phi29 dna polymerase  (New England Biolabs)


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    Name:
    PhiX174 Virion DNA
    Description:
    PhiX174 Virion DNA 250 ug
    Catalog Number:
    N3023L
    Price:
    312
    Size:
    250 ug
    Category:
    Genomic DNA
    Score:
    85
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    Structured Review

    New England Biolabs phi29 dna polymerase
    PhiX174 Virion DNA
    PhiX174 Virion DNA 250 ug
    https://www.bioz.com/result/phi29 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 0 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase - by Bioz Stars, 2019-10
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    Images

    1) Product Images from "A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples"

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01731

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Figure Legend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Techniques Used: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.
    Figure Legend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Techniques Used: Plasmid Preparation, Electroporation, Amplification

    2) Product Images from "Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA"

    Article Title: Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003361

    UNG inhibition decreases the replication activity of DHBV cccDNA in the presence of A3G expression. (A) RCA products from the cccDNAs. Expression vectors of A3G, UGI, and GFP were used for transfection of LMH cells together with the pCSD3.5ΔS replicon plasmid. After 7 days of cultivation, cccDNAs were purified from the nuclear fraction by Hirt extraction and treated with DpnI to digest transfected plasmids. The cccDNAs were amplified with phi29 DNA polymerase. The DHBV replicon plasmid (pCSD3.5ΔS) was also reacted as a control. RCA concatemeric products (indicated by an arrow) were digested with EcoRI and electrophoresed on agarose gel to verify successful amplification of the 3.0-kb full-length DHBV genomic DNA (left side). The 4.7-kb fragment represents the pCSD3.5 backbone (see Figure S3A for the plasmid construct). (B) qPCR analysis to assess replication activity of reconstructed replicon plasmids. The amplified full-length genomes from cccDNA were cloned into a pCSD3.5 backbone. Resulting reconstructed clones were used to transfect LMH cells without any other vectors (see Figure S5 for the experimental design). DHBV NC-DNA was purified and quantified 3 days later. The graph shows the relative DHBV DNA level; the level of GFP transfectants was set as 1. ***P
    Figure Legend Snippet: UNG inhibition decreases the replication activity of DHBV cccDNA in the presence of A3G expression. (A) RCA products from the cccDNAs. Expression vectors of A3G, UGI, and GFP were used for transfection of LMH cells together with the pCSD3.5ΔS replicon plasmid. After 7 days of cultivation, cccDNAs were purified from the nuclear fraction by Hirt extraction and treated with DpnI to digest transfected plasmids. The cccDNAs were amplified with phi29 DNA polymerase. The DHBV replicon plasmid (pCSD3.5ΔS) was also reacted as a control. RCA concatemeric products (indicated by an arrow) were digested with EcoRI and electrophoresed on agarose gel to verify successful amplification of the 3.0-kb full-length DHBV genomic DNA (left side). The 4.7-kb fragment represents the pCSD3.5 backbone (see Figure S3A for the plasmid construct). (B) qPCR analysis to assess replication activity of reconstructed replicon plasmids. The amplified full-length genomes from cccDNA were cloned into a pCSD3.5 backbone. Resulting reconstructed clones were used to transfect LMH cells without any other vectors (see Figure S5 for the experimental design). DHBV NC-DNA was purified and quantified 3 days later. The graph shows the relative DHBV DNA level; the level of GFP transfectants was set as 1. ***P

    Techniques Used: Inhibition, Activity Assay, Expressing, Transfection, Plasmid Preparation, Purification, Amplification, Agarose Gel Electrophoresis, Construct, Real-time Polymerase Chain Reaction, Clone Assay

    3) Product Images from "Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion"

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion

    Journal: Scientific Reports

    doi: 10.1038/srep14979

    Schematic diagram of the mass amplification of tripodna with adhesive 5′-ends. The template oligodeoxynucleotides (template 1) were designed to satisfy the following requirements: ( a ) the polypodna automatically forms by self-assembly; ( b ) each pod of the polypodna contains a 9 base long TspRI restriction digest site; ( c ) Each 5′-terminal end is phosphorylated in order to ligate with 3′-terminal within the polypodna body, and ( d ) connecting chain is added to the 3′-terminal of the polypodna to allow polypodna to be connected to one another. The designed templates were amplified via the following steps. ( 1 ) The template ssODNs were circularized using T4 DNA ligase. ( 2 ) After annealing the primer (primer 1), the DNA template was amplified through rolling circle amplification technique using Phi29 polymerase. ( 3 ) Before enzyme digestion, the RCA product was treated with EDTA and folded. ( 4 ) Long single-stranded DNAs were digested using restriction enzyme. ( 5 ) The target sequences were purified by size chromatography. ( 6 ) The resultant DNAs self-assembled after annealing, and they formed a hydrogel.
    Figure Legend Snippet: Schematic diagram of the mass amplification of tripodna with adhesive 5′-ends. The template oligodeoxynucleotides (template 1) were designed to satisfy the following requirements: ( a ) the polypodna automatically forms by self-assembly; ( b ) each pod of the polypodna contains a 9 base long TspRI restriction digest site; ( c ) Each 5′-terminal end is phosphorylated in order to ligate with 3′-terminal within the polypodna body, and ( d ) connecting chain is added to the 3′-terminal of the polypodna to allow polypodna to be connected to one another. The designed templates were amplified via the following steps. ( 1 ) The template ssODNs were circularized using T4 DNA ligase. ( 2 ) After annealing the primer (primer 1), the DNA template was amplified through rolling circle amplification technique using Phi29 polymerase. ( 3 ) Before enzyme digestion, the RCA product was treated with EDTA and folded. ( 4 ) Long single-stranded DNAs were digested using restriction enzyme. ( 5 ) The target sequences were purified by size chromatography. ( 6 ) The resultant DNAs self-assembled after annealing, and they formed a hydrogel.

