q5  (New England Biolabs)


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    • 99
    Name:
    KLD Enzyme Mix
    Description:
    KLD Enzyme Mix 25 rxns
    Catalog Number:
    M0554S
    Price:
    312
    Category:
    PCR Mutagenesis Kits
    Size:
    25 rxns
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    Structured Review

    New England Biolabs q5
    KLD Enzyme Mix
    KLD Enzyme Mix 25 rxns
    https://www.bioz.com/result/q5/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    q5 - by Bioz Stars, 2021-05
    99/100 stars

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    Related Articles

    Sequencing:

    Article Title: An engineered ScCas9 with broad PAM range and high specificity and activity.
    Article Snippet: CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. .. CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. .. CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing.

    Polymerase Chain Reaction:

    Article Title: An engineered ScCas9 with broad PAM range and high specificity and activity.
    Article Snippet: CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. .. CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. .. CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing.

    Article Title: A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
    Article Snippet: An expression plasmid for VIM-20, which harbors the H229R mutation, was generated by site-directed mutagenesis using a forward primer (5′-TATTCAGCAACGTTACCCGGAAGCGCAATTC-3′), a reverse primer (5′-CGTTCGATGCTGGTCGGC-3′), and the pET-28a(+)-TEV-VIM-2 plasmid as the template. .. After the PCR, the amplified sequences were added directly to a kinase-ligase-DpnI enzyme mix (NEB) for rapid, room-temperature circularization and template removal. .. A 2-μl aliquot of the ligation mixture was used to transform 30 μl of E. coli DH5α chemically competent cells (Lucigen), and the transformation mixture was spread onto a lysogeny broth (LB) plate containing 50 μg/ml kanamycin.

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: Cloning Modified pET28a plasmids encoding for ybbR-His-XylanaseT6(T129C) (Geobacillus stearothermophilus )-Doc3 (R. flavefaciens ), ybbR-His-sfGFP-DocI (Clostridium thermocellum ), and Titin-Ig domains (repeats 27 to 32, repeat 34, human) were used as templates for polymerase chain reaction (PCR) with subsequent reconstitution by Gibson assembly. .. The previously reported d59 sortase(P94R/D160N/D165A/K190E/K196T) mutant was created by introducing the mutations via overlap extension PCR followed by ligating the linearized plasmid using Kinase–Ligase–DpnI (KLD) enzyme mix and KLD reaction buffer from the Q5 site-directed mutagenesis kit (New England Biolabs, MA, USA). .. The chemically competent E. coli DH5-α cells were transformed [Life Technologies GmbH, Frankfurt, Germany; 30 min on ice, 30 s heat shock at 42 °C followed by 37 °C for 1 h in a super optimal broth with catabolite repression medium] and plated on kanamycin-supplemented agar plates.

    Article Title: Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy
    Article Snippet: For insertion of the (GS)4 and (GS)8 linkers into the Doc domain, exponential amplification with primers bearing coding sequences for the inserts at their 5’-ends was performed with a Phusion High-Fidelity DNA polymerase (New England Biolabs, MA). .. PCR products were then blunt end ligated using KLD Enzyme Mix and KLD Reaction Buffer from the Q5 site directed mutagenesis kit (New England Biolabs, MA). .. The modified DNA constructs were used to transform Escherichia coli DH5-alpha cells, grown on kanamycin-containing agar plates and subsequently screened.

    Article Title: The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation
    Article Snippet: Briefly, the fliD complementation plasmid was amplified by PCR with divergent primers containing targeted nucleotide substitutions in the forward primer (listed in Supplementary Table ). .. An aliquot of the linear PCR product was treated with the KLD enzyme mix to circularize the mutated plasmid while degrading any residual template. .. The treated plasmids were transformed into E. coli DH5ɑ and transformants selected by chloramphenicol resistance.

    Amplification:

    Article Title: An engineered ScCas9 with broad PAM range and high specificity and activity.
    Article Snippet: CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. .. CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. .. CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing.

    Article Title: A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
    Article Snippet: An expression plasmid for VIM-20, which harbors the H229R mutation, was generated by site-directed mutagenesis using a forward primer (5′-TATTCAGCAACGTTACCCGGAAGCGCAATTC-3′), a reverse primer (5′-CGTTCGATGCTGGTCGGC-3′), and the pET-28a(+)-TEV-VIM-2 plasmid as the template. .. After the PCR, the amplified sequences were added directly to a kinase-ligase-DpnI enzyme mix (NEB) for rapid, room-temperature circularization and template removal. .. A 2-μl aliquot of the ligation mixture was used to transform 30 μl of E. coli DH5α chemically competent cells (Lucigen), and the transformation mixture was spread onto a lysogeny broth (LB) plate containing 50 μg/ml kanamycin.

    Construct:

    Article Title: Development of a Formaldehyde Biosensor with Application to Synthetic Methylotrophy
    Article Snippet: Pathway optimization assays were conducted in E. coli MG1655(DE3) ( ). pBbS2k-RFP was a gift from Jay Keasling (Addgene plasmid #35330). pETM6-mCherry was a gift from Mattheos Koffas (Addgene plasmid #66534). .. Plasmids were constructed using USER cloning, Gibson Assembly Master Mix, or KLD Enzyme Mix (NEB). .. DNA fragments were generated by PCR with the primers listed in .

    Clone Assay:

    Article Title: Development of a Formaldehyde Biosensor with Application to Synthetic Methylotrophy
    Article Snippet: Pathway optimization assays were conducted in E. coli MG1655(DE3) ( ). pBbS2k-RFP was a gift from Jay Keasling (Addgene plasmid #35330). pETM6-mCherry was a gift from Mattheos Koffas (Addgene plasmid #66534). .. Plasmids were constructed using USER cloning, Gibson Assembly Master Mix, or KLD Enzyme Mix (NEB). .. DNA fragments were generated by PCR with the primers listed in .

    Mutagenesis:

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: Cloning Modified pET28a plasmids encoding for ybbR-His-XylanaseT6(T129C) (Geobacillus stearothermophilus )-Doc3 (R. flavefaciens ), ybbR-His-sfGFP-DocI (Clostridium thermocellum ), and Titin-Ig domains (repeats 27 to 32, repeat 34, human) were used as templates for polymerase chain reaction (PCR) with subsequent reconstitution by Gibson assembly. .. The previously reported d59 sortase(P94R/D160N/D165A/K190E/K196T) mutant was created by introducing the mutations via overlap extension PCR followed by ligating the linearized plasmid using Kinase–Ligase–DpnI (KLD) enzyme mix and KLD reaction buffer from the Q5 site-directed mutagenesis kit (New England Biolabs, MA, USA). .. The chemically competent E. coli DH5-α cells were transformed [Life Technologies GmbH, Frankfurt, Germany; 30 min on ice, 30 s heat shock at 42 °C followed by 37 °C for 1 h in a super optimal broth with catabolite repression medium] and plated on kanamycin-supplemented agar plates.

    Article Title: Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy
    Article Snippet: For insertion of the (GS)4 and (GS)8 linkers into the Doc domain, exponential amplification with primers bearing coding sequences for the inserts at their 5’-ends was performed with a Phusion High-Fidelity DNA polymerase (New England Biolabs, MA). .. PCR products were then blunt end ligated using KLD Enzyme Mix and KLD Reaction Buffer from the Q5 site directed mutagenesis kit (New England Biolabs, MA). .. The modified DNA constructs were used to transform Escherichia coli DH5-alpha cells, grown on kanamycin-containing agar plates and subsequently screened.

    Plasmid Preparation:

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: Cloning Modified pET28a plasmids encoding for ybbR-His-XylanaseT6(T129C) (Geobacillus stearothermophilus )-Doc3 (R. flavefaciens ), ybbR-His-sfGFP-DocI (Clostridium thermocellum ), and Titin-Ig domains (repeats 27 to 32, repeat 34, human) were used as templates for polymerase chain reaction (PCR) with subsequent reconstitution by Gibson assembly. .. The previously reported d59 sortase(P94R/D160N/D165A/K190E/K196T) mutant was created by introducing the mutations via overlap extension PCR followed by ligating the linearized plasmid using Kinase–Ligase–DpnI (KLD) enzyme mix and KLD reaction buffer from the Q5 site-directed mutagenesis kit (New England Biolabs, MA, USA). .. The chemically competent E. coli DH5-α cells were transformed [Life Technologies GmbH, Frankfurt, Germany; 30 min on ice, 30 s heat shock at 42 °C followed by 37 °C for 1 h in a super optimal broth with catabolite repression medium] and plated on kanamycin-supplemented agar plates.

    Article Title: The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation
    Article Snippet: Briefly, the fliD complementation plasmid was amplified by PCR with divergent primers containing targeted nucleotide substitutions in the forward primer (listed in Supplementary Table ). .. An aliquot of the linear PCR product was treated with the KLD enzyme mix to circularize the mutated plasmid while degrading any residual template. .. The treated plasmids were transformed into E. coli DH5ɑ and transformants selected by chloramphenicol resistance.

    Transformation Assay:

    Article Title: Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase
    Article Snippet: Kinase, Ligase & DpnI (KLD) treatment: 1 μL of PCR product was mixed with 5 μL of 2X KLD Reaction buffer, 1 μL of 10X KLD Enzyme Mix (both from NEB) and 3 μL of nuclease-free water. .. The KLD reaction mixture was used to transform E . coli DH-5α chemically competent cells (NEB) using the following protocol: 5 μL of KLD reaction mix were added to a tube of thawed DH-5α competent E . coli cells on ice, and mixed gently for a few seconds; after transformation, the bacteria were incubated on ice for 30 minutes, heat shocked at 42 °C for 30 seconds and incubated on ice again for 5 minutes. ..

    Incubation:

    Article Title: Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase
    Article Snippet: Kinase, Ligase & DpnI (KLD) treatment: 1 μL of PCR product was mixed with 5 μL of 2X KLD Reaction buffer, 1 μL of 10X KLD Enzyme Mix (both from NEB) and 3 μL of nuclease-free water. .. The KLD reaction mixture was used to transform E . coli DH-5α chemically competent cells (NEB) using the following protocol: 5 μL of KLD reaction mix were added to a tube of thawed DH-5α competent E . coli cells on ice, and mixed gently for a few seconds; after transformation, the bacteria were incubated on ice for 30 minutes, heat shocked at 42 °C for 30 seconds and incubated on ice again for 5 minutes. ..

    Inverse PCR:

    Article Title: Engineered systems of inducible anti-repressors for the next generation of biological programming
    Article Snippet: .. This was performed with inverse PCR followed by treatment with KLD Enzyme Mix (NEB). .. Reporter system and series-parallel genetic architecture The GFP reporter plasmid system developed by Rondon and Wilson was utilized for the single TF studies with a proximal operator and was also used as the series-parallel proximal architecture.

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    • neb q5  (New England Biolabs)
    99
    New England Biolabs neb q5
    Macerprepped DNA is good template for PCR. 1-3, PCR product by <t>NEB</t> Q5® HiFi polymerase. 4-6, PCR product by NEB Onetaq polymerase. I, 4, Miraprepped DNA. 2, 5, Macerprepped DNA with Solution 3 process. 3, 6, Macerprepped DNA with Solution N3 process. M, marker.
    Neb Q5, 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/neb q5/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    neb q5 - by Bioz Stars, 2021-05
    99/100 stars
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    high fidelity dna polymerase  (New England Biolabs)
    99
    New England Biolabs high fidelity dna polymerase
    Cloning strategy to insert HAs into the pDsRed- 24MS2 plasmid for one-step CRISPR (A) Detailed view of the target sequence that starts with a 5′G and ends with the NGG PAM site and the gRNA. HAs sit on either site of the Cas9 cleavage site, which is located in the target sequence, 3 nt away from the PAM sequence. (B) The pDsRed-24MS2 plasmid contains two MCSs for HA insertion, the DsRed marker gene flanked by loxP sites and 24xMS2 stem-loops. (C) <t>HA1</t> is PCR amplified from genomic <t>DNA</t> (1). The plasmid backbone is linearized using restriction sites in the 5′ MCS (2) and ligated with the HA1 fragment (3). The resulting pDsRed-24MS2+HA1 plasmid is linearized by restriction digest (4) and the HA2 fragment is amplified from genomic DNA (5). HA2 is ligated into the linearized vector (6) and purified for microinjection (7). (D) The finished plasmid pDsRed-24MS2+HA1+HA2 is used as a dsDNA donor for CRISPR engineering.
    High Fidelity Dna Polymerase, 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/high fidelity dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    high fidelity dna polymerase - by Bioz Stars, 2021-05
    99/100 stars
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    Image Search Results


