bst dna polymerase large fragment  (New England Biolabs)


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    Name:
    Bsu DNA Polymerase Large Fragment
    Description:
    Bsu DNA Polymerase Large Fragment 1 000 units
    Catalog Number:
    M0330L
    Price:
    266
    Category:
    Thermostable DNA Polymerases
    Size:
    1 000 units
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    New England Biolabs bst dna polymerase large fragment
    Bsu DNA Polymerase Large Fragment
    Bsu DNA Polymerase Large Fragment 1 000 units
    https://www.bioz.com/result/bst dna polymerase large fragment/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Loop-mediated isothermal amplification of DNA"

    Article Title: Loop-mediated isothermal amplification of DNA

    Journal: Nucleic Acids Research

    doi:

    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).
    Figure Legend Snippet: Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Techniques Used: Amplification, Incubation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).
    Figure Legend Snippet: Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Techniques Used: Southern Blot, Hybridization, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Staining, Marker

    2) Product Images from "Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli"

    Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0230881

    Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).
    Figure Legend Snippet: Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Techniques Used: Amplification, Selection, Incubation, Concentration Assay, Marker, Negative Control

    3) Product Images from "MSH2-MSH3 promotes DNA end resection during HR and blocks TMEJ through interaction with SMARCAD1 and EXO1"

    Article Title: MSH2-MSH3 promotes DNA end resection during HR and blocks TMEJ through interaction with SMARCAD1 and EXO1

    Journal: bioRxiv

    doi: 10.1101/2021.04.23.441074

    MSH2-MSH3 inhibits POLQ extension from a mismatched primer. Increasing amounts of POLQ (0.3, 0.6, 1.3 nM) were incubated in the presence of indicated amount of MSH2-MSH3 with the 5′- 32 P-labeled primer templates described on top of the gel in the presence of all 4 nt at 37°C for 10 minutes. The first lane (-) contained no enzyme. (A) The percentage (%) of the product extension from the primer is shown below each lane. 2 mismatched base pairs were placed at the 3 rd and 4 th bp from the primer-template junction. (B) The termination probability at position N3 is defined as the band density at N3 divided by the intensity of ≥ N3. (C) The amount of full-length extension products is defined as the fully extended band density divided by the intensity of ≥ N0 (Primer position). The effects of MSH2-MSH3 on non-mismatched substrate are measured similarly (D, E, F). (G) POLQ movements to MI induced DSB sites were monitored in U2OS cells transfected with control or MSH2 siRNA after incubation with 10 μM of BrdU. (H) Mutation signatures at the CEL locus upon CRISPR-Cas9 induced DSB were compared in control and MSH2 knockdown HEK293T cells. Boxplot showing frequency of deletion mutations harboring microhomology longer than two nucleotides at the DNA junction out of total deletion mutations induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by unpaired two-tailed t-test (n=7) (left). DNA deletion spectrum associated microhomology longer than 4 nucleotides induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by paired two-tailed t-test (n=7) (right).
    Figure Legend Snippet: MSH2-MSH3 inhibits POLQ extension from a mismatched primer. Increasing amounts of POLQ (0.3, 0.6, 1.3 nM) were incubated in the presence of indicated amount of MSH2-MSH3 with the 5′- 32 P-labeled primer templates described on top of the gel in the presence of all 4 nt at 37°C for 10 minutes. The first lane (-) contained no enzyme. (A) The percentage (%) of the product extension from the primer is shown below each lane. 2 mismatched base pairs were placed at the 3 rd and 4 th bp from the primer-template junction. (B) The termination probability at position N3 is defined as the band density at N3 divided by the intensity of ≥ N3. (C) The amount of full-length extension products is defined as the fully extended band density divided by the intensity of ≥ N0 (Primer position). The effects of MSH2-MSH3 on non-mismatched substrate are measured similarly (D, E, F). (G) POLQ movements to MI induced DSB sites were monitored in U2OS cells transfected with control or MSH2 siRNA after incubation with 10 μM of BrdU. (H) Mutation signatures at the CEL locus upon CRISPR-Cas9 induced DSB were compared in control and MSH2 knockdown HEK293T cells. Boxplot showing frequency of deletion mutations harboring microhomology longer than two nucleotides at the DNA junction out of total deletion mutations induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by unpaired two-tailed t-test (n=7) (left). DNA deletion spectrum associated microhomology longer than 4 nucleotides induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by paired two-tailed t-test (n=7) (right).