    Techniques Used: Amplification, Purification, Chromatography

    4) Product Images from "Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e"

    Article Title: Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e

    Journal: Chemical Science

    doi: 10.1039/c8sc03121e

    Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.
    Figure Legend Snippet: Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.

    Techniques Used: Amplification, Incubation, Labeling

    5) Product Images from "Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein"

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl350

    Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.
    Figure Legend Snippet: Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Techniques Used: Hybridization

    Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).
    Figure Legend Snippet: Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Techniques Used: Molecular Weight

    6) Product Images from "A transcription and translation-coupled DNA replication system using rolling-circle replication"

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    Journal: Scientific Reports

    doi: 10.1038/srep10404

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.
    Figure Legend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Autoradiography, Marker, Purification

    Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.
    Figure Legend Snippet: Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Techniques Used: Synthesized, Incubation, SDS Page, Autoradiography, Produced

    DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.
    Figure Legend Snippet: DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Techniques Used: Purification, Plasmid Preparation

    Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.
    Figure Legend Snippet: Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Techniques Used: Incubation, Sequencing

    7) Product Images from "Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein"

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl350

    Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.
    Figure Legend Snippet: Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Techniques Used: Hybridization

    Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).
    Figure Legend Snippet: Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Techniques Used: Molecular Weight

    Related Articles

    Diagnostic Assay:

    Article Title: hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity
    Article Snippet: Clones of the correct orientation were selected following a diagnostic digest with Eco RV. .. Virion phiX174 was supplied by New England Biolabs.

    Clone Assay:

    Article Title: hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity
    Article Snippet: Clones of the correct orientation were selected following a diagnostic digest with Eco RV. .. Virion phiX174 was supplied by New England Biolabs.

    Expressing:

    Article Title: hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity
    Article Snippet: Full-length hSSB1 and truncations were cloned into bacterial expression vectors encoding a His-tag (pET28c). .. Virion phiX174 was supplied by New England Biolabs.

    Synthesized:

    Article Title: In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Article Snippet: By doing so, one can specifically monitor leading or lagging strand synthesis depending on the radioactive deoxyribonucleoside triphosphate (dNTP) used in the assay. .. Razor blade 1.57 mm OD polyethylene tubing ( e.g ., Clay Adams® Intramedic® , BD, catalog number: 427431) Sephadex microcentrifuge columns (Illustra Microspin G-25) (GE Healthcare, catalog number: 27-5325-01) Plastic wrap ( e.g. , Fisherbrand Clear Plastic Wrap, Fisher Scientific, catalog number: 22-305654) C-fold paper towels ( e.g. , Scott paper towels, KCWW, Kimberly-Clark, catalog number: 01510) Positively charged nylon DNA blotting membrane (Hybond-N+, 30.0×50.0 cm) (GE Healthcare, catalog number: RPN3050B) Chromatography transfer paper (Whatman 3MM, 46.0×57.0 cm) (GE Healthcare, catalog number: 3030-917) Syringe tip ( e.g ., B-D 18 G 1 ½ PrecisionGlide® Needle) (BD, catalog number: 305196) phiX174 virion DNA, 1 mg/ml (New England Biolabs, catalog number: N3023L) Phi29 DNA polymerase (New England Biolabs, catalog number: M0269S) 100 mM dNTP (deoxynucleotide triphosphate) set (Thermo Fisher Scientific, Thermo Scientific™, catalog number: R0181) 1 µM CMG ( C dc45 M cm2–7 G ins) helicase (see for purification details) pUC19, 1 mg/ml (New England Biolabs, catalog number: N3041L) Bsa I-HF with CutSmart buffer (New England Biolabs, catalog number: R3535L) ‘blockLd’ oligo* ‘blockLg’ oligo* ‘Pr1B’ oligo* ‘160Ld’ oligo* ‘91Lg’ oligo* ‘Fork primer’ oligo* Nucleotide-biased template (synthesized by Biomatik, Wilmington DE)* *Note: See . .. T4 ligase, including 10× ligase buffer (New England Biolabs, catalog number: M0202M) 100 mM ATP (GE Healthcare, catalog number: 27-2056-01) 0.5 M EDTA, disodium salt (Sigma-Aldrich, catalog number: E5134) 5 M NaCl (Sigma-Aldrich, catalog number: S9888) Sepharose 4B size exclusion chromatography resin (GE Healthcare, catalog number: 17012001) 1 kb MW marker (New England Biolabs, catalog number: N3232L) Ethidium bromide (EthBr, 10 mg/ml) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15585011) T4 kinase and 10× T4 kinase buffer (New England Biolabs, catalog number: M0201L) 32 P-γ-ATP, 3,000 Ci/mmol, 3.3 µM (PerkinElmer, catalog number: BLU002A) Type XI low-melt agarose (Sigma-Aldrich, catalog number: A3038) Note: This product has been discontinued .

