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:
    261
    Size:
    1 000 units
    Category:
    Thermostable DNA Polymerases
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    Structured Review

    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 90 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase large fragment - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) 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

    2) 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

    3) Product Images from "Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay"

    Article Title: Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.00279

    Optimization of Bst DNA polymerase concentration for the LAMP reaction of the bar transgene. (A) LAMP products detected by 1000 × SYBR Green I. (B) The amplification curves obtained in real-time LAMP based on different dosage of Bst DNA polymerase. (A) Tubes 1, 2, 7, 8, 13, 14, 19, and 20: ddH 2 O. Tubes 3, 4, 9, 10, 15, 16, 21, and 22: FN95-1702 (negative control, CK). Tubes 5, 6, 11, 12, 17, 18, 23 and 24: the plasmid 1Ac0229. Tubes 1–6, 7–12, 13–18, and 19–24: Bst DNA polymerase concentrations of 2.0, 4.0, 6.0, and 8.0 U, respectively, two repeats. (B) Curves separately represent the dosage of Bst DNA polymerase of 8.0, 6.0, 4.0, and 2.0 U, from left to right. The colored line at the very bottom indicates the blank and negative controls.
    Figure Legend Snippet: Optimization of Bst DNA polymerase concentration for the LAMP reaction of the bar transgene. (A) LAMP products detected by 1000 × SYBR Green I. (B) The amplification curves obtained in real-time LAMP based on different dosage of Bst DNA polymerase. (A) Tubes 1, 2, 7, 8, 13, 14, 19, and 20: ddH 2 O. Tubes 3, 4, 9, 10, 15, 16, 21, and 22: FN95-1702 (negative control, CK). Tubes 5, 6, 11, 12, 17, 18, 23 and 24: the plasmid 1Ac0229. Tubes 1–6, 7–12, 13–18, and 19–24: Bst DNA polymerase concentrations of 2.0, 4.0, 6.0, and 8.0 U, respectively, two repeats. (B) Curves separately represent the dosage of Bst DNA polymerase of 8.0, 6.0, 4.0, and 2.0 U, from left to right. The colored line at the very bottom indicates the blank and negative controls.

    Techniques Used: Concentration Assay, SYBR Green Assay, Amplification, Negative Control, Plasmid Preparation

    4) Product Images from "Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane"

    Article Title: Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

    Journal: Scientific Reports

    doi: 10.1038/srep04912

    The detection results based on different concentrations of Bst DNA polymerase. (A) LAMP products detected by 1,000× SYBR Green I. (B) Detection of LAMP products by agarose gel electrophoresis stained by EB. Tube and lane 1: ddH 2 O. Tube and lane 2: FN95-1702 (negative control, CK). Tubes and lanes 3–4, 5–6, 7–8 and 9–10: Bst DNA polymerase concentrations 2.0 U, 4.0 U, 6.0 U and 8.0 U, respectively, two repeats. Lane M: DL 15,000+2,000 DNA marker.
    Figure Legend Snippet: The detection results based on different concentrations of Bst DNA polymerase. (A) LAMP products detected by 1,000× SYBR Green I. (B) Detection of LAMP products by agarose gel electrophoresis stained by EB. Tube and lane 1: ddH 2 O. Tube and lane 2: FN95-1702 (negative control, CK). Tubes and lanes 3–4, 5–6, 7–8 and 9–10: Bst DNA polymerase concentrations 2.0 U, 4.0 U, 6.0 U and 8.0 U, respectively, two repeats. Lane M: DL 15,000+2,000 DNA marker.

    Techniques Used: SYBR Green Assay, Agarose Gel Electrophoresis, Staining, Negative Control, Marker

    5) 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

    6) 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

    Amplification:

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    Article Snippet: Paragraph title: PSR amplification methods ... The PSR reactions were carried out in a 25 μl reaction mixture containing the following components: 1.0 μl Bst DNA polymerase large fragment (New England Biolabs), 2.5 μl 10 × ThermolPol reaction buffer (New England Biolabs, including 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween 20), 0.8 M Betaine (Sigma), 6 mM MgSO4 , 1.4 mM each deoxynucleoside triphosphate.