    Techniques Used: Incubation, Labeling, Transfection, Mutagenesis, CRISPR, Two Tailed Test

    4) Product Images from "Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification"

    Article Title: Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-56225-0

    The relationship between reaction time and the absorbance of the reaction tubes. Colorimetric dye can be used with pyrophosphatase. Pyrophosphate, a by-product of the LAMP reaction, attenuates the activity of DNA polymerase. The addition of pyrophosphatase can increase the LAMP reaction speed. *, Bst DNA Polymerase (New England Biolabs, Ipswich, MS, USA) conventional LAMP reagent; **, Isothermal Master Mix (no dye; Canon Medical Systems Corporation, Tochigi, Japan); PC, positive control; NC, negative control.
    Figure Legend Snippet: The relationship between reaction time and the absorbance of the reaction tubes. Colorimetric dye can be used with pyrophosphatase. Pyrophosphate, a by-product of the LAMP reaction, attenuates the activity of DNA polymerase. The addition of pyrophosphatase can increase the LAMP reaction speed. *, Bst DNA Polymerase (New England Biolabs, Ipswich, MS, USA) conventional LAMP reagent; **, Isothermal Master Mix (no dye; Canon Medical Systems Corporation, Tochigi, Japan); PC, positive control; NC, negative control.

    Techniques Used: Activity Assay, Mass Spectrometry, Positive Control, Negative Control

    5) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    Temperature profile (a), electric power profile (b), and frequency profile (c) of MW-RCA using Bst DNA polymerase-LF at 60°C.
    Figure Legend Snippet: Temperature profile (a), electric power profile (b), and frequency profile (c) of MW-RCA using Bst DNA polymerase-LF at 60°C.

    Techniques Used:

    Electrophoresis and fluorescence intensity of DNA synthesized by four DNA polymerases under conventional and microwave heating. MW = microwave. (a) Bst DNA polymerase-LF, 10–60-min rolling circle amplification (RCA) and10–30-min microwave-assisted (MW)-RCA. (b) Bst DNA polymerase, 10–60-min RCA and 10–30-min MW-RCA (c) Csa DNA polymerase, 10–90-min RCA and 10–30-min MW-RCA. (d) 96–7 DNA polymerase, 10–120-min RCA and 10–30 min MW-RCA.
    Figure Legend Snippet: Electrophoresis and fluorescence intensity of DNA synthesized by four DNA polymerases under conventional and microwave heating. MW = microwave. (a) Bst DNA polymerase-LF, 10–60-min rolling circle amplification (RCA) and10–30-min microwave-assisted (MW)-RCA. (b) Bst DNA polymerase, 10–60-min RCA and 10–30-min MW-RCA (c) Csa DNA polymerase, 10–90-min RCA and 10–30-min MW-RCA. (d) 96–7 DNA polymerase, 10–120-min RCA and 10–30 min MW-RCA.

    Techniques Used: Electrophoresis, Fluorescence, Synthesized, Amplification

    6) Product Images from "Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli"

    Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0230881

    Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).
    Figure Legend Snippet: Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Techniques Used: Amplification, Selection, Incubation, Concentration Assay, Marker, Negative Control

    7) Product Images from "Loop-mediated isothermal amplification of DNA"

    Article Title: Loop-mediated isothermal amplification of DNA

    Journal: Nucleic Acids Research

    doi:

    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).
    Figure Legend Snippet: Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Techniques Used: Amplification, Incubation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).
    Figure Legend Snippet: Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Techniques Used: Southern Blot, Hybridization, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Staining, Marker

    8) Product Images from "DNA synthesis from diphosphate substrates by DNA polymerases"

    Article Title: DNA synthesis from diphosphate substrates by DNA polymerases

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1712193115

    Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).
    Figure Legend Snippet: Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

    Techniques Used: Polymerase Chain Reaction, Amplification

    9) Product Images from "Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array-CGH"