    Blocking Assay:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: Commercial RFI ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L) (see ). .. Commercial RFI ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L) (see ).

    Real-time Polymerase Chain Reaction:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. DNA was isolated from four replicates as in , except that the pH of the phenol solution was adjusted to 8 using Tris alkaline buffer and purified DNA was diluted 1:1000.

    Incubation:

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
    Article Snippet: Assay of Endo IV activity was performed by incubation of enzyme and substrate for 30 min at 37°C in a reaction mixture (20 μl) containing 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 1 mM DTT and BSA (0.1 mg/ml). .. Purified Endo IV was diluted in a solution containing 10 mM Tris–HCl (pH 8.0), 1 mM DTT and BSA (0.1 mg/ml) and was included in the reaction mixture in a volume of 1 μl. phiX174 replicative form I (RFI) circular dsDNA (New England BioLabs), phiX174 RFI linear dsDNA prepared by PstI digestion, phiX174 virion circular ssDNA (New England BioLabs), linear ssRNA used for synthesis of the GST-Endo IV fusion protein, and heat-denatured T4 or T4dC genomic dsDNA were used as substrates at a final concentration of 5 μg/ml.

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: Alignments of deduced amino acid sequence were generated with DNAMAN program. .. To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. .. Six hours later, 10 μl of the reaction solution was analyzed by electrophoresis on a 1.0 % agarose gel.

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S). .. Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S).

    Activity Assay:

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
    Article Snippet: Assay of Endo IV activity was performed by incubation of enzyme and substrate for 30 min at 37°C in a reaction mixture (20 μl) containing 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 1 mM DTT and BSA (0.1 mg/ml). .. Purified Endo IV was diluted in a solution containing 10 mM Tris–HCl (pH 8.0), 1 mM DTT and BSA (0.1 mg/ml) and was included in the reaction mixture in a volume of 1 μl. phiX174 replicative form I (RFI) circular dsDNA (New England BioLabs), phiX174 RFI linear dsDNA prepared by PstI digestion, phiX174 virion circular ssDNA (New England BioLabs), linear ssRNA used for synthesis of the GST-Endo IV fusion protein, and heat-denatured T4 or T4dC genomic dsDNA were used as substrates at a final concentration of 5 μg/ml.

    Cell Culture:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: PCC 7002 (WT and ΔA2542) were cultured overnight in 20 ml media A+ in bubble tubes and diluted to an OD730 nm = 0.01 in 150 ml media A+ in 250 ml pyrex bottle with glass rod for bubbling (in quintuple). .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    Shift Assay:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Paragraph title: Degradation assay, nuclei spin down assay, and phosphorylation shift assay ... Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S).

    Recombinase Polymerase Amplification:

    Article Title: In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Article Snippet: Razor blade 1.57 mm OD polyethylene tubing ( e.g ., Clay Adams® Intramedic® , BD, catalog number: 427431) Sephadex microcentrifuge columns (Illustra Microspin G-25) (GE Healthcare, catalog number: 27-5325-01) Plastic wrap ( e.g. , Fisherbrand Clear Plastic Wrap, Fisher Scientific, catalog number: 22-305654) C-fold paper towels ( e.g. , Scott paper towels, KCWW, Kimberly-Clark, catalog number: 01510) Positively charged nylon DNA blotting membrane (Hybond-N+, 30.0×50.0 cm) (GE Healthcare, catalog number: RPN3050B) Chromatography transfer paper (Whatman 3MM, 46.0×57.0 cm) (GE Healthcare, catalog number: 3030-917) Syringe tip ( e.g ., B-D 18 G 1 ½ PrecisionGlide® Needle) (BD, catalog number: 305196) phiX174 virion DNA, 1 mg/ml (New England Biolabs, catalog number: N3023L) Phi29 DNA polymerase (New England Biolabs, catalog number: M0269S) 100 mM dNTP (deoxynucleotide triphosphate) set (Thermo Fisher Scientific, Thermo Scientific™, catalog number: R0181) 1 µM CMG ( C dc45 M cm2–7 G ins) helicase (see for purification details) pUC19, 1 mg/ml (New England Biolabs, catalog number: N3041L) Bsa I-HF with CutSmart buffer (New England Biolabs, catalog number: R3535L) ‘blockLd’ oligo* ‘blockLg’ oligo* ‘Pr1B’ oligo* ‘160Ld’ oligo* ‘91Lg’ oligo* ‘Fork primer’ oligo* Nucleotide-biased template (synthesized by Biomatik, Wilmington DE)* *Note: See . .. T4 ligase, including 10× ligase buffer (New England Biolabs, catalog number: M0202M) 100 mM ATP (GE Healthcare, catalog number: 27-2056-01) 0.5 M EDTA, disodium salt (Sigma-Aldrich, catalog number: E5134) 5 M NaCl (Sigma-Aldrich, catalog number: S9888) Sepharose 4B size exclusion chromatography resin (GE Healthcare, catalog number: 17012001) 1 kb MW marker (New England Biolabs, catalog number: N3232L) Ethidium bromide (EthBr, 10 mg/ml) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15585011) T4 kinase and 10× T4 kinase buffer (New England Biolabs, catalog number: M0201L) 32 P-γ-ATP, 3,000 Ci/mmol, 3.3 µM (PerkinElmer, catalog number: BLU002A) Type XI low-melt agarose (Sigma-Aldrich, catalog number: A3038) Note: This product has been discontinued .

