phi29 dna polymerase  (New England Biolabs)


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    Name:
    phi29 DNA Polymerase
    Description:
    phi29 DNA Polymerase 1 250 units
    Catalog Number:
    m0269l
    Price:
    228
    Size:
    1 250 units
    Category:
    DNA Polymerases
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    Structured Review

    New England Biolabs phi29 dna polymerase
    phi29 DNA Polymerase
    phi29 DNA Polymerase 1 250 units
    https://www.bioz.com/result/phi29 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase - by Bioz Stars, 2020-09
    99/100 stars

    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 "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

    3) 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, 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

    4) 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

    5) 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

    6) Product Images from "Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains"

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    Journal: Langmuir : the ACS journal of surfaces and colloids

    doi: 10.1021/acs.langmuir.8b01812

    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.
    Figure Legend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Techniques Used: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry

    7) Product Images from "Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes"

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13719-9

    Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.
    Figure Legend Snippet: Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.

    Techniques Used: Isolation, Mutagenesis, Amplification, Produced, Sequencing

    Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .
    Figure Legend Snippet: Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .

    Techniques Used: Whole Genome Amplification, Sequencing, Amplification, Ligation

    8) Product Images from "Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System"

    Article Title: Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System

    Journal: Chembiochem

    doi: 10.1002/cbic.201900299

    Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.
    Figure Legend Snippet: Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.

    Techniques Used: Plasmid Preparation, Fluorescence, Binding Assay, Incubation, Staining, Agarose Gel Electrophoresis

    9) Product Images from "DeNAno: selectable deoxyribonucleic acid nanoparticle libraries"

    Article Title: DeNAno: selectable deoxyribonucleic acid nanoparticle libraries

    Journal: Journal of biotechnology

    doi: 10.1016/j.jbiotec.2009.12.002

    DNA nanoparticle iterative selection scheme. ssDNA libraries are ligated with T4 ligase and polymerized with phi29 DNA polymerase. 3'–5' exonuclease activity of phi29 DNA polymerase ensures nanoparticle purity from extraneous DNA. Immature DCs
    Figure Legend Snippet: DNA nanoparticle iterative selection scheme. ssDNA libraries are ligated with T4 ligase and polymerized with phi29 DNA polymerase. 3'–5' exonuclease activity of phi29 DNA polymerase ensures nanoparticle purity from extraneous DNA. Immature DCs

    Techniques Used: Selection, Activity Assay

    10) Product Images from "Electrochemical Biosensors Combined with Isothermal Amplification for Quantitative Detection of Nucleic Acids"

    Article Title: Electrochemical Biosensors Combined with Isothermal Amplification for Quantitative Detection of Nucleic Acids

    Journal: Biosensors and Biodetection

    doi: 10.1007/978-1-4939-6911-1_10

    RCA-CC biosensor for microRNA detection. ( a ) Integrated gold electrodes were fabricated on polystyrene substrate by chemical plating. ( b ) Schematic illustration of three-gold electrode substrate. ( c ) After immobilization of DNA probe 1 on the working electrode, microRNA and DNA probe 2, which are partly complementary, hybridize to it, and then solid-phase RCA is initiated by phi29 DNA polymerase at 30 °C. If no hybridization takes place, the RCA reaction does not proceed
    Figure Legend Snippet: RCA-CC biosensor for microRNA detection. ( a ) Integrated gold electrodes were fabricated on polystyrene substrate by chemical plating. ( b ) Schematic illustration of three-gold electrode substrate. ( c ) After immobilization of DNA probe 1 on the working electrode, microRNA and DNA probe 2, which are partly complementary, hybridize to it, and then solid-phase RCA is initiated by phi29 DNA polymerase at 30 °C. If no hybridization takes place, the RCA reaction does not proceed

    Techniques Used: Hybridization

    11) 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

    12) 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

    13) 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

    Related Articles

    Amplification:

    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
    Article Snippet: .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. ..

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion
    Article Snippet: .. The resultant mixture (10 μM) was amplified by incubating at 30 °C for 16 h in a solution containing 2.5 U/μL phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM (NH4 )2 SO4 , 4 mM DTT, 200 μg/ml BSA, and 2.5 mM dNTP (Invitrogen, Carlsbad, CA, USA). .. Polypodna production by restriction digestion The highly viscous RCA product was incubated in 2 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C for 15 min to solubilize the product.