    Article Title: Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array-CGH

    Journal: Genome Research

    doi: 10.1101/gr.377203

    Evaluation of DNA amplification bias using array–CGH on human cDNA microarrays. ( A ) To assess the extent of dynamic range compression, three microarray–CGH experiments were performed in duplicate using genomic female DNA (46, XX) versus genomic male DNA, or DNA from cell lines containing 3 X-chromosomes (47, XXX) and 5 X-chromosomes (49, XXXXX) with a normal number of autosomes against genomic male DNA. Autosomal genes located in Chromosomes 1 and 2 are compared with genes located in Chromosome X for the same set of experiments. Ratio values correspond to the average of two independent experiments, and are displayed in a log 2 scale. Averaging the log 2 ratios for the X-linked probes in the three different experiments gives values of 0.234 ± 0.143 (1.176 in linear scale, average of 112 probes), 0.423 ± 0.220 (1.341 in linear scale, average of 113 probes), and 0.681 ± 0.290 (1.603 in linear scale, average of 115 probes) for the 2X, 3X, and 5X experiments, respectively. Dynamically compressed ratios can be converted to actual ratios by fitting log 2 . ( B ) Comparison of DNA polymerase-induced representational distortion using human DNA samples. Normal human DNA was amplified with either φ29 or Bst DNA polymerase, labeled with Cy3, and hybridized against similarly amplified human DNA labeled with Cy5. Plots for Chromosomes 1 and 2 are shown in the same scale as the plot in A . ( C ) Confidence limits for array–CGH analysis of human DNA. Plots correspond to unamplified human female versus male DNAs and whole genome Bst -amplified human female versus Bst -amplified male DNAs. Average log 2 fluorescence ratios for replicate spots are ordered according to the chromosome number and the position in the chromosome. Ratio values for X-linked genes show a similar distribution to that observed for the 2X dosage in A . Confidence limits (horizontal dashed lines) for 99.9% of data for autosomal genes are between −0.262 and 0.262 (0.833 and 1.199 when expressed as linear ratios) for the unamplified experiment. The same confidence bounds calculated for the unamplified experiment are replicated in the plot of ratios generated by microarray analysis of amplified DNA.
    Figure Legend Snippet: Evaluation of DNA amplification bias using array–CGH on human cDNA microarrays. ( A ) To assess the extent of dynamic range compression, three microarray–CGH experiments were performed in duplicate using genomic female DNA (46, XX) versus genomic male DNA, or DNA from cell lines containing 3 X-chromosomes (47, XXX) and 5 X-chromosomes (49, XXXXX) with a normal number of autosomes against genomic male DNA. Autosomal genes located in Chromosomes 1 and 2 are compared with genes located in Chromosome X for the same set of experiments. Ratio values correspond to the average of two independent experiments, and are displayed in a log 2 scale. Averaging the log 2 ratios for the X-linked probes in the three different experiments gives values of 0.234 ± 0.143 (1.176 in linear scale, average of 112 probes), 0.423 ± 0.220 (1.341 in linear scale, average of 113 probes), and 0.681 ± 0.290 (1.603 in linear scale, average of 115 probes) for the 2X, 3X, and 5X experiments, respectively. Dynamically compressed ratios can be converted to actual ratios by fitting log 2 . ( B ) Comparison of DNA polymerase-induced representational distortion using human DNA samples. Normal human DNA was amplified with either φ29 or Bst DNA polymerase, labeled with Cy3, and hybridized against similarly amplified human DNA labeled with Cy5. Plots for Chromosomes 1 and 2 are shown in the same scale as the plot in A . ( C ) Confidence limits for array–CGH analysis of human DNA. Plots correspond to unamplified human female versus male DNAs and whole genome Bst -amplified human female versus Bst -amplified male DNAs. Average log 2 fluorescence ratios for replicate spots are ordered according to the chromosome number and the position in the chromosome. Ratio values for X-linked genes show a similar distribution to that observed for the 2X dosage in A . Confidence limits (horizontal dashed lines) for 99.9% of data for autosomal genes are between −0.262 and 0.262 (0.833 and 1.199 when expressed as linear ratios) for the unamplified experiment. The same confidence bounds calculated for the unamplified experiment are replicated in the plot of ratios generated by microarray analysis of amplified DNA.