    Spin Down Assay:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Paragraph title: Degradation assay, nuclei spin down assay, and phosphorylation shift assay ... Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S).

    Chromatography:

    Article Title: In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Article Snippet: By doing so, one can specifically monitor leading or lagging strand synthesis depending on the radioactive deoxyribonucleoside triphosphate (dNTP) used in the assay. .. Razor blade 1.57 mm OD polyethylene tubing ( e.g ., Clay Adams® Intramedic® , BD, catalog number: 427431) Sephadex microcentrifuge columns (Illustra Microspin G-25) (GE Healthcare, catalog number: 27-5325-01) Plastic wrap ( e.g. , Fisherbrand Clear Plastic Wrap, Fisher Scientific, catalog number: 22-305654) C-fold paper towels ( e.g. , Scott paper towels, KCWW, Kimberly-Clark, catalog number: 01510) Positively charged nylon DNA blotting membrane (Hybond-N+, 30.0×50.0 cm) (GE Healthcare, catalog number: RPN3050B) Chromatography transfer paper (Whatman 3MM, 46.0×57.0 cm) (GE Healthcare, catalog number: 3030-917) Syringe tip ( e.g ., B-D 18 G 1 ½ PrecisionGlide® Needle) (BD, catalog number: 305196) phiX174 virion DNA, 1 mg/ml (New England Biolabs, catalog number: N3023L) Phi29 DNA polymerase (New England Biolabs, catalog number: M0269S) 100 mM dNTP (deoxynucleotide triphosphate) set (Thermo Fisher Scientific, Thermo Scientific™, catalog number: R0181) 1 µM CMG ( C dc45 M cm2–7 G ins) helicase (see for purification details) pUC19, 1 mg/ml (New England Biolabs, catalog number: N3041L) Bsa I-HF with CutSmart buffer (New England Biolabs, catalog number: R3535L) ‘blockLd’ oligo* ‘blockLg’ oligo* ‘Pr1B’ oligo* ‘160Ld’ oligo* ‘91Lg’ oligo* ‘Fork primer’ oligo* Nucleotide-biased template (synthesized by Biomatik, Wilmington DE)* *Note: See . .. T4 ligase, including 10× ligase buffer (New England Biolabs, catalog number: M0202M) 100 mM ATP (GE Healthcare, catalog number: 27-2056-01) 0.5 M EDTA, disodium salt (Sigma-Aldrich, catalog number: E5134) 5 M NaCl (Sigma-Aldrich, catalog number: S9888) Sepharose 4B size exclusion chromatography resin (GE Healthcare, catalog number: 17012001) 1 kb MW marker (New England Biolabs, catalog number: N3232L) Ethidium bromide (EthBr, 10 mg/ml) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15585011) T4 kinase and 10× T4 kinase buffer (New England Biolabs, catalog number: M0201L) 32 P-γ-ATP, 3,000 Ci/mmol, 3.3 µM (PerkinElmer, catalog number: BLU002A) Type XI low-melt agarose (Sigma-Aldrich, catalog number: A3038) Note: This product has been discontinued .

    Atomic Absorption Spectroscopy:

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form. .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Dissection:

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues
    Article Snippet: Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA). .. Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA).

    Infection:

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues
    Article Snippet: Probes and accession annotations are available in the Gene Expression Omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ). .. Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA). .. Plasmid minipreps were prepared from pBR322 subclones carrying JC or BK polyomavirus genomes (J. C. Alwine, University of Pennsylvania, Philadelphia, PA) and from pUC19 carrying the human papillomavirus 16 (HPV16) genome (obtained from Peter Howley, Harvard Medical School, Boston, MA).

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease
    Article Snippet: M13mp18 replicative form (RF) was purified from infected E. coli UT481 [Δ( lac-pro ) hsdS (r− m− ) lacIq lacZ ] cells using the Qiagen maxi plasmid kit. .. Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Sequencing:

    Article Title: Designing a Bio-responsive Robot from DNA Origami
    Article Snippet: Paragraph title: 8. Choose Scaffold Sequence ... Another option is to use pre-digested ssDNA, such as the phiX174 virion ssDNA HaeIII digest, available from New England Biolabs.

    Binding Assay:

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: MutL protein was a gift from F. J. Lopez de Saro and M. ODonnell (The Rockefeller University, New York) and MutL and single-strand binding protein were obtained from United States Biological (Cleveland, OH). .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Radioactivity:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L). .. Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L).