    Article Title: Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein
    Article Snippet: .. Materials and Methods Amplification reactions were performed in 50 mM Tris-Acetate, pH 7.9, 50 mM potassium acetate, 7 mM magnesium acetate, 2 mM DTT, 0.6 mM each dNTP, 1.0 μM each forward and reverse primer, 0.33 U/μL Bsu DNA Polymerase, Large Fragment (New England Biolabs (NEB)) and 300–456 ng/μl (9.0–13.6 µM) T4 gene 32 protein (NEB). ..

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C. .. The purified DNA was then amplified using 25 μl Kapa 2G robust hot start ready mix (Kapa Biosystems), 1 μl 50× Nextera primer cocktail (Illumina – compatible) and 1 μl barcoded Illumina-compatible adaptor 2 (8-bp index) on a thermocycler with the following parameters: 1 min at 95 °C followed by 10-15 cycles of 20 s at 95 °C, 30 s at 60 °C, 45 s at 72 °C.

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    Article Snippet: .. The isothermal amplification was performed in a 25-μL volume containing 1 μL of ligation product mixture, 1.2 μM of each primer-1 and -2, 100 μM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100, 6% dimethyl sulfoxide, 6 μL of 1× SYBR Green I dye (Bio-Vision), and 6.4 U Bst DNA polymerase large fragment (New England BioLabs). .. The reactions were incubated at 65°C in a 96-well plate and were real-time monitored for 100 cycles with a 25-sec period by an Opticon2 DNA Engine (Bio-Rad).

    Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem
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    Lambda DNA Preparation:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: The purified DNA (∼ 0.5 ng, spiked with 5 pg of unmethylated lambda DNA) was then incubated with 4 μl Nextera HMW Buffer (Epicentre-Illumina), 16 μl nuclease-free water (Ambion), and 4 μl prepared Tn5mC transposome complex for 12 min at 55 °C followed by purification using 36 μl (1.8×) Agencourt AMPure XP magnetic beads; and the DNA was eluted in 14 μl EB buffer (Qiagen). .. An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C.

    Construct:

    Article Title: Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method
    Article Snippet: PSR amplification methods The template target used in this study was the E. coli pGEX-NDM-BL21 containing bla NDM-1 gene constructed by our laboratory previously . .. The PSR reactions were carried out in a 25 μl reaction mixture containing the following components: 1.0 μl Bst DNA polymerase large fragment (New England Biolabs), 2.5 μl 10 × ThermolPol reaction buffer (New England Biolabs, including 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween 20), 0.8 M Betaine (Sigma), 6 mM MgSO4 , 1.4 mM each deoxynucleoside triphosphate.

    Real-time Polymerase Chain Reaction:

    Article Title: Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei
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    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C. .. The prepared libraries were analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by Quantitative PCR (qPCR) and then used for cluster generation and pair-end sequencing with 90 bp reads (PE90) on Illumina Hiseq 2000 (Illumina).

    Primer Extension Assay:

    Article Title: Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale
    Article Snippet: Paragraph title: Single nucleotide primer extension assay ... For each reaction, 100 nM of 32 P-primer, 130 nM of template, 800 μM of one of the dNTPs and 0.5 U of Bsu DNA polymerase large fragment (NEB) were used in 10 μL 1 × NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9).

    Incubation:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: .. An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C. .. Each reaction was spiked with 200 ng of sonicated unmethylated lambda DNA (200-400 bp) (Takara) and then subject to bisulfite conversion using a Zymo EZ DNA Methylation Kit (Zymo Research) following manufacturer's protocols involving a 14 h 50 °C incubation at dark and 22 μl (2 × 11 μl) elution.

    Article Title: Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification
    Article Snippet: The isothermal amplification was performed in a 25-μL volume containing 1 μL of ligation product mixture, 1.2 μM of each primer-1 and -2, 100 μM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100, 6% dimethyl sulfoxide, 6 μL of 1× SYBR Green I dye (Bio-Vision), and 6.4 U Bst DNA polymerase large fragment (New England BioLabs). .. The reactions were incubated at 65°C in a 96-well plate and were real-time monitored for 100 cycles with a 25-sec period by an Opticon2 DNA Engine (Bio-Rad).

    Article Title: Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)
    Article Snippet: The closed tube LAMP assay was performed as described earlier [ ].The reaction was performed in 25 μl of reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol each of F3 and B3 primers,20 pmol each of the FLP and BLP ( )] , 1.4 mM of each deoxynucleoside triphosphate, 0.8 M betaine, 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% TritonX-100, 8 units of Bst DNA polymerase large Fragment (New England Biolabs, Ipswich, MA, USA), and 2 μl of DNA sample. .. The closed tube was then incubated on a dry bath at 65°C for 30 minutes.

    Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem
    Article Snippet: Chromatin immunoprecipitation, DNA amplification and sequencing A minimum of 3 μg E. grandis DSX chromatin was incubated with 1 μg anti-H3K4me3 antibody (Millipore #07-473), or 1 μg naïve mouse IgG2a (sc-3878, Santa Cruz Biotechnology, CA) as negative control, overnight at 4°C. .. We replaced the use of Sequenase v.2.0 DNA polymerase (Affymetrix, CA) with Bsu DNA polymerase, large fragment (NEB, MA), and substituted the corresponding Sequenase reaction buffer with NEB Buffer 2.

    Article Title: Loop-mediated isothermal amplification of DNA
    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. .. 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: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: Reactions were incubated at 37°C for 60 min, followed by enzyme inactivation at 80°C for 20 min. Linearized fragment was isolated via resolution by agarose gel and gel extraction (QIAGEN). .. Biotinylated pUC18 used in was prepared by incubating the Hin dIII fragment with 1 mM dATP, dCTP, biotin-11-dGTP (Perkin Elmer), and dTTP and 10 units of Bsu DNA polymerase large fragment (NEB) in the supplied buffer at 37°C for 60 min, followed by enzyme inactivation at 75°C for 20 min.

    Ligation:

    Article Title: Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification
    Article Snippet: .. The isothermal amplification was performed in a 25-μL volume containing 1 μL of ligation product mixture, 1.2 μM of each primer-1 and -2, 100 μM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100, 6% dimethyl sulfoxide, 6 μL of 1× SYBR Green I dye (Bio-Vision), and 6.4 U Bst DNA polymerase large fragment (New England BioLabs). .. The reactions were incubated at 65°C in a 96-well plate and were real-time monitored for 100 cycles with a 25-sec period by an Opticon2 DNA Engine (Bio-Rad).

    Generated:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: T-WGBS library construction and sequencing Transposome complex was first generated by incubating 2.5 μl of 10 μM annealed adaptors with 2.5 μl 100% glycerol and 5 μl Ez-Tn5 transposase (Epicentre, Illumina) for 30 min at 25 °C. .. An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C.

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: Large linear substrates The 2.7, 7 kb, and 12 kb linear DNA substrates were generated by digesting pUC18, pEAE399, or pEAE99 with Hin dIII (NEB) according to the manufacturer’s instructions. .. Biotinylated pUC18 used in was prepared by incubating the Hin dIII fragment with 1 mM dATP, dCTP, biotin-11-dGTP (Perkin Elmer), and dTTP and 10 units of Bsu DNA polymerase large fragment (NEB) in the supplied buffer at 37°C for 60 min, followed by enzyme inactivation at 75°C for 20 min.

    other:

    Article Title: Development of Loop-Mediated Isothermal Amplification for Detection of Leifsonia xyli subsp. xyli in Sugarcane
    Article Snippet: The LAMP reaction used in further experimentation was carried out in a 25 μ L reaction mixture system containing 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 5.75 mM MgSO4 , 0.1% Triton X-100, 0.2 μ M each F3 and B3, 0.8 μ M each FIP and BIP, 8 U Bst DNA polymerase large fragment (New England Biolabs), and 1.4 mM dNTPs.

    Article Title: Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid ▿
    Article Snippet: The reaction mixture (25 μl) contained 1.6 μM concentrations (each) of FIP and BIP, 0.2 μM concentrations (each) of F3 and B3, 0.4 μM LF, 8 U of Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA), 1.4 mM deoxynucleoside triphosphates, 0.8 M betaine (Sigma, St. Louis, MO), 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 (2 μl).