    Techniques Used: Amplification, Microarray, Labeling, Fluorescence, Generated

    Evaluation of amplification bias using array–CGH on yeast cDNA microarrays. Microarrays contained 6135 unique yeast ORFs. Fluorescence ratios were measured and plotted against the order of the genes in the genome, starting from Chromosome I to Chromosome XVI. ( Upper left panel) Analysis of a microarray hybridized with the same DNA, labeled with Cy3 and Cy5. ( Upper right panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Lower left panel) DNA from the yeast KO strain was amplified using Bst DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center left panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase for only 2 h, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center right panel) Equivalent experiment using Bst DNA polymerase. ( Lower right panel) DNAs from the two different yeast strains were amplified to the same extent using Bst and hybridized together. The three genes known to be deleted appear as outlier data points indicated by arrows. The other two outlier data points, near genes GIN4 and CLA4 , have abnormally low area values of 48 and 21 according to the Spot analysis software, compared with the average of 255 for all the spots in the array. This abnormality could be produced by a fluorescent speckle over the spot, resulting in unreliable ratios.
    Figure Legend Snippet: Evaluation of amplification bias using array–CGH on yeast cDNA microarrays. Microarrays contained 6135 unique yeast ORFs. Fluorescence ratios were measured and plotted against the order of the genes in the genome, starting from Chromosome I to Chromosome XVI. ( Upper left panel) Analysis of a microarray hybridized with the same DNA, labeled with Cy3 and Cy5. ( Upper right panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Lower left panel) DNA from the yeast KO strain was amplified using Bst DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center left panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase for only 2 h, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center right panel) Equivalent experiment using Bst DNA polymerase. ( Lower right panel) DNAs from the two different yeast strains were amplified to the same extent using Bst and hybridized together. The three genes known to be deleted appear as outlier data points indicated by arrows. The other two outlier data points, near genes GIN4 and CLA4 , have abnormally low area values of 48 and 21 according to the Spot analysis software, compared with the average of 255 for all the spots in the array. This abnormality could be produced by a fluorescent speckle over the spot, resulting in unreliable ratios.

    Techniques Used: Amplification, Fluorescence, Microarray, Labeling, Software, Produced

    Gel electrophoresis analysis of amplified DNA. ( A ) Control reactions were incubated for 5 h in a 30-μL volume containing no DNA, or 7.5 ng of human DNA. Samples representing 5% of the reaction were denatured in alkaline buffer and analyzed on a 0.5% alkaline agarose gel, and stained with SYBR-green II (Molecular Probes). Lanes labeled M contained phage λ DNA digested with restriction endonuclease Hin dIII. Reactions catalyzed by φ29 polymerase were incubated with (+) or without (−) input of denatured human DNA. Random heptamers contained standard (−) DNA, or were modified by the addition of two nitroindole groups (+) at the 5′ end. ( B ) Time-course reactions for φ29 and Bst DNA polymerases. Reactions were performed using nitroindole-modified primers. Every hour (from 1–5 h), 1.5 μL was removed, denatured in alkaline buffer, and analyzed in 0.5% alkaline agarose gel. Lanes labeled C correspond to control samples incubated for 5 h without input DNA. The lane labeled G corresponds to a gel load of genomic DNA equivalent to 100× the original DNA input of the amplification reactions. ( C ) Plots under gel images display the time course (fold amplification vs. time) of both polymerase reactions, generated by quantification of DNA yield with the PicoGreen Quantitation Kit. Background fluorescence at time 0 was subtracted for all time points. Each point represents the mean (±1 SD) of four independent analyses.
    Figure Legend Snippet: Gel electrophoresis analysis of amplified DNA. ( A ) Control reactions were incubated for 5 h in a 30-μL volume containing no DNA, or 7.5 ng of human DNA. Samples representing 5% of the reaction were denatured in alkaline buffer and analyzed on a 0.5% alkaline agarose gel, and stained with SYBR-green II (Molecular Probes). Lanes labeled M contained phage λ DNA digested with restriction endonuclease Hin dIII. Reactions catalyzed by φ29 polymerase were incubated with (+) or without (−) input of denatured human DNA. Random heptamers contained standard (−) DNA, or were modified by the addition of two nitroindole groups (+) at the 5′ end. ( B ) Time-course reactions for φ29 and Bst DNA polymerases. Reactions were performed using nitroindole-modified primers. Every hour (from 1–5 h), 1.5 μL was removed, denatured in alkaline buffer, and analyzed in 0.5% alkaline agarose gel. Lanes labeled C correspond to control samples incubated for 5 h without input DNA. The lane labeled G corresponds to a gel load of genomic DNA equivalent to 100× the original DNA input of the amplification reactions. ( C ) Plots under gel images display the time course (fold amplification vs. time) of both polymerase reactions, generated by quantification of DNA yield with the PicoGreen Quantitation Kit. Background fluorescence at time 0 was subtracted for all time points. Each point represents the mean (±1 SD) of four independent analyses.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, Labeling, Modification, Generated, Quantitation Assay, Fluorescence