    Isolation:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    Labeling:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Degradation assays were performed as previously described [ ] with the following modifications. .. Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S). .. The reactions were analyzed by PhosphorImager and quantitation was performed using ImageQuant™ software (Molecular Dynamics).

    Purification:

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
    Article Snippet: Assay of Endo IV activity was performed by incubation of enzyme and substrate for 30 min at 37°C in a reaction mixture (20 μl) containing 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 1 mM DTT and BSA (0.1 mg/ml). .. Purified Endo IV was diluted in a solution containing 10 mM Tris–HCl (pH 8.0), 1 mM DTT and BSA (0.1 mg/ml) and was included in the reaction mixture in a volume of 1 μl. phiX174 replicative form I (RFI) circular dsDNA (New England BioLabs), phiX174 RFI linear dsDNA prepared by PstI digestion, phiX174 virion circular ssDNA (New England BioLabs), linear ssRNA used for synthesis of the GST-Endo IV fusion protein, and heat-denatured T4 or T4dC genomic dsDNA were used as substrates at a final concentration of 5 μg/ml. .. The reaction was stopped by the addition of 20 μl of 25 mM EDTA (pH 8.0), and the reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold (Invitrogen).

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: Alignments of deduced amino acid sequence were generated with DNAMAN program. .. To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. .. Six hours later, 10 μl of the reaction solution was analyzed by electrophoresis on a 1.0 % agarose gel.

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues
    Article Snippet: Probes and accession annotations are available in the Gene Expression Omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ). .. Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA). .. Plasmid minipreps were prepared from pBR322 subclones carrying JC or BK polyomavirus genomes (J. C. Alwine, University of Pennsylvania, Philadelphia, PA) and from pUC19 carrying the human papillomavirus 16 (HPV16) genome (obtained from Peter Howley, Harvard Medical School, Boston, MA).

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease
    Article Snippet: M13mp18 ssDNA was purified from M13 phage-infected UT481 cells according to standard protocols. .. Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: MutSΔ680 protein was purified as described ( ). .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Article Title: In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Article Snippet: By doing so, one can specifically monitor leading or lagging strand synthesis depending on the radioactive deoxyribonucleoside triphosphate (dNTP) used in the assay. .. Razor blade 1.57 mm OD polyethylene tubing ( e.g ., Clay Adams® Intramedic® , BD, catalog number: 427431) Sephadex microcentrifuge columns (Illustra Microspin G-25) (GE Healthcare, catalog number: 27-5325-01) Plastic wrap ( e.g. , Fisherbrand Clear Plastic Wrap, Fisher Scientific, catalog number: 22-305654) C-fold paper towels ( e.g. , Scott paper towels, KCWW, Kimberly-Clark, catalog number: 01510) Positively charged nylon DNA blotting membrane (Hybond-N+, 30.0×50.0 cm) (GE Healthcare, catalog number: RPN3050B) Chromatography transfer paper (Whatman 3MM, 46.0×57.0 cm) (GE Healthcare, catalog number: 3030-917) Syringe tip ( e.g ., B-D 18 G 1 ½ PrecisionGlide® Needle) (BD, catalog number: 305196) phiX174 virion DNA, 1 mg/ml (New England Biolabs, catalog number: N3023L) Phi29 DNA polymerase (New England Biolabs, catalog number: M0269S) 100 mM dNTP (deoxynucleotide triphosphate) set (Thermo Fisher Scientific, Thermo Scientific™, catalog number: R0181) 1 µM CMG ( C dc45 M cm2–7 G ins) helicase (see for purification details) pUC19, 1 mg/ml (New England Biolabs, catalog number: N3041L) Bsa I-HF with CutSmart buffer (New England Biolabs, catalog number: R3535L) ‘blockLd’ oligo* ‘blockLg’ oligo* ‘Pr1B’ oligo* ‘160Ld’ oligo* ‘91Lg’ oligo* ‘Fork primer’ oligo* Nucleotide-biased template (synthesized by Biomatik, Wilmington DE)* *Note: See . .. T4 ligase, including 10× ligase buffer (New England Biolabs, catalog number: M0202M) 100 mM ATP (GE Healthcare, catalog number: 27-2056-01) 0.5 M EDTA, disodium salt (Sigma-Aldrich, catalog number: E5134) 5 M NaCl (Sigma-Aldrich, catalog number: S9888) Sepharose 4B size exclusion chromatography resin (GE Healthcare, catalog number: 17012001) 1 kb MW marker (New England Biolabs, catalog number: N3232L) Ethidium bromide (EthBr, 10 mg/ml) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15585011) T4 kinase and 10× T4 kinase buffer (New England Biolabs, catalog number: M0201L) 32 P-γ-ATP, 3,000 Ci/mmol, 3.3 µM (PerkinElmer, catalog number: BLU002A) Type XI low-melt agarose (Sigma-Aldrich, catalog number: A3038) Note: This product has been discontinued .

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    Degradation Assay:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Paragraph title: Degradation assay, nuclei spin down assay, and phosphorylation shift assay ... Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S).