    Macerprepped DNA is good template for PCR. 1-3, PCR product by NEB Q5® HiFi polymerase. 4-6, PCR product by NEB Onetaq polymerase. I, 4, Miraprepped DNA. 2, 5, Macerprepped DNA with Solution 3 process. 3, 6, Macerprepped DNA with Solution N3 process. M, marker.

    Journal: bioRxiv

    Article Title: The Macerprep: a minimalist kit- and enzyme-free high-yield miniprep utilising alkaline lysis and alkaline hydrolysis principles

    doi: 10.1101/2020.08.13.249607

    Figure Lengend Snippet: Macerprepped DNA is good template for PCR. 1-3, PCR product by NEB Q5® HiFi polymerase. 4-6, PCR product by NEB Onetaq polymerase. I, 4, Miraprepped DNA. 2, 5, Macerprepped DNA with Solution 3 process. 3, 6, Macerprepped DNA with Solution N3 process. M, marker.

    Article Snippet: According to the NEB calculator (NEB, online tool), Tm for NEB Q5 and NEB Onetaq was determined to be 52°C and 61°C.

    Techniques: Polymerase Chain Reaction, Marker

    DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions. ( A ) Schematic of DropSynth 2.0. ( B ) Comparison of percent perfect assemblies (minimum 100 assembly barcodes) of a 384-gene library assembled using DropSynth with three different polymerases (KAPA Robust, NEB Q5 or KAPA HiFi) with or without MutS-based enzymatic error correction. ( C ) Comparison of total assemblies represented with at least one assembly barcode for all conditions. Two codon versions of the 384-gene library were assembled for each condition, and representation is improved when combining across both codon usages. ( D ) A 2% agarose gel of 384-gene assembly product following bulk amplification with standard PCR or using single-primer suppression PCR; yield of assembled product is noticeably higher using single-primer suppression PCR.

    Journal: Nucleic Acids Research

    Article Title: DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions

    doi: 10.1093/nar/gkaa600

    Figure Lengend Snippet: DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions. ( A ) Schematic of DropSynth 2.0. ( B ) Comparison of percent perfect assemblies (minimum 100 assembly barcodes) of a 384-gene library assembled using DropSynth with three different polymerases (KAPA Robust, NEB Q5 or KAPA HiFi) with or without MutS-based enzymatic error correction. ( C ) Comparison of total assemblies represented with at least one assembly barcode for all conditions. Two codon versions of the 384-gene library were assembled for each condition, and representation is improved when combining across both codon usages. ( D ) A 2% agarose gel of 384-gene assembly product following bulk amplification with standard PCR or using single-primer suppression PCR; yield of assembled product is noticeably higher using single-primer suppression PCR.

    Article Snippet: Though MutS appeared to marginally improve fidelity in assemblies using KAPA Robust and NEB Q5, it did not have a statistically significant effect on assemblies using KAPA HiFi (Figure , and ).