    Related Articles

    Concentration Assay:

    Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli
    Article Snippet: Assay reactions To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT, Guangzhou Hua-feng Biological Co., Ltd, Guangdong, China) was used as a separate containment control for evaluating the results (visualization using ultraviolet light). .. The CPA assay was conducted in a reaction mixture of 30 μL total volume containing 6 μL 1s (10 mM), 1.5 μL 2a/3a (10 mM), 0.5 μL 4s/5a (10 mM), 2.5 μL (1.0 mM) dNTPs (Takara Bio, Dalian, CN), 3 μL 10 × Thermol-Pol reaction buffer, 1.5 μL Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA, USA), 3 μL (1 mM) MgCl2 (Sigma-Aldrich, St. Louis, MO, USA), 2 μL (0.8 M) betaine (Sigma-Aldrich) and 2 μL of an appropriate concentration of DNA template (E . coli C83920). .. The CPA reaction was then performed at 63°C for 1 h and then terminated by incubation at 85°C for 3 min. For the IMSA assay, the reaction was also performed in a 30 μL mixture, which contained 1 μL (10 mM) outer primers DsF/DsR, 2.5 μL (10 mM) FIT/RIT and SteF/SteR, respectively, 3 μL 10 × Thermol-Pol reaction buffer, 2 μL (16 U) Bst DNA polymerase large fragment, 3 μL (1 mM) MgCl2 , 2 μL (0.8 M) betaine, 2.5 μL (1.0 mM) dNTPs and 2 μL of target DNA, the reaction system supplemented with sterilized deionized water.

    Incubation:

    Article Title: Loop-mediated isothermal amplification of DNA
    Article Snippet: LAMP was carried out in a total 25 µl reaction mixture containing 0.8 µM each FIP and BIP, 0.2 µM each F3 and B3, 400 µM each dNTP, 1 M betaine (Sigma), 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 and the specified amounts of double-stranded target DNA. .. The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction. .. Aliquots of 5 µl of LAMP products and 1 µl of the products digested with restriction enzymes were electrophoresed in 2% agarose gels (0.5× TBE) followed by staining with SYBR Green I (Molecular Probes Inc.).

    Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli
    Article Snippet: The CPA assay was conducted in a reaction mixture of 30 μL total volume containing 6 μL 1s (10 mM), 1.5 μL 2a/3a (10 mM), 0.5 μL 4s/5a (10 mM), 2.5 μL (1.0 mM) dNTPs (Takara Bio, Dalian, CN), 3 μL 10 × Thermol-Pol reaction buffer, 1.5 μL Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA, USA), 3 μL (1 mM) MgCl2 (Sigma-Aldrich, St. Louis, MO, USA), 2 μL (0.8 M) betaine (Sigma-Aldrich) and 2 μL of an appropriate concentration of DNA template (E . coli C83920). .. The CPA reaction was then performed at 63°C for 1 h and then terminated by incubation at 85°C for 3 min. For the IMSA assay, the reaction was also performed in a 30 μL mixture, which contained 1 μL (10 mM) outer primers DsF/DsR, 2.5 μL (10 mM) FIT/RIT and SteF/SteR, respectively, 3 μL 10 × Thermol-Pol reaction buffer, 2 μL (16 U) Bst DNA polymerase large fragment, 3 μL (1 mM) MgCl2 , 2 μL (0.8 M) betaine, 2.5 μL (1.0 mM) dNTPs and 2 μL of target DNA, the reaction system supplemented with sterilized deionized water. .. The LAMP and real-time PCR assays followed the initial reaction using conditions confirmed by previous respective studies.