    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
    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. .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Mutagenesis:

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein
    Article Snippet: .. The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase. ..

    Purification:

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination
    Article Snippet: .. TE buffer: 10m M Tris–HCl [pH 8.0]; 0.1m M EDTA RAD51 buffer: 40m M Tris–HCl [pH 8.0]; 1m M MgCl2 ; 5m M CaCl2 ; 100m M KCl; 1m M DTT; 1m M ATP; 0.2 mgmL−1 BSA; 1m M Trolox (Sigma-Aldrich); 1.0% glucose (w/v); 500units catalase (Sigma-Aldrich); 70units glucose oxidase (Sigma-Aldrich) 10× T4 DNA ligase reaction buffer (B0202S; NEB) T4 DNA ligase (M0202; NEB) Primer oligo (/Biosg/TC TCC TCC TTC T—HPLC purified; Integrated DNA Technologies) Template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG—PAGE purified; Integrated DNA Technologies) Nuclease-free water BSA, Molecular Biology Grade (B9000S; NEB) Thermocycler (Mastercycler pro S; Eppendorf ) 10× phi29 DNA polymerase reaction buffer (B0269S; NEB) phi29 DNA polymerase (homemade 5 μ M stock) Deoxynucleotide (dNTP) solution set (N0446S; NEB) .. Prepare a 49 μL ligation reaction containing: (i) 5 μL 10× T4 ligase reaction buffer; (ii) 2 μL template oligo (10 μ M stock in TE buffer); (iii) 1.8 μL primer oligo (10 μ M stock in TE buffer); and (iv) 40.2 μL nuclease-free water.

    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
    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. .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication
    Article Snippet: .. Assay of DNA replication by purified phi29 DNA polymerase in a standard buffer 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). .. In the experiment shown in , the indicated concentration of NTP mixture (ATP:GTP:CTP:UTP:magnesium acetate = 3:2:1:1:7.2), E. coli tRNA mixture, or T7 RNA polymerase was added.

    Incubation:

    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
    Article Snippet: .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. ..

    other:

    Article Title: Effects of acetaldehyde-induced DNA lesions on DNA metabolism
    Article Snippet: Enzymes and chemicals Phi29 DNA polymerase, restriction enzymes (Mlu CI, Hae III, Msp I, Hha I) and 6x Gel loading Dye were purchased from New England Biolabs (NEB: Ipswich, MA, USA).

    Plasmid Preparation:

    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
    Article Snippet: .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. ..

    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
    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. .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication
    Article Snippet: .. Assay of DNA replication by purified phi29 DNA polymerase in a standard buffer 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). .. In the experiment shown in , the indicated concentration of NTP mixture (ATP:GTP:CTP:UTP:magnesium acetate = 3:2:1:1:7.2), E. coli tRNA mixture, or T7 RNA polymerase was added.

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    New England Biolabs phi29 polymerase
    Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using <t>Phi29</t> polymerase followed by electroporation into E.coli bacteria.
    Phi29 Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.

    Journal: Nucleic Acids Research

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions

    doi: 10.1093/nar/gkl605

    Figure Lengend Snippet: Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.

    Article Snippet: CONCLUSIONS Our results demonstrate that Phi29 polymerase can amplify ligation reactions in vitro without detectable biases, yielding sub-milligram quantities of DNA and potentially increasing the number of transformants in excess of 106 -fold.

    Techniques: Ligation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Electroporation

    In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.

    Journal: Nucleic Acids Research

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions

    doi: 10.1093/nar/gkl605

    Figure Lengend Snippet: In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.

    Article Snippet: CONCLUSIONS Our results demonstrate that Phi29 polymerase can amplify ligation reactions in vitro without detectable biases, yielding sub-milligram quantities of DNA and potentially increasing the number of transformants in excess of 106 -fold.

    Techniques: In Vitro, Amplification, Ligation, Recombinant

    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: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    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: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Techniques: Plasmid Preparation, Electroporation, Amplification

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkl350

    Figure Lengend 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.

    Article Snippet: The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase.

    Techniques: 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).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkl350

    Figure Lengend 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).

    Article Snippet: The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase.

    Techniques: Molecular Weight