    Techniques: Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

    Cloning strategy to insert HAs into the pDsRed- 24MS2 plasmid for one-step CRISPR (A) Detailed view of the target sequence that starts with a 5′G and ends with the NGG PAM site and the gRNA. HAs sit on either site of the Cas9 cleavage site, which is located in the target sequence, 3 nt away from the PAM sequence. (B) The pDsRed-24MS2 plasmid contains two MCSs for HA insertion, the DsRed marker gene flanked by loxP sites and 24xMS2 stem-loops. (C) HA1 is PCR amplified from genomic DNA (1). The plasmid backbone is linearized using restriction sites in the 5′ MCS (2) and ligated with the HA1 fragment (3). The resulting pDsRed-24MS2+HA1 plasmid is linearized by restriction digest (4) and the HA2 fragment is amplified from genomic DNA (5). HA2 is ligated into the linearized vector (6) and purified for microinjection (7). (D) The finished plasmid pDsRed-24MS2+HA1+HA2 is used as a dsDNA donor for CRISPR engineering.

    Journal: STAR Protocols

    Article Title: CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos

    doi: 10.1016/j.xpro.2021.100380

    Figure Lengend Snippet: Cloning strategy to insert HAs into the pDsRed- 24MS2 plasmid for one-step CRISPR (A) Detailed view of the target sequence that starts with a 5′G and ends with the NGG PAM site and the gRNA. HAs sit on either site of the Cas9 cleavage site, which is located in the target sequence, 3 nt away from the PAM sequence. (B) The pDsRed-24MS2 plasmid contains two MCSs for HA insertion, the DsRed marker gene flanked by loxP sites and 24xMS2 stem-loops. (C) HA1 is PCR amplified from genomic DNA (1). The plasmid backbone is linearized using restriction sites in the 5′ MCS (2) and ligated with the HA1 fragment (3). The resulting pDsRed-24MS2+HA1 plasmid is linearized by restriction digest (4) and the HA2 fragment is amplified from genomic DNA (5). HA2 is ligated into the linearized vector (6) and purified for microinjection (7). (D) The finished plasmid pDsRed-24MS2+HA1+HA2 is used as a dsDNA donor for CRISPR engineering.

    Article Snippet: In short: a.Day 1: HA1 and 2 are amplified using a high-fidelity DNA polymerase and digested with restriction enzymes that recognisethe sequences that were added to the HA primers ( C, steps 1 and 5). b. HA1 is inserted into the linearized pTVCherry vector ( C, steps 2 and 3). c.Days 2–5.

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

    Cloning strategy to insert HAs into the pTV cherry plasmid for two-step CRISPR (A) The pTV cherry plasmid contains two MCSs to insert HA sequences for HDR, a mini-white marker gene, loxP and FRT sites, and an attP site. It also contains a mCherry gene (not shown). (B) Detailed view of the target sequence, starting with a 5′G and ending with the NGG PAM site. The Cas9 cleavage site is located 3 nt away from the PAM sequence and the 5′ HA ends upstream of the cleavage site. (C) HA1 is amplified from genomic DNA (1), the pTV cherry plasmid is linearized using restriction sites in the 5′ MCS (2) and both are ligated (3). The pTV cherry +HA1 plasmid is linearized using restriction sites present in the 3′ MCS (4) and the HA2 insert is PCR amplified from genomic DNA (5) before it is ligated into the plasmid backbone (6). The plasmid containing both HAs is purified for microinjection (7). (D) The resulting pTV cherry +HA1+HA2 plasmid can be used as a dsDNA donor plasmid during CRISPR engineering.

    Journal: STAR Protocols

    Article Title: CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos

    doi: 10.1016/j.xpro.2021.100380

    Figure Lengend Snippet: Cloning strategy to insert HAs into the pTV cherry plasmid for two-step CRISPR (A) The pTV cherry plasmid contains two MCSs to insert HA sequences for HDR, a mini-white marker gene, loxP and FRT sites, and an attP site. It also contains a mCherry gene (not shown). (B) Detailed view of the target sequence, starting with a 5′G and ending with the NGG PAM site. The Cas9 cleavage site is located 3 nt away from the PAM sequence and the 5′ HA ends upstream of the cleavage site. (C) HA1 is amplified from genomic DNA (1), the pTV cherry plasmid is linearized using restriction sites in the 5′ MCS (2) and both are ligated (3). The pTV cherry +HA1 plasmid is linearized using restriction sites present in the 3′ MCS (4) and the HA2 insert is PCR amplified from genomic DNA (5) before it is ligated into the plasmid backbone (6). The plasmid containing both HAs is purified for microinjection (7). (D) The resulting pTV cherry +HA1+HA2 plasmid can be used as a dsDNA donor plasmid during CRISPR engineering.

