England Biolabs (Beverly, MA).

    Spectrophotometry:

    Article Title: Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus
    Article Snippet: Total DNA was prepared using the Wizard Genomic DNA purification kit (Promega, Madison, WI) and quantified by spectrophotometry (NanoDrop, Wilmington, DE). .. PCR amplification was performed with 50-μl volumes containing 1× PCR buffer containing 1.5 mM MgCl2 (New England Biolabs, Herts, United Kingdom), 100 mM deoxynucleoside triphosphates, 0.1 μM of each primer (MWG Biotech AG, Ebersberg, Germany), 2.5 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom), 100 ng template DNA, and PCR-grade water (Invitrogen, CA).

    Produced:

    Article Title: Direct Selection of RNA Beacon Aptamers
    Article Snippet: The original RNA pool was produced by in vitro transcription. .. Reactions contained 0.125 μM DM001, 1 μM DM003, 1 μM DM004 , 200 μM dNTPs, 1X buffer (provided with enzyme) and 12.5 U/mL Taq DNA polymerase (New England Biolabs).

    Concentration Assay:

    Article Title: Promoter Activation by CII, a Potent Transcriptional Activator from Bacteriophage 186
    Article Snippet: CII145 was amplified from pZS45-CII145 by high fidelity PCR using primers 289 and RSP (see for sequences). .. The resulting PCR product was used as template for error-prone PCR with Taq DNA polymerase (New England Biolabs) and MgCl2 supplemented to a total concentration of 6 m m . .. The product of error-prone PCR was subcloned back into pZS45-CII145 via BamHI and XhoI restriction sites and transformed into screening strain IM18 by electroporation.

    DNA Purification:

    Article Title: Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus
    Article Snippet: Total DNA was prepared using the Wizard Genomic DNA purification kit (Promega, Madison, WI) and quantified by spectrophotometry (NanoDrop, Wilmington, DE). .. PCR amplification was performed with 50-μl volumes containing 1× PCR buffer containing 1.5 mM MgCl2 (New England Biolabs, Herts, United Kingdom), 100 mM deoxynucleoside triphosphates, 0.1 μM of each primer (MWG Biotech AG, Ebersberg, Germany), 2.5 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom), 100 ng template DNA, and PCR-grade water (Invitrogen, CA).

    Recombinant:

    Article Title: A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production
    Article Snippet: E. coli transformation, plasmid isolation, and recombinant techniques were performed using standard methods ( ). .. Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were purchased from NEB.

    Article Title: The Alternative Sigma Factor ?H Is Required for Toxin Gene Expression by Bacillus anthracis
    Article Snippet: Preparation of plasmid DNA from E. coli , transformation of E. coli , and recombinant DNA techniques were performed using standard procedures ( ). .. Restriction enzymes and T4 DNA ligase were purchased from Promega (Madison, WI) and Fisher Scientific, and Taq DNA polymerase was purchased from New England Biolabs (Beverly, MA).

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  • 99
    New England Biolabs taq dna polymerase supertaq
    <t>DNA</t> modification and bypass during bisulfite treatment. ( A ) Scheme of the chemical reaction of cytidine: Treatment with bisulfite generates the non-aromatic, non-planar 5,6-dihydrouridine-6-sulfonate (dhU6S), which decomposes to uracil upon treatment with base (and heat). ( B ) Primer extension activity of different polymerases <t>(Taq,</t> 5D4, 3A10, E10, TgoT) on template T1 either unmodified (C), bisulfite-treated and desulfonated (Reagent 1, 80°C, 20 min) (converting dC to dU) (D) or bisulfite-treated (Reagent 1) but not desulphonated (converting dC to dhU6S) (S). Polymerases 5D4 and 3A10 are able to generate full-length (+20) products even from the non-desulphonated template (S). ( C ) Time-course comparison of primer extension activity of Taq and 5D4 on T1 either unmodified (C) or bisulfite-treated with (D) or without desulphonation (S). (P: primer).
    Taq Dna Polymerase Supertaq, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase supertaq/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase supertaq - by Bioz Stars, 2019-10
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    97
    New England Biolabs taq dna polymerase
    CE analysis of processing synthetic <t>DNA</t> by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized <t>Taq</t> DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.
    Taq Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/New England Biolabs
    Average 97 stars, based on 927 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2019-10
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    99
    New England Biolabs taq dna ligase
    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly <t>enzymes–Taq</t> <t>DNA</t> polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Taq Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna ligase/product/New England Biolabs
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    DNA modification and bypass during bisulfite treatment. ( A ) Scheme of the chemical reaction of cytidine: Treatment with bisulfite generates the non-aromatic, non-planar 5,6-dihydrouridine-6-sulfonate (dhU6S), which decomposes to uracil upon treatment with base (and heat). ( B ) Primer extension activity of different polymerases (Taq, 5D4, 3A10, E10, TgoT) on template T1 either unmodified (C), bisulfite-treated and desulfonated (Reagent 1, 80°C, 20 min) (converting dC to dU) (D) or bisulfite-treated (Reagent 1) but not desulphonated (converting dC to dhU6S) (S). Polymerases 5D4 and 3A10 are able to generate full-length (+20) products even from the non-desulphonated template (S). ( C ) Time-course comparison of primer extension activity of Taq and 5D4 on T1 either unmodified (C) or bisulfite-treated with (D) or without desulphonation (S). (P: primer).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: DNA modification and bypass during bisulfite treatment. ( A ) Scheme of the chemical reaction of cytidine: Treatment with bisulfite generates the non-aromatic, non-planar 5,6-dihydrouridine-6-sulfonate (dhU6S), which decomposes to uracil upon treatment with base (and heat). ( B ) Primer extension activity of different polymerases (Taq, 5D4, 3A10, E10, TgoT) on template T1 either unmodified (C), bisulfite-treated and desulfonated (Reagent 1, 80°C, 20 min) (converting dC to dU) (D) or bisulfite-treated (Reagent 1) but not desulphonated (converting dC to dhU6S) (S). Polymerases 5D4 and 3A10 are able to generate full-length (+20) products even from the non-desulphonated template (S). ( C ) Time-course comparison of primer extension activity of Taq and 5D4 on T1 either unmodified (C) or bisulfite-treated with (D) or without desulphonation (S). (P: primer).

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Modification, Activity Assay

    Amplification of 24 individual human genomic loci. Comparison of PCR performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic DNA. Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: Amplification of 24 individual human genomic loci. Comparison of PCR performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic DNA. Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Amplification, Polymerase Chain Reaction

    PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Gas Chromatography, Molecular Weight

    Degree of methylation of individual CpG sites. Promoter regions of four genes ( A — prkcdbp , B — dab2ip , C — ptgs2 , D — ezh2) were amplified with either Taq ot 5D4/Taq blends, using bisulfide-treated genomic DNA from normal cells or LNCapP cells as a template and subjected to deep sequencing. Cyan—methylated CpGs, orange—unmethylated CpGs. Individual CpGs are numbered starting from the 5′ end of the amplicon.

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: Degree of methylation of individual CpG sites. Promoter regions of four genes ( A — prkcdbp , B — dab2ip , C — ptgs2 , D — ezh2) were amplified with either Taq ot 5D4/Taq blends, using bisulfide-treated genomic DNA from normal cells or LNCapP cells as a template and subjected to deep sequencing. Cyan—methylated CpGs, orange—unmethylated CpGs. Individual CpGs are numbered starting from the 5′ end of the amplicon.

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Methylation, Amplification, Sequencing

    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Labeling, Gas Chromatography, Incubation, Negative Control

    Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Gas Chromatography, Polymerase Chain Reaction, Sequencing, Generated, Produced, Purification, Incubation

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Gas Chromatography, Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation, Activated Clotting Time Assay

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Journal: PLoS ONE

    Article Title: Cellular reagents for diagnostics and synthetic biology

    doi: 10.1371/journal.pone.0201681

    Figure Lengend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Article Snippet: Assemblies using pure enzymes contained 0.08 units of T5 exonuclease (NEB), 0.5 units of Phusion DNA polymerase (NEB) and 80 units of Taq DNA ligase (NEB).

    Techniques: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    RNA detection by two-step reverse transcription TaqMan qPCR using cellular reagents for MMLV RT and Taq DNA polymerase. Indicated copies of synthetic RNA template derived from Zika virus genomic sequence were tested using 2 x 10 7 cells each of MMLV RT and Taq DNA polymerase lyophilized cellular reagents. Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are presented. Color of the traces indicate presence (black traces) or absence (red traces) of MMLV RT cellular reagents, or the absence of templates (blue traces). The corresponding derivation of template copies from Cq analyses are tabulated. Cq values were converted to template copies using standard curve analyses of the same RNA samples with commercial qRT-PCR master mix ( S8 Fig ).

