phi29  (New England Biolabs)


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

    New England Biolabs phi29
    phi29 DNA Polymerase
    phi29 DNA Polymerase 1 250 units
    https://www.bioz.com/result/phi29/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phi29 - by Bioz Stars, 2021-03
    98/100 stars

    Images

    1) Product Images from "High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky296

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: Standard Deviation

    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.
    Figure Legend 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.

    Techniques Used: Sequencing, 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).
    Figure Legend 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).

    Techniques Used: Sequencing, Standard Deviation

    Related Articles

    Ligation:

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions
    Article Snippet: The off-rates for these single antibody domains are therefore comparable to those reported for single chain Fv fragments isolated from a large repertoire ( > 1010 clones) ( ). .. 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. ..

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions
    Article Snippet: A dilution series was prepared from the ligation reaction and used as a template for Phi29 amplification. .. Hexamer primers and Phi29 polymerase were added and the reactions incubated isothermally at 30°C over night, allowing circular ligation products to be amplified through a rolling-circle mechanism ( ). .. After amplification the resulting linear concatemers were cut with restriction endonuclease, and the plasmids were re-circularised by self-ligation at low DNA concentrations.

    In Vitro:

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions
    Article Snippet: The off-rates for these single antibody domains are therefore comparable to those reported for single chain Fv fragments isolated from a large repertoire ( > 1010 clones) ( ). .. 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. ..

    Incubation:

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions
    Article Snippet: A dilution series was prepared from the ligation reaction and used as a template for Phi29 amplification. .. Hexamer primers and Phi29 polymerase were added and the reactions incubated isothermally at 30°C over night, allowing circular ligation products to be amplified through a rolling-circle mechanism ( ). .. After amplification the resulting linear concatemers were cut with restriction endonuclease, and the plasmids were re-circularised by self-ligation at low DNA concentrations.

    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: TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells
    Article Snippet: Reconstituted DNA was digested with HinfI and RsaI (New England Biolabs) and again purified by phenol:chloroform extraction. .. 500 ng of digested DNA were incubated with 7.5 U of phi29 DNA polymerase (New England Biolabs) in presence of dATP, dTTP and dGTP (1 mM each) at 30°C for 8 h, followed by heat-inactivation at 65°C for 20 min. Amplification products were dot-blotted onto nylon membranes (GE Healthcare) and hybridized at 55°C overnight with a double-stranded telomeric probe (Telo2 probe), radioactively labeled using Klenow fragment (New England Biolabs) and [α-32P]dCTP. ..

    Amplification:

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions
    Article Snippet: A dilution series was prepared from the ligation reaction and used as a template for Phi29 amplification. .. Hexamer primers and Phi29 polymerase were added and the reactions incubated isothermally at 30°C over night, allowing circular ligation products to be amplified through a rolling-circle mechanism ( ). .. After amplification the resulting linear concatemers were cut with restriction endonuclease, and the plasmids were re-circularised by self-ligation at low DNA concentrations.

    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: TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells
    Article Snippet: Reconstituted DNA was digested with HinfI and RsaI (New England Biolabs) and again purified by phenol:chloroform extraction. .. 500 ng of digested DNA were incubated with 7.5 U of phi29 DNA polymerase (New England Biolabs) in presence of dATP, dTTP and dGTP (1 mM each) at 30°C for 8 h, followed by heat-inactivation at 65°C for 20 min. Amplification products were dot-blotted onto nylon membranes (GE Healthcare) and hybridized at 55°C overnight with a double-stranded telomeric probe (Telo2 probe), radioactively labeled using Klenow fragment (New England Biolabs) and [α-32P]dCTP. ..

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

    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: 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 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.

    Labeling:

    Article Title: TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells
    Article Snippet: Reconstituted DNA was digested with HinfI and RsaI (New England Biolabs) and again purified by phenol:chloroform extraction. .. 500 ng of digested DNA were incubated with 7.5 U of phi29 DNA polymerase (New England Biolabs) in presence of dATP, dTTP and dGTP (1 mM each) at 30°C for 8 h, followed by heat-inactivation at 65°C for 20 min. Amplification products were dot-blotted onto nylon membranes (GE Healthcare) and hybridized at 55°C overnight with a double-stranded telomeric probe (Telo2 probe), radioactively labeled using Klenow fragment (New England Biolabs) and [α-32P]dCTP. ..

    Purification:

    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: DISCUSSION A thermophilic SSB protein and its mutant form from T.thermophilus HB8 were purified and its effects on the activity of phi29 DNA polymerases were assayed. .. The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase. ..

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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: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 polymerase/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
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    phi29 polymerase - by Bioz Stars, 2021-03
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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

    C-circle analysis in T-TALE cells. ( a ) C-circle assay analysis of genomic DNA from the indicated cells treated with dox for up to 72 hours. Reaction products were dot-blotted and hybridized to a radiolabeled telomeric probe. Control reactions were performed in absence of phi29 polymerase (-Φ29). ( b ) Quantifications of C-circle signals from experiments as in a . Bars and error bars are means and SEMs from 3 independent experiments. Circles are single data points.

    Journal: bioRxiv

    Article Title: TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells

    doi: 10.1101/2021.02.18.431840

    Figure Lengend Snippet: C-circle analysis in T-TALE cells. ( a ) C-circle assay analysis of genomic DNA from the indicated cells treated with dox for up to 72 hours. Reaction products were dot-blotted and hybridized to a radiolabeled telomeric probe. Control reactions were performed in absence of phi29 polymerase (-Φ29). ( b ) Quantifications of C-circle signals from experiments as in a . Bars and error bars are means and SEMs from 3 independent experiments. Circles are single data points.

    Article Snippet: 500 ng of digested DNA were incubated with 7.5 U of phi29 DNA polymerase (New England Biolabs) in presence of dATP, dTTP and dGTP (1 mM each) at 30°C for 8 h, followed by heat-inactivation at 65°C for 20 min. Amplification products were dot-blotted onto nylon membranes (GE Healthcare) and hybridized at 55°C overnight with a double-stranded telomeric probe (Telo2 probe), radioactively labeled using Klenow fragment (New England Biolabs) and [α-32P]dCTP.

    Techniques: