phi29 dna polymerase reaction buffer  (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 reaction buffer
    phi29 DNA Polymerase
    phi29 DNA Polymerase 1 250 units
    https://www.bioz.com/result/phi29 dna polymerase reaction buffer/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase reaction buffer - by Bioz Stars, 2020-11
    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 "Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination"

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.03.011

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.
    Figure Legend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry

    3) 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

    4) 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

    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: 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: 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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  • 99
    New England Biolabs phi29 dna polymerase reaction buffer
    Plasmid <t>DNA</t> from the cecal sample after amplification with <t>phi29</t> polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Phi29 Dna Polymerase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase reaction buffer/product/New England Biolabs
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase reaction buffer - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    Image Search Results


    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

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Journal: Methods in enzymology

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    doi: 10.1016/bs.mie.2017.03.011

    Figure Lengend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    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)

    Techniques: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry

    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.

    Journal: Langmuir : the ACS journal of surfaces and colloids

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

    doi: 10.1021/acs.langmuir.8b01812

    Figure Lengend 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.

    Article Snippet: Low-complexity ssDNA was synthesized in 1× phi29 DNA polymerase reaction buffer (NEB M0269S), 500 μ M dCTP and dTTP (NEB N0446S), 0.2 mg mL−1 BSA (NEB B9000S), 10 nM annealed circles, and 100 nM phi29 DNAP (purified in-house).

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

    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.

    Journal: Chemical Science

    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

    doi: 10.1039/c8sc03121e

    Figure Lengend 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.

    Article Snippet: Then, 21.5 U of SplintR DNA ligase (New England Biolabs) prepared in 5 μl BUFFER-1 was added to the mixture and incubated at 37 °C for 1 h. Afterwards, 15 μl phi29 DNA polymerase reaction buffer (BUFFER-2, 1× buffer components: 50 mM Tris-Cl, 10 mM MgCl2, 10 mM (NH4 )2 SO4 , 4 mM DTT, pH 7.4), which contained 5 U of phi29 DNA polymerase (New England Biolabs) and 0.5 mM dNTP (New England Biolabs), was added and incubated at 37 °C for 3 h. Before termination of the polymerization process, 2.5 nM Tb probe and 2.5 nM Cy5.5 probe prepared in 120 μl hybridization buffer (BUFFER-3, 20 mM Tris-Cl, 500 mM NaCl, 0.1% BSA, pH 8.0) were added and then incubated in a thermal cycler with a temperature control program (65 °C for 10 min → decreased from 65 °C to 22 °C with a 2°C min–1 speed → 22 °C for 10 min).

    Techniques: Amplification, Incubation, Labeling