    Article Snippet: In short: a.Day 1: HA1 and 2 are amplified using a high-fidelity DNA polymerase and digested with restriction enzymes that recognisethe sequences that were added to the HA primers ( C, steps 1 and 5). b. HA1 is inserted into the linearized pTVCherry vector ( C, steps 2 and 3). c.Days 2–5.

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

    Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) A representative quantification of dTTP using a published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31–0.08 pmol) are not shown. ( B ) A representative quantification of dTTP using Q5 DNA polymerase, 197-nt template and EvaGreen detection chemistry. ( C , D ) dTTP signal generated by the fluorometric methods from a high concentration mouse liver extract with and without 0.5 pmol dTTP spike-in calibrant. The extract volume was adjusted to 2 μl per mg of initial tissue weight. The extract comprised half of the reaction. ( E , F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of a polynomial curve fit.

    Journal: Nucleic Acids Research

    Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

    doi: 10.1093/nar/gkaa516

    Figure Lengend Snippet: Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) A representative quantification of dTTP using a published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31–0.08 pmol) are not shown. ( B ) A representative quantification of dTTP using Q5 DNA polymerase, 197-nt template and EvaGreen detection chemistry. ( C , D ) dTTP signal generated by the fluorometric methods from a high concentration mouse liver extract with and without 0.5 pmol dTTP spike-in calibrant. The extract volume was adjusted to 2 μl per mg of initial tissue weight. The extract comprised half of the reaction. ( E , F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of a polynomial curve fit.

    Article Snippet: We found that the Q5 DNA polymerase, which we mainly used in this work, retains specificity in the presence of rNTP-to-dNTP ratios typically present in cultured cells.

    Techniques: Hydrolysis Assay, Generated, Concentration Assay, Fluorescence

    Optimization of EvaGreen- and Q5 DNA polymerase-based dNTP quantification. ( A–F ) Each critical reaction component and reaction temperature was varied, and the outcome evaluated. ( G ) The end-point fluorescence was measured at 66°C and after raising the temperature to 75°C to dissociate unused primers. ( H ) Assay conditions in graphs A to G. The standard curves were generated from the end-point baseline-corrected fluorescence values. The assessments were performed in triplicates. Supplementary Figure S3B shows the effect of increasing EvaGreen concentration above 1.25 μM. Supplementary Figure S3C-D shows the effect of different DNA polymerase concentrations on the dTTP and dCTP quantification.

    Journal: Nucleic Acids Research

    Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

    doi: 10.1093/nar/gkaa516

    Figure Lengend Snippet: Optimization of EvaGreen- and Q5 DNA polymerase-based dNTP quantification. ( A–F ) Each critical reaction component and reaction temperature was varied, and the outcome evaluated. ( G ) The end-point fluorescence was measured at 66°C and after raising the temperature to 75°C to dissociate unused primers. ( H ) Assay conditions in graphs A to G. The standard curves were generated from the end-point baseline-corrected fluorescence values. The assessments were performed in triplicates. Supplementary Figure S3B shows the effect of increasing EvaGreen concentration above 1.25 μM. Supplementary Figure S3C-D shows the effect of different DNA polymerase concentrations on the dTTP and dCTP quantification.

    Article Snippet: We found that the Q5 DNA polymerase, which we mainly used in this work, retains specificity in the presence of rNTP-to-dNTP ratios typically present in cultured cells.

    Techniques: Fluorescence, Generated, Concentration Assay