    Journal: PLoS ONE

    Article Title: Cellular reagents for diagnostics and synthetic biology

    doi: 10.1371/journal.pone.0201681

    Figure Lengend Snippet: RNA detection by two-step reverse transcription TaqMan qPCR using cellular reagents for MMLV RT and Taq DNA polymerase. Indicated copies of synthetic RNA template derived from Zika virus genomic sequence were tested using 2 x 10 7 cells each of MMLV RT and Taq DNA polymerase lyophilized cellular reagents. Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are presented. Color of the traces indicate presence (black traces) or absence (red traces) of MMLV RT cellular reagents, or the absence of templates (blue traces). The corresponding derivation of template copies from Cq analyses are tabulated. Cq values were converted to template copies using standard curve analyses of the same RNA samples with commercial qRT-PCR master mix ( S8 Fig ).

    Article Snippet: Assemblies using pure enzymes contained 0.08 units of T5 exonuclease (NEB), 0.5 units of Phusion DNA polymerase (NEB) and 80 units of Taq DNA ligase (NEB).

    Techniques: RNA Detection, Real-time Polymerase Chain Reaction, Derivative Assay, Sequencing, Amplification, Fluorescence, Generated, Software, Quantitative RT-PCR

    TaqMan qPCR analysis using lyophilized Taq DNA polymerase cellular reagents. Indicated copies of synthetic DNA templates derived from Zika virus genomic sequence were amplified using 2.5 units of pure commercial Taq DNA polymerase (panels a and b) or 2 x 10 7 cells of rehydrated cellular reagents expressing Taq DNA polymerase (panels c and d). Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are depicted in panels a and c. Amplification curve colors distinguish starting template copies–yellow: 60,000 template copies; green: 6000 template copies; blue: 600 template copies; red: 60 template copies; and gray: no template control. These curves depict the real-time kinetics of PCR amplification mediated by pure versus cellular reagents. The corresponding standard curve analyses performed using the “Abs quant” protocol in the LightCycler 96 software are depicted in panels b and d, respectively. Standard curve analyses data for comparing amplification efficiency, linearity, and error are tabulated as insets.

    Journal: PLoS ONE

    Article Title: Cellular reagents for diagnostics and synthetic biology

    doi: 10.1371/journal.pone.0201681

    Figure Lengend Snippet: TaqMan qPCR analysis using lyophilized Taq DNA polymerase cellular reagents. Indicated copies of synthetic DNA templates derived from Zika virus genomic sequence were amplified using 2.5 units of pure commercial Taq DNA polymerase (panels a and b) or 2 x 10 7 cells of rehydrated cellular reagents expressing Taq DNA polymerase (panels c and d). Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are depicted in panels a and c. Amplification curve colors distinguish starting template copies–yellow: 60,000 template copies; green: 6000 template copies; blue: 600 template copies; red: 60 template copies; and gray: no template control. These curves depict the real-time kinetics of PCR amplification mediated by pure versus cellular reagents. The corresponding standard curve analyses performed using the “Abs quant” protocol in the LightCycler 96 software are depicted in panels b and d, respectively. Standard curve analyses data for comparing amplification efficiency, linearity, and error are tabulated as insets.

    Article Snippet: Assemblies using pure enzymes contained 0.08 units of T5 exonuclease (NEB), 0.5 units of Phusion DNA polymerase (NEB) and 80 units of Taq DNA ligase (NEB).

    Techniques: Real-time Polymerase Chain Reaction, Derivative Assay, Sequencing, Amplification, Expressing, Fluorescence, Generated, Software, Polymerase Chain Reaction

    Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Journal: Scientific Reports

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    doi: 10.1038/srep05052

    Figure Lengend Snippet: Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Article Snippet: Even the use of a non-proofreading but robust DNA polymerase, Taq (NE Biolabs) failed to amplify only the desired fragment ( ).

    Techniques: Binding Assay, Amplification, Polymerase Chain Reaction

    Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material .

    Journal: Scientific Reports

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    doi: 10.1038/srep05052

    Figure Lengend Snippet: Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material .

    Article Snippet: Even the use of a non-proofreading but robust DNA polymerase, Taq (NE Biolabs) failed to amplify only the desired fragment ( ).

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation

    PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3 . Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material .

    Journal: Scientific Reports

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    doi: 10.1038/srep05052

    Figure Lengend Snippet: PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3 . Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material .

    Article Snippet: Even the use of a non-proofreading but robust DNA polymerase, Taq (NE Biolabs) failed to amplify only the desired fragment ( ).

    Techniques: Polymerase Chain Reaction, Generated, Amplification, Plasmid Preparation, Binding Assay

    Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.

    Journal: Scientific Reports

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    doi: 10.1038/srep05052

    Figure Lengend Snippet: Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.

    Article Snippet: Even the use of a non-proofreading but robust DNA polymerase, Taq (NE Biolabs) failed to amplify only the desired fragment ( ).

    Techniques: Clone Assay, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Amplification