    Polymerase Chain Reaction:

    Article Title: Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method
    Article Snippet: The PSR reactions were carried out in a 25 μl reaction mixture containing the following components: 1.0 μl Bst DNA polymerase large fragment (New England Biolabs), 2.5 μl 10 × ThermolPol reaction buffer (New England Biolabs, including 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween 20), 0.8 M Betaine (Sigma), 6 mM MgSO4 , 1.4 mM each deoxynucleoside triphosphate. .. Primers of PCR assay for bla NDM-1 was NDM-F (5’-ATGGAATTGCCCAATATTATGCA-3’, upstream primer) and NDM-R (5’-TCAGCGCAGCTTGTCGGCCATGC-3’, downstream primer).

    Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem
    Article Snippet: We replaced the use of Sequenase v.2.0 DNA polymerase (Affymetrix, CA) with Bsu DNA polymerase, large fragment (NEB, MA), and substituted the corresponding Sequenase reaction buffer with NEB Buffer 2. .. Both the pre-amplification and PCR reactions were supplemented with 50 ng/μl tRNA.

    Article Title: Linear nicking endonuclease-mediated strand displacement DNA amplification
    Article Snippet: DNA polymerase large fragment), Bst DNA polymerase large fragment, μ29 DNA polymerase, 9°Nm DNA polymerase, Vent exo- DNA polymerase, and BSA (bovine serum albumin) were acquired from New England Biolabs (NEB). .. PCR was performed using a Phusion PCR kit from Finnzymes.

    Sonication:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C. .. Each reaction was spiked with 200 ng of sonicated unmethylated lambda DNA (200-400 bp) (Takara) and then subject to bisulfite conversion using a Zymo EZ DNA Methylation Kit (Zymo Research) following manufacturer's protocols involving a 14 h 50 °C incubation at dark and 22 μl (2 × 11 μl) elution.

    Binding Assay:

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: .. For radiolabeled linear substrates used in filter binding reactions, pUC18, pEAE399, or pEAE99 Hin dIII fragments were extended by Bsu DNA polymerase large fragment using α 32 -dATP by a method analogous to that used to biotinylate linear DNA fragment above. ..

    Article Title: Linear nicking endonuclease-mediated strand displacement DNA amplification
    Article Snippet: DNA polymerase large fragment), Bst DNA polymerase large fragment, μ29 DNA polymerase, 9°Nm DNA polymerase, Vent exo- DNA polymerase, and BSA (bovine serum albumin) were acquired from New England Biolabs (NEB). .. Sequenase version 2.0, an engineered T7 DNA polymerase [ ], and E. coli . single-stranded DNA binding protein (SSB) were purchased from United States Biochemicals.

    Magnetic Beads:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: The purified DNA (∼ 0.5 ng, spiked with 5 pg of unmethylated lambda DNA) was then incubated with 4 μl Nextera HMW Buffer (Epicentre-Illumina), 16 μl nuclease-free water (Ambion), and 4 μl prepared Tn5mC transposome complex for 12 min at 55 °C followed by purification using 36 μl (1.8×) Agencourt AMPure XP magnetic beads; and the DNA was eluted in 14 μl EB buffer (Qiagen). .. An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C.

    Isolation:

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: Reactions were incubated at 37°C for 60 min, followed by enzyme inactivation at 80°C for 20 min. Linearized fragment was isolated via resolution by agarose gel and gel extraction (QIAGEN). .. Biotinylated pUC18 used in was prepared by incubating the Hin dIII fragment with 1 mM dATP, dCTP, biotin-11-dGTP (Perkin Elmer), and dTTP and 10 units of Bsu DNA polymerase large fragment (NEB) in the supplied buffer at 37°C for 60 min, followed by enzyme inactivation at 75°C for 20 min.

    Electrophoretic Mobility Shift Assay:

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: Biotinylated pUC18 used in was prepared by incubating the Hin dIII fragment with 1 mM dATP, dCTP, biotin-11-dGTP (Perkin Elmer), and dTTP and 10 units of Bsu DNA polymerase large fragment (NEB) in the supplied buffer at 37°C for 60 min, followed by enzyme inactivation at 75°C for 20 min. .. The presence of streptavidin was confirmed by gel shift.