    Plasmid Preparation:

    Article Title: MSH2-MSH3 promotes DNA end resection during HR and blocks TMEJ through interaction with SMARCAD1 and EXO1
    Article Snippet: Fractionated protein peak of each step was confirmed by SDS-PAGE and concentrations of proteins were measured by Bradford assay. .. DNA polymerase assays An active DNA polymerase fragment of POLQ was expressed from the Sumo3 POLQM1 plasmid and purified as described ( ). .. Klenow Fragment (3′→5′ exo-) was purchased from NEB.

    Purification:

    Article Title: MSH2-MSH3 promotes DNA end resection during HR and blocks TMEJ through interaction with SMARCAD1 and EXO1
    Article Snippet: Fractionated protein peak of each step was confirmed by SDS-PAGE and concentrations of proteins were measured by Bradford assay. .. DNA polymerase assays An active DNA polymerase fragment of POLQ was expressed from the Sumo3 POLQM1 plasmid and purified as described ( ). .. Klenow Fragment (3′→5′ exo-) was purchased from NEB.

    other:

    Article Title: Loop-mediated isothermal amplification of DNA
    Article Snippet: The LAMP reaction is then initiated by addition of the Bst DNA polymerase large fragment and carried out at 65°C for 1 h. The mechanism and expected reaction steps of LAMP are illustrated in Figure .

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    New England Biolabs bst dna polymerase large fragment
    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 <t>DNA</t> amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U <t>Bst</t> polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).
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    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Journal: Nucleic Acids Research

    Article Title: Loop-mediated isothermal amplification of DNA

    doi:

    Figure Lengend Snippet: Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

    Techniques: Amplification, Incubation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Journal: Nucleic Acids Research

    Article Title: Loop-mediated isothermal amplification of DNA

    doi:

    Figure Lengend Snippet: Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

    Techniques: Southern Blot, Hybridization, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Staining, Marker

    Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Journal: PLoS ONE

    Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli

    doi: 10.1371/journal.pone.0230881

    Figure Lengend Snippet: Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Article Snippet: The CPA reaction was then performed at 63°C for 1 h and then terminated by incubation at 85°C for 3 min. For the IMSA assay, the reaction was also performed in a 30 μL mixture, which contained 1 μL (10 mM) outer primers DsF/DsR, 2.5 μL (10 mM) FIT/RIT and SteF/SteR, respectively, 3 μL 10 × Thermol-Pol reaction buffer, 2 μL (16 U) Bst DNA polymerase large fragment, 3 μL (1 mM) MgCl2 , 2 μL (0.8 M) betaine, 2.5 μL (1.0 mM) dNTPs and 2 μL of target DNA, the reaction system supplemented with sterilized deionized water.

    Techniques: Amplification, Selection, Incubation, Concentration Assay, Marker, Negative Control

    MSH2-MSH3 inhibits POLQ extension from a mismatched primer. Increasing amounts of POLQ (0.3, 0.6, 1.3 nM) were incubated in the presence of indicated amount of MSH2-MSH3 with the 5′- 32 P-labeled primer templates described on top of the gel in the presence of all 4 nt at 37°C for 10 minutes. The first lane (-) contained no enzyme. (A) The percentage (%) of the product extension from the primer is shown below each lane. 2 mismatched base pairs were placed at the 3 rd and 4 th bp from the primer-template junction. (B) The termination probability at position N3 is defined as the band density at N3 divided by the intensity of ≥ N3. (C) The amount of full-length extension products is defined as the fully extended band density divided by the intensity of ≥ N0 (Primer position). The effects of MSH2-MSH3 on non-mismatched substrate are measured similarly (D, E, F). (G) POLQ movements to MI induced DSB sites were monitored in U2OS cells transfected with control or MSH2 siRNA after incubation with 10 μM of BrdU. (H) Mutation signatures at the CEL locus upon CRISPR-Cas9 induced DSB were compared in control and MSH2 knockdown HEK293T cells. Boxplot showing frequency of deletion mutations harboring microhomology longer than two nucleotides at the DNA junction out of total deletion mutations induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by unpaired two-tailed t-test (n=7) (left). DNA deletion spectrum associated microhomology longer than 4 nucleotides induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by paired two-tailed t-test (n=7) (right).