    Periodic Counter-current Chromatography:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: PCC 7002 (WT and ΔA2542) were cultured overnight in 20 ml media A+ in bubble tubes and diluted to an OD730 nm = 0.01 in 150 ml media A+ in 250 ml pyrex bottle with glass rod for bubbling (in quintuple). .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    ATPase Assay:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: ATPase assay buffer: 66 mM Tris-HCl, pH 7.5, 26 mM MgCl2 , 3.6 mM DTT, 2 mM ATP, 180 μg/ml BSA. .. Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L).

    SDS Page:

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form. .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Plasmid Preparation:

    Article Title: hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity
    Article Snippet: The HisRad50 was excised from the pVL1392 vector (TP11) (gift from Tanya Paull) using EcoR1 restriction enzyme and then cloned directly into an Eco R1 digested pFastBac1 vector. .. Virion phiX174 was supplied by New England Biolabs.

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: Alignments of deduced amino acid sequence were generated with DNAMAN program. .. To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. .. Six hours later, 10 μl of the reaction solution was analyzed by electrophoresis on a 1.0 % agarose gel.

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease
    Article Snippet: M13mp18 replicative form (RF) was purified from infected E. coli UT481 [Δ( lac-pro ) hsdS (r− m− ) lacIq lacZ ] cells using the Qiagen maxi plasmid kit. .. Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: Paragraph title: Plasmid copy number quantification ... A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    Recombinant:

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: Alignments of deduced amino acid sequence were generated with DNAMAN program. .. To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. .. Six hours later, 10 μl of the reaction solution was analyzed by electrophoresis on a 1.0 % agarose gel.

    Sample Prep:

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues
    Article Snippet: Paragraph title: Sample preparation. ... Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA).

    Ethanol Precipitation:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: Supernatant was removed, and samples were stored at –80°C until processing (10 days). .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. The spike-in was added immediately after addition of the TE (with lysozyme) to the frozen cell pellets at a concentration of 10 copies per cell.

    Concentration Assay:

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
    Article Snippet: Assay of Endo IV activity was performed by incubation of enzyme and substrate for 30 min at 37°C in a reaction mixture (20 μl) containing 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 1 mM DTT and BSA (0.1 mg/ml). .. Purified Endo IV was diluted in a solution containing 10 mM Tris–HCl (pH 8.0), 1 mM DTT and BSA (0.1 mg/ml) and was included in the reaction mixture in a volume of 1 μl. phiX174 replicative form I (RFI) circular dsDNA (New England BioLabs), phiX174 RFI linear dsDNA prepared by PstI digestion, phiX174 virion circular ssDNA (New England BioLabs), linear ssRNA used for synthesis of the GST-Endo IV fusion protein, and heat-denatured T4 or T4dC genomic dsDNA were used as substrates at a final concentration of 5 μg/ml. .. The reaction was stopped by the addition of 20 μl of 25 mM EDTA (pH 8.0), and the reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold (Invitrogen).

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: The concentration of RecA was determined by the ninhydrin protein assay ( ). .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Thin Layer Chromatography:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: Paragraph title: 2.2.2. Thin-layer chromatography (TLC) method ... Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L).

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    New England Biolabs phi29 dna polymerase
    Transcription- and translation-coupled <t>DNA</t> (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding <t>phi29</t> DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.
    Phi29 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 74/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/New England Biolabs
    Average 74 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase - by Bioz Stars, 2019-10
    74/100 stars
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    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Article Snippet: The standard reaction buffer contained the template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each), [32 P]-dCTP (3.3 μM), phi29 Tris-HCl (50 mM, pH 7.8), magnesium chloride (5 mM), potassium chloride (7.5 mM), dithiothreitol (0.1 mM), and purified phi29 DNA polymerase (1 U/μl, NEB).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Autoradiography, Marker, Purification

    Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Article Snippet: The standard reaction buffer contained the template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each), [32 P]-dCTP (3.3 μM), phi29 Tris-HCl (50 mM, pH 7.8), magnesium chloride (5 mM), potassium chloride (7.5 mM), dithiothreitol (0.1 mM), and purified phi29 DNA polymerase (1 U/μl, NEB).

    Techniques: Synthesized, Incubation, SDS Page, Autoradiography, Produced

    DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Article Snippet: The standard reaction buffer contained the template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each), [32 P]-dCTP (3.3 μM), phi29 Tris-HCl (50 mM, pH 7.8), magnesium chloride (5 mM), potassium chloride (7.5 mM), dithiothreitol (0.1 mM), and purified phi29 DNA polymerase (1 U/μl, NEB).

    Techniques: Purification, Plasmid Preparation

    Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Article Snippet: The standard reaction buffer contained the template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each), [32 P]-dCTP (3.3 μM), phi29 Tris-HCl (50 mM, pH 7.8), magnesium chloride (5 mM), potassium chloride (7.5 mM), dithiothreitol (0.1 mM), and purified phi29 DNA polymerase (1 U/μl, NEB).