    Purification:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: The purified DNA (∼ 0.5 ng, spiked with 5 pg of unmethylated lambda DNA) was then incubated with 4 μl Nextera HMW Buffer (Epicentre-Illumina), 16 μl nuclease-free water (Ambion), and 4 μl prepared Tn5mC transposome complex for 12 min at 55 °C followed by purification using 36 μl (1.8×) Agencourt AMPure XP magnetic beads; and the DNA was eluted in 14 μl EB buffer (Qiagen). .. An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C.

    Article Title: DNA synthesis from diphosphate substrates by DNA polymerases
    Article Snippet: Taq DNA polymerase, Phusion High-Fidelity DNA polymerase, DeepVent DNA polymerase, Vent DNA polymerase, Q5 DNA High-Fidelity polymerase, Bst polymerase, Bsu DNA polymerase (large fragment), and ThermoPol Reaction Buffer [1×: 20 mM Tris⋅HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton–X–100, pH 8.8 at 25 °C] were purchased from New England Biolabs. .. Pfu , DeepVent (used in primer extension reaction presented in ), KOD, and 9°N DNA polymerases were expressed and purified in-house ( ).

    Sequencing:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: Paragraph title: T-WGBS library construction and sequencing ... An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C.

    Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem
    Article Snippet: Paragraph title: Chromatin immunoprecipitation, DNA amplification and sequencing ... We replaced the use of Sequenase v.2.0 DNA polymerase (Affymetrix, CA) with Bsu DNA polymerase, large fragment (NEB, MA), and substituted the corresponding Sequenase reaction buffer with NEB Buffer 2.

    Labeling:

    Article Title: Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale
    Article Snippet: Primer was labeled with [γ32 P]-ATP according to the standard protocol. .. For each reaction, 100 nM of 32 P-primer, 130 nM of template, 800 μM of one of the dNTPs and 0.5 U of Bsu DNA polymerase large fragment (NEB) were used in 10 μL 1 × NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale
    Article Snippet: For each reaction, 100 nM of 32 P-primer, 130 nM of template, 800 μM of one of the dNTPs and 0.5 U of Bsu DNA polymerase large fragment (NEB) were used in 10 μL 1 × NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9). .. The quenched reaction mixtures were analyzed by 12% denaturing PAGE containing 8M urea.

    Chromatin Immunoprecipitation:

    Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem
    Article Snippet: Paragraph title: Chromatin immunoprecipitation, DNA amplification and sequencing ... We replaced the use of Sequenase v.2.0 DNA polymerase (Affymetrix, CA) with Bsu DNA polymerase, large fragment (NEB, MA), and substituted the corresponding Sequenase reaction buffer with NEB Buffer 2.

    Plasmid Preparation:

    Article Title: Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein
    Article Snippet: Materials and Methods Amplification reactions were performed in 50 mM Tris-Acetate, pH 7.9, 50 mM potassium acetate, 7 mM magnesium acetate, 2 mM DTT, 0.6 mM each dNTP, 1.0 μM each forward and reverse primer, 0.33 U/μL Bsu DNA Polymerase, Large Fragment (New England Biolabs (NEB)) and 300–456 ng/μl (9.0–13.6 µM) T4 gene 32 protein (NEB). .. DNA templates (NEB) were pUC19 plasmid DNA and M13mp18 single-stranded DNA.

    SYBR Green Assay:

    Article Title: Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification
    Article Snippet: .. The isothermal amplification was performed in a 25-μL volume containing 1 μL of ligation product mixture, 1.2 μM of each primer-1 and -2, 100 μM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100, 6% dimethyl sulfoxide, 6 μL of 1× SYBR Green I dye (Bio-Vision), and 6.4 U Bst DNA polymerase large fragment (New England BioLabs). .. The reactions were incubated at 65°C in a 96-well plate and were real-time monitored for 100 cycles with a 25-sec period by an Opticon2 DNA Engine (Bio-Rad).

    Article Title: Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)
    Article Snippet: The closed tube LAMP assay was performed as described earlier [ ].The reaction was performed in 25 μl of reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol each of F3 and B3 primers,20 pmol each of the FLP and BLP ( )] , 1.4 mM of each deoxynucleoside triphosphate, 0.8 M betaine, 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% TritonX-100, 8 units of Bst DNA polymerase large Fragment (New England Biolabs, Ipswich, MA, USA), and 2 μl of DNA sample. .. 1 μl of 1:10 diluted SYBR green I (Molecular Probes, Eugene, OR, USA) was placed on the inner side of the tube.

