nb bsm i  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    Name:
    Nb BsmI
    Description:
    Nb BsmI 1 000 units
    Catalog Number:
    r0706l
    Price:
    70
    Size:
    1 000 units
    Category:
    Restriction Enzymes
    Buy from Supplier


    Structured Review

    New England Biolabs nb bsm i
    Nb BsmI
    Nb BsmI 1 000 units
    https://www.bioz.com/result/nb bsm i/product/New England Biolabs
    Average 96 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    nb bsm i - by Bioz Stars, 2020-09
    96/100 stars

    Images

    1) Product Images from "Measurements of DNA-loop formation via Cre-mediated recombination"

    Article Title: Measurements of DNA-loop formation via Cre-mediated recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks430

    Method for generating doubly labeled plasmid DNAs. ( A ) We engineered novel-plasmid DNA constructs in which pairs of tandem BbvC I and Bsm I restriction sites flank respective loxP sites. The minor distance between centers of the loxP sites is 65 bp. ( B ) Tandem nicks were generated at BbvC I and Bsm I sites by treatment with mutant endonucleases Nb.BbvCI and Nb.BsmI. ( C ) Fluorophore labels (donor in green; acceptor in red) were incorporated by displacing the single-stranded fragments released by the tandem nicks, which were subsequently sealed with T4 DNA ligase. ( D ) The covalently closed plasmid was separated from residual nicked DNA and linearized by treatment with Pst I.
    Figure Legend Snippet: Method for generating doubly labeled plasmid DNAs. ( A ) We engineered novel-plasmid DNA constructs in which pairs of tandem BbvC I and Bsm I restriction sites flank respective loxP sites. The minor distance between centers of the loxP sites is 65 bp. ( B ) Tandem nicks were generated at BbvC I and Bsm I sites by treatment with mutant endonucleases Nb.BbvCI and Nb.BsmI. ( C ) Fluorophore labels (donor in green; acceptor in red) were incorporated by displacing the single-stranded fragments released by the tandem nicks, which were subsequently sealed with T4 DNA ligase. ( D ) The covalently closed plasmid was separated from residual nicked DNA and linearized by treatment with Pst I.

    Techniques Used: Labeling, Plasmid Preparation, Construct, Generated, Mutagenesis

    2) Product Images from "Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification"

    Article Title: Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr909

    Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.
    Figure Legend Snippet: Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.

    Techniques Used: Incubation, Generated, Marker

    3) Product Images from "Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification"

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1014

    Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.
    Figure Legend Snippet: Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.

    Techniques Used: Concentration Assay

    PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.
    Figure Legend Snippet: PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.

    Techniques Used: Concentration Assay, Serial Dilution, Amplification

    Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.
    Figure Legend Snippet: Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.

    Techniques Used: Concentration Assay, Serial Dilution, Amplification

    Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.
    Figure Legend Snippet: Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.

    Techniques Used: Agarose Gel Electrophoresis, Marker

    Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.
    Figure Legend Snippet: Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Techniques Used: Sequencing, Concentration Assay

    Related Articles

    other:

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification
    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Plasmid Preparation:

    Article Title: RNA polymerase I passage through nucleosomes depends on its lobe binding subunits
    Article Snippet: .. Biotinylated oligos downstream of the tail in reverse direction (oligos 4018 and 4220 for 1 kb and 2 kb transcripts, respectively) and an oligo containing a Nb.BsmI (NEB) nicking site (oligo 4220a) were used to generate templates of 1 and 2 kb using plasmid 2316 as template. .. PCR products were cut with Nb.BsmI and heat inactivated at 80°C for 20 min. After 10 min, a competitor oligo 4220a with the same sequence as the 24 nt 3’overhang was added in excess to anneal with released cleaved 5’Nb.BsmI fragment.

    Article Title: Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress
    Article Snippet: .. For plasmid DNA catenation analysis of pRS316, purified DNA was nicked with Nb.Bsm1 (New England Biolabs, R0706S) according to manufacturer’s instructions. .. For fork pausing analysis of pRS416-RFB, purified DNA was digested with BamHI-HF (New England Biolabs, R3136S) and SnaBI (New England Biolabs, R0130L) according to manufacturer’s instructions.

    Article Title: Measurements of DNA-loop formation via Cre-mediated recombination
    Article Snippet: .. The purified plasmid was treated with the site-specific nicking endonucleases Nb.BbvC I and Nb.Bsm I ( ) (New England Biolabs, Ipswich, MA, USA) at 37 and 65°C, respectively. ..