    Journal: bioRxiv

    Article Title: MSH2-MSH3 promotes DNA end resection during HR and blocks TMEJ through interaction with SMARCAD1 and EXO1

    doi: 10.1101/2021.04.23.441074

    Figure Lengend Snippet: MSH2-MSH3 inhibits POLQ extension from a mismatched primer. Increasing amounts of POLQ (0.3, 0.6, 1.3 nM) were incubated in the presence of indicated amount of MSH2-MSH3 with the 5′- 32 P-labeled primer templates described on top of the gel in the presence of all 4 nt at 37°C for 10 minutes. The first lane (-) contained no enzyme. (A) The percentage (%) of the product extension from the primer is shown below each lane. 2 mismatched base pairs were placed at the 3 rd and 4 th bp from the primer-template junction. (B) The termination probability at position N3 is defined as the band density at N3 divided by the intensity of ≥ N3. (C) The amount of full-length extension products is defined as the fully extended band density divided by the intensity of ≥ N0 (Primer position). The effects of MSH2-MSH3 on non-mismatched substrate are measured similarly (D, E, F). (G) POLQ movements to MI induced DSB sites were monitored in U2OS cells transfected with control or MSH2 siRNA after incubation with 10 μM of BrdU. (H) Mutation signatures at the CEL locus upon CRISPR-Cas9 induced DSB were compared in control and MSH2 knockdown HEK293T cells. Boxplot showing frequency of deletion mutations harboring microhomology longer than two nucleotides at the DNA junction out of total deletion mutations induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by unpaired two-tailed t-test (n=7) (left). DNA deletion spectrum associated microhomology longer than 4 nucleotides induced by CRISPR-Cas9 targeting CEL locus in control and MSH2 knockdown HEK293T cells. P-value was calculated by paired two-tailed t-test (n=7) (right).

    Article Snippet: DNA polymerase assays An active DNA polymerase fragment of POLQ was expressed from the Sumo3 POLQM1 plasmid and purified as described ( ).

    Techniques: Incubation, Labeling, Transfection, Mutagenesis, CRISPR, Two Tailed Test

    The relationship between reaction time and the absorbance of the reaction tubes. Colorimetric dye can be used with pyrophosphatase. Pyrophosphate, a by-product of the LAMP reaction, attenuates the activity of DNA polymerase. The addition of pyrophosphatase can increase the LAMP reaction speed. *, Bst DNA Polymerase (New England Biolabs, Ipswich, MS, USA) conventional LAMP reagent; **, Isothermal Master Mix (no dye; Canon Medical Systems Corporation, Tochigi, Japan); PC, positive control; NC, negative control.

    Journal: Scientific Reports

    Article Title: Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification

    doi: 10.1038/s41598-019-56225-0

    Figure Lengend Snippet: The relationship between reaction time and the absorbance of the reaction tubes. Colorimetric dye can be used with pyrophosphatase. Pyrophosphate, a by-product of the LAMP reaction, attenuates the activity of DNA polymerase. The addition of pyrophosphatase can increase the LAMP reaction speed. *, Bst DNA Polymerase (New England Biolabs, Ipswich, MS, USA) conventional LAMP reagent; **, Isothermal Master Mix (no dye; Canon Medical Systems Corporation, Tochigi, Japan); PC, positive control; NC, negative control.

    Article Snippet: Briefly, we carried out LAMP in a reaction mixture consisting of 1.6 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 0.4 µM of LF/LB, 8 U of Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA, USA), 1.4 mM deoxynucleoside triphosphates, 0.8 M betaine (Sigma, St. Louis, MO, USA), 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween 20, and template DNA, and the final volume was adjusted to 25 µL with distilled water.

    Techniques: Activity Assay, Mass Spectrometry, Positive Control, Negative Control