    Negative Control:

    Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem
    Article Snippet: Chromatin immunoprecipitation, DNA amplification and sequencing A minimum of 3 μg E. grandis DSX chromatin was incubated with 1 μg anti-H3K4me3 antibody (Millipore #07-473), or 1 μg naïve mouse IgG2a (sc-3878, Santa Cruz Biotechnology, CA) as negative control, overnight at 4°C. .. We replaced the use of Sequenase v.2.0 DNA polymerase (Affymetrix, CA) with Bsu DNA polymerase, large fragment (NEB, MA), and substituted the corresponding Sequenase reaction buffer with NEB Buffer 2.

    Agarose Gel Electrophoresis:

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: Reactions were incubated at 37°C for 60 min, followed by enzyme inactivation at 80°C for 20 min. Linearized fragment was isolated via resolution by agarose gel and gel extraction (QIAGEN). .. Biotinylated pUC18 used in was prepared by incubating the Hin dIII fragment with 1 mM dATP, dCTP, biotin-11-dGTP (Perkin Elmer), and dTTP and 10 units of Bsu DNA polymerase large fragment (NEB) in the supplied buffer at 37°C for 60 min, followed by enzyme inactivation at 75°C for 20 min.

    DNA Methylation Assay:

    Article Title: De novo DNA methylation during monkey pre-implantation embryogenesis
    Article Snippet: An extension step was then performed by adding 2 μl of 10× Thermopol reaction buffer (New England Biolabs), 2 μl 10 mM 5mC dNTP Mix (Zymo Research), 1 μl of Bst DNA polymerase large fragment (New England Biolabs) to each reaction mixture and incubated for 20 min at 65 °C. .. Each reaction was spiked with 200 ng of sonicated unmethylated lambda DNA (200-400 bp) (Takara) and then subject to bisulfite conversion using a Zymo EZ DNA Methylation Kit (Zymo Research) following manufacturer's protocols involving a 14 h 50 °C incubation at dark and 22 μl (2 × 11 μl) elution.

    Lamp Assay:

    Article Title: Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)
    Article Snippet: .. The closed tube LAMP assay was performed as described earlier [ ].The reaction was performed in 25 μl of reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol each of F3 and B3 primers,20 pmol each of the FLP and BLP ( )] , 1.4 mM of each deoxynucleoside triphosphate, 0.8 M betaine, 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% TritonX-100, 8 units of Bst DNA polymerase large Fragment (New England Biolabs, Ipswich, MA, USA), and 2 μl of DNA sample. .. 1 μl of 1:10 diluted SYBR green I (Molecular Probes, Eugene, OR, USA) was placed on the inner side of the tube.

    DNA Purification:

    Article Title: Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method
    Article Snippet: Then the genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Co. USA). .. The PSR reactions were carried out in a 25 μl reaction mixture containing the following components: 1.0 μl Bst DNA polymerase large fragment (New England Biolabs), 2.5 μl 10 × ThermolPol reaction buffer (New England Biolabs, including 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween 20), 0.8 M Betaine (Sigma), 6 mM MgSO4 , 1.4 mM each deoxynucleoside triphosphate.

    Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem
    Article Snippet: After crosslink reversal and DNA purification, the ChIP DNA was quantified with the Qubit HS dsDNA kit (Invitrogen). .. We replaced the use of Sequenase v.2.0 DNA polymerase (Affymetrix, CA) with Bsu DNA polymerase, large fragment (NEB, MA), and substituted the corresponding Sequenase reaction buffer with NEB Buffer 2.

    Gel Extraction:

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: Reactions were incubated at 37°C for 60 min, followed by enzyme inactivation at 80°C for 20 min. Linearized fragment was isolated via resolution by agarose gel and gel extraction (QIAGEN). .. Biotinylated pUC18 used in was prepared by incubating the Hin dIII fragment with 1 mM dATP, dCTP, biotin-11-dGTP (Perkin Elmer), and dTTP and 10 units of Bsu DNA polymerase large fragment (NEB) in the supplied buffer at 37°C for 60 min, followed by enzyme inactivation at 75°C for 20 min.

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