    Purification:

    Article Title: Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress
    Article Snippet: .. For plasmid DNA catenation analysis of pRS316, purified DNA was nicked with Nb.Bsm1 (New England Biolabs, R0706S) according to manufacturer’s instructions. .. For fork pausing analysis of pRS416-RFB, purified DNA was digested with BamHI-HF (New England Biolabs, R3136S) and SnaBI (New England Biolabs, R0130L) according to manufacturer’s instructions.

    Article Title: Measurements of DNA-loop formation via Cre-mediated recombination
    Article Snippet: .. The purified plasmid was treated with the site-specific nicking endonucleases Nb.BbvC I and Nb.Bsm I ( ) (New England Biolabs, Ipswich, MA, USA) at 37 and 65°C, respectively. ..

    Article Title: Folding DNA origami from a double-stranded source of scaffold
    Article Snippet: .. To avoid potential complications associated with the impossibility of complete separation between strands that are topologically interwound, the DNA was nicked at a single site with Nb.BsmI (New England Biolabs) and subsequently gel purified. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    New England Biolabs nb bsm i
    Method for generating doubly labeled plasmid DNAs. ( A ) We engineered novel-plasmid DNA constructs in which pairs of tandem <t>BbvC</t> I and <t>Bsm</t> I restriction sites flank respective loxP sites. The minor distance between centers of the loxP sites is 65 bp. ( B ) Tandem nicks were generated at BbvC I and Bsm I sites by treatment with mutant endonucleases Nb.BbvCI and Nb.BsmI. ( C ) Fluorophore labels (donor in green; acceptor in red) were incorporated by displacing the single-stranded fragments released by the tandem nicks, which were subsequently sealed with T4 DNA ligase. ( D ) The covalently closed plasmid was separated from residual nicked DNA and linearized by treatment with Pst I.
    Nb Bsm I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nb bsm i/product/New England Biolabs
    Average 96 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    nb bsm i - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    Image Search Results


    Method for generating doubly labeled plasmid DNAs. ( A ) We engineered novel-plasmid DNA constructs in which pairs of tandem BbvC I and Bsm I restriction sites flank respective loxP sites. The minor distance between centers of the loxP sites is 65 bp. ( B ) Tandem nicks were generated at BbvC I and Bsm I sites by treatment with mutant endonucleases Nb.BbvCI and Nb.BsmI. ( C ) Fluorophore labels (donor in green; acceptor in red) were incorporated by displacing the single-stranded fragments released by the tandem nicks, which were subsequently sealed with T4 DNA ligase. ( D ) The covalently closed plasmid was separated from residual nicked DNA and linearized by treatment with Pst I.

    Journal: Nucleic Acids Research

    Article Title: Measurements of DNA-loop formation via Cre-mediated recombination

    doi: 10.1093/nar/gks430

    Figure Lengend Snippet: Method for generating doubly labeled plasmid DNAs. ( A ) We engineered novel-plasmid DNA constructs in which pairs of tandem BbvC I and Bsm I restriction sites flank respective loxP sites. The minor distance between centers of the loxP sites is 65 bp. ( B ) Tandem nicks were generated at BbvC I and Bsm I sites by treatment with mutant endonucleases Nb.BbvCI and Nb.BsmI. ( C ) Fluorophore labels (donor in green; acceptor in red) were incorporated by displacing the single-stranded fragments released by the tandem nicks, which were subsequently sealed with T4 DNA ligase. ( D ) The covalently closed plasmid was separated from residual nicked DNA and linearized by treatment with Pst I.

    Article Snippet: The purified plasmid was treated with the site-specific nicking endonucleases Nb.BbvC I and Nb.Bsm I ( ) (New England Biolabs, Ipswich, MA, USA) at 37 and 65°C, respectively.

    Techniques: Labeling, Plasmid Preparation, Construct, Generated, Mutagenesis

    Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.

    Journal: Nucleic Acids Research

    Article Title: Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

    doi: 10.1093/nar/gkr909

    Figure Lengend Snippet: Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI and exonuclease III were purchased from New England Biolabs.

    Techniques: Incubation, Generated, Marker

    Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Concentration Assay

    PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Concentration Assay, Serial Dilution, Amplification

    Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Concentration Assay, Serial Dilution, Amplification

    Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Agarose Gel Electrophoresis, Marker

    Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Sequencing, Concentration Assay