    Techniques: Incubation, Sequencing

    DNA polymerase error preferences. Heat maps reflect nucleotide substitution and single nucleotide deletion preferences of ( A ) Dpo4, ( B ) Taq, ( C ) Sequenase 2.0, ( D ) AMV RT and ( E ) Phi29 copying in ‘T’, ‘A’, ‘C’ and ‘G’ template contexts. Error fraction was determined by normalizing individual error subtype frequencies to the total error rate measured at the lowest [dRTP] tested (10 −7 μM) for each template context. Values are the average of two experiments.

    Journal: Nucleic Acids Research

    Article Title: High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing

    doi: 10.1093/nar/gky296

    Figure Lengend Snippet: DNA polymerase error preferences. Heat maps reflect nucleotide substitution and single nucleotide deletion preferences of ( A ) Dpo4, ( B ) Taq, ( C ) Sequenase 2.0, ( D ) AMV RT and ( E ) Phi29 copying in ‘T’, ‘A’, ‘C’ and ‘G’ template contexts. Error fraction was determined by normalizing individual error subtype frequencies to the total error rate measured at the lowest [dRTP] tested (10 −7 μM) for each template context. Values are the average of two experiments.

    Article Snippet: Purified Taq polymerase, Avian Myeloblastosis Virus (AMV) RT, Phi29 and Sulfolobus islandicus Dpo4 were purchased through New England Biolabs.

    Techniques:

    MagNIFi assay validation. ( A ) Fidelity dose response curves of Sequenase 2.0, AMV RT, Phi29, Dpo4 and Taq copying in ‘T’ template contexts. Values are the average of two experiments. Standard deviation error bars ( n = 2) are smaller than data points. Curves show qualitative agreement between DNA polymerase FC 50 values (indicated by black dotted line) and the expected rank order of natural DNA polymerase error rates ( Supplementary Table S4 ). In general, fidelity increases from right to left. ( B ) Calibration between error rate and [FC 50 ]. We show a calibration curve relating multiple reported error rates per DNA polymerase ( Supplementary Table S4 ) and the average FC 50 value of each DNA polymerase ( Supplementary Table S3 ). Nonlinear fitting on a log–log plot (line of best fit in grey) revealed the following equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$y = 10^{(2.063log(x) + 1.557)}$\end{document} , RMSE = 0.0008998 errors/bp.

    Journal: Nucleic Acids Research

    Article Title: High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing

    doi: 10.1093/nar/gky296

    Figure Lengend Snippet: MagNIFi assay validation. ( A ) Fidelity dose response curves of Sequenase 2.0, AMV RT, Phi29, Dpo4 and Taq copying in ‘T’ template contexts. Values are the average of two experiments. Standard deviation error bars ( n = 2) are smaller than data points. Curves show qualitative agreement between DNA polymerase FC 50 values (indicated by black dotted line) and the expected rank order of natural DNA polymerase error rates ( Supplementary Table S4 ). In general, fidelity increases from right to left. ( B ) Calibration between error rate and [FC 50 ]. We show a calibration curve relating multiple reported error rates per DNA polymerase ( Supplementary Table S4 ) and the average FC 50 value of each DNA polymerase ( Supplementary Table S3 ). Nonlinear fitting on a log–log plot (line of best fit in grey) revealed the following equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$y = 10^{(2.063log(x) + 1.557)}$\end{document} , RMSE = 0.0008998 errors/bp.

    Article Snippet: Purified Taq polymerase, Avian Myeloblastosis Virus (AMV) RT, Phi29 and Sulfolobus islandicus Dpo4 were purchased through New England Biolabs.

    Techniques: Standard Deviation, Flow Cytometry

    Assessing MagNIFi assay potential. ( A ) Simulation of the minimum number of sequencing reads required to accurately measure different error rates. Black line denotes a CV of 15%. ( B ) Sensitivity of FC 50 values to sampling error and DNA polymerase stochasticity. Distribution of calculated FC 50 values for 1000 simulated rare base titration experiments, with each rare base condition receiving 50 simulated reads. Each condition was simulated by drawing 50 samples from a Bernoulli process with an underlying error rate equal to the experimentally derived error rate. FC 50 values were determined using the fitting procedure described in Methods. Histograms are shown for simulations based on Dpo4 (blue) and Phi29 (pink) error rates in a ‘T’ template context. ( C ) Determination of MagNIFi assay sample variability across different read counts. Error rate data collected for Dpo4 copying in all four template contexts reveal that error rate differences between 36 sets of biological replicates ( n = 2) do not vary with read count differences between the same set of biological replicates. ( D ) Resolving power of the MagNIFi assay based on FC 50 sensitivity. Minimum detectable fold change in FC 50 was determined based on the 95% confidence interval of the fitted FC 50 (see Supplementary Table S3 for values). The ratio of the Upper Bound 95% CI: Fitted FC 50 was determined for Dpo4 copying in ‘T’, ‘A’, ‘C’ and ‘G’ template contexts.

