phi29 dna polymerase  (New England Biolabs)


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    Structured Review

    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: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) 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

    Related Articles

    Concentration Assay:

    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.

    Plasmid Preparation:

    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.

    Purification:

    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 phi 29 polymerase
    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 <t>phi-29</t> 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.
    Phi 29 Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 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

    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