    Journal: Nucleic Acids Research

    Article Title: High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing

    doi: 10.1093/nar/gky296

    Figure Lengend Snippet: Assessing MagNIFi assay potential. ( A ) Simulation of the minimum number of sequencing reads required to accurately measure different error rates. Black line denotes a CV of 15%. ( B ) Sensitivity of FC 50 values to sampling error and DNA polymerase stochasticity. Distribution of calculated FC 50 values for 1000 simulated rare base titration experiments, with each rare base condition receiving 50 simulated reads. Each condition was simulated by drawing 50 samples from a Bernoulli process with an underlying error rate equal to the experimentally derived error rate. FC 50 values were determined using the fitting procedure described in Methods. Histograms are shown for simulations based on Dpo4 (blue) and Phi29 (pink) error rates in a ‘T’ template context. ( C ) Determination of MagNIFi assay sample variability across different read counts. Error rate data collected for Dpo4 copying in all four template contexts reveal that error rate differences between 36 sets of biological replicates ( n = 2) do not vary with read count differences between the same set of biological replicates. ( D ) Resolving power of the MagNIFi assay based on FC 50 sensitivity. Minimum detectable fold change in FC 50 was determined based on the 95% confidence interval of the fitted FC 50 (see Supplementary Table S3 for values). The ratio of the Upper Bound 95% CI: Fitted FC 50 was determined for Dpo4 copying in ‘T’, ‘A’, ‘C’ and ‘G’ template contexts.

    Article Snippet: Purified Taq polymerase, Avian Myeloblastosis Virus (AMV) RT, Phi29 and Sulfolobus islandicus Dpo4 were purchased through New England Biolabs.

    Techniques: Sequencing, Flow Cytometry, Sampling, Titration, Derivative Assay

    Local sequence context effects on DNA polymerase fidelity. ( A ) The identity and position of template bases neighboring a ‘T’ EES impact the measured FC 50 of Phi29 copying in a VVVTVVV template context. Change in logFC 50 (logFC 50 _Average – logFC 50 _Fixed Template Base) was calculated for each base identity/position. Positive ΔlogFC 50 values pertain to an increase in fidelity whereas negative values signify a decrease in fidelity. ( B ) The identity and position of template bases flanking ‘C’ and ‘G’ EESs impact the % total error Phi29 creates at 10 −7 μM dRTP when copying in DDDCDDD and HHHGHHH template contexts, respectively. A gray dotted line represents the average % total error made by Phi29 in a given context. ( C ) Base identity at the +1 template position in DDDCDDD, VVVTVVV, and BBBABBB contexts impacts the distribution of Dpo4 error preferences (nucleotide substitutions and single nucleotide deletions). Error preference was determined by normalizing error subtype frequency to the total error rate measured at 10 −7 μM dRTP. For all graphs in (A)–(C), values represent the average of two experiments. Error bars signify standard deviation ( n = 2).

    Journal: Nucleic Acids Research

    Article Title: High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing

    doi: 10.1093/nar/gky296

    Figure Lengend Snippet: Local sequence context effects on DNA polymerase fidelity. ( A ) The identity and position of template bases neighboring a ‘T’ EES impact the measured FC 50 of Phi29 copying in a VVVTVVV template context. Change in logFC 50 (logFC 50 _Average – logFC 50 _Fixed Template Base) was calculated for each base identity/position. Positive ΔlogFC 50 values pertain to an increase in fidelity whereas negative values signify a decrease in fidelity. ( B ) The identity and position of template bases flanking ‘C’ and ‘G’ EESs impact the % total error Phi29 creates at 10 −7 μM dRTP when copying in DDDCDDD and HHHGHHH template contexts, respectively. A gray dotted line represents the average % total error made by Phi29 in a given context. ( C ) Base identity at the +1 template position in DDDCDDD, VVVTVVV, and BBBABBB contexts impacts the distribution of Dpo4 error preferences (nucleotide substitutions and single nucleotide deletions). Error preference was determined by normalizing error subtype frequency to the total error rate measured at 10 −7 μM dRTP. For all graphs in (A)–(C), values represent the average of two experiments. Error bars signify standard deviation ( n = 2).

    Article Snippet: Purified Taq polymerase, Avian Myeloblastosis Virus (AMV) RT, Phi29 and Sulfolobus islandicus Dpo4 were purchased through New England Biolabs.

    Techniques: Sequencing, Flow Cytometry, Standard Deviation

    Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Plasmid Preparation, Isolation, Clone Assay, Transformation Assay, Amplification, Marker, Functional Assay

    (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Polymerase Chain Reaction, Clone Assay, Random Primed, Amplification, Agarose Gel Electrophoresis, Diagnostic Assay, Plasmid Preparation

    ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Southern Blot, Sequencing, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Diagnostic Assay, Amplification, BAC Assay

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Article Snippet: Plasmid DNA was amplified by adding 1 μL of 10 μM Exo-Resistant Random Primer (Thermo Scientific), 2 μL phi29 DNA Polymerase Reaction Buffer (New England Biolabs) and 8.2 μL of MilliQ water to 5 μL of the purified treated DNA.

    Techniques: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Article Snippet: Plasmid DNA was amplified by adding 1 μL of 10 μM Exo-Resistant Random Primer (Thermo Scientific), 2 μL phi29 DNA Polymerase Reaction Buffer (New England Biolabs) and 8.2 μL of MilliQ water to 5 μL of the purified treated DNA.

    Techniques: Plasmid Preparation, Electroporation, Amplification