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Roche faststart taq dna polymerase
Efficiency of hl-dsDNase treatment. 1.3 pg of λ <t>DNA</t> was incubated with various quantities of hl-dsDNase in 90% <t>Taq</t> DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.
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1) Product Images from "An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications"

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013042

Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.
Figure Legend Snippet: Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.

Techniques Used: Incubation, Real-time Polymerase Chain Reaction

2) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

3) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

4) Product Images from "An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications"

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013042

Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.
Figure Legend Snippet: Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.

Techniques Used: Incubation, Real-time Polymerase Chain Reaction

5) Product Images from "A comprehensive look at transcription factor gene expression changes in colorectal adenomas"

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-46

Methylation analysis of the CpG island in the DACH1 promoter. (A) : Schematic depiction of the CpG islands located respectively 5’ upstream from the DACH1 transcription start site (CpG I) and in the first intron of the DACH1 gene (CpG II). (B) : Examples of CpG I COBRA analysis in colorectal cancers with intense (red), patchy (green), or no (blue) DACH1 protein immunostaining and in 4 colon cancer cell lines characterized by low (HT29 and Caco2) or very low (HCT116 and CO115) DACH1 expression (based on microarray-documented DACH1 mRNA expression levels - see also Additional file 9 ). Asterisks indicate Taq α I -digested DNA fragments representing methylated alleles; slower-migrating fragments correspond to undigested, unmethylated DNA. MW, molecular weight; bp, base pair.
Figure Legend Snippet: Methylation analysis of the CpG island in the DACH1 promoter. (A) : Schematic depiction of the CpG islands located respectively 5’ upstream from the DACH1 transcription start site (CpG I) and in the first intron of the DACH1 gene (CpG II). (B) : Examples of CpG I COBRA analysis in colorectal cancers with intense (red), patchy (green), or no (blue) DACH1 protein immunostaining and in 4 colon cancer cell lines characterized by low (HT29 and Caco2) or very low (HCT116 and CO115) DACH1 expression (based on microarray-documented DACH1 mRNA expression levels - see also Additional file 9 ). Asterisks indicate Taq α I -digested DNA fragments representing methylated alleles; slower-migrating fragments correspond to undigested, unmethylated DNA. MW, molecular weight; bp, base pair.

Techniques Used: Methylation, Combined Bisulfite Restriction Analysis Assay, Immunostaining, Expressing, Microarray, Molecular Weight

6) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

7) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

8) Product Images from "Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition"

Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077771

Reduction of PCR Inhibition by Increased Amounts of Taq Polymerase. qPCR with HMW and template DNA from FFPE tissue using different amounts of Taq polymerase. PCR-amplification of increasing amounts of genomic HMW template DNA (2.5-3,840 ng) using (A) 2 U Taq , (B) 4 U Taq and PCR-amplification of template DNA from FFPE tissue using (C) 2 U Taq , (D) 4 U Taq . Shown are the mean values (± standard deviations) from triplicate measurements.
Figure Legend Snippet: Reduction of PCR Inhibition by Increased Amounts of Taq Polymerase. qPCR with HMW and template DNA from FFPE tissue using different amounts of Taq polymerase. PCR-amplification of increasing amounts of genomic HMW template DNA (2.5-3,840 ng) using (A) 2 U Taq , (B) 4 U Taq and PCR-amplification of template DNA from FFPE tissue using (C) 2 U Taq , (D) 4 U Taq . Shown are the mean values (± standard deviations) from triplicate measurements.

Techniques Used: Polymerase Chain Reaction, Inhibition, Real-time Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded, Amplification

Inhibitory Effect of Template DNA from FFPE Tissues on PCR Performance. qPCR with 1 µg of genomic HMW template DNA and increasing amounts (60-1,440 ng) of spiked genomic template DNA from FFPE tissue Genomic DNA from FFPE tissue was treated beforehand with active DNase I (+) and heat-inactivated DNase I (-), respectively. qPCR was performed using a 150-bp fragment and 1 U Taq polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure Legend Snippet: Inhibitory Effect of Template DNA from FFPE Tissues on PCR Performance. qPCR with 1 µg of genomic HMW template DNA and increasing amounts (60-1,440 ng) of spiked genomic template DNA from FFPE tissue Genomic DNA from FFPE tissue was treated beforehand with active DNase I (+) and heat-inactivated DNase I (-), respectively. qPCR was performed using a 150-bp fragment and 1 U Taq polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.

Techniques Used: Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Successful PCR Amplification of Larger Fragments by Overcoming PCR Inhibition. PCR-amplified DNA fragments of different sizes within the PITX2 gene locus using template DNA from FFPE tissue. The PCR was carried out using 1 µg (upper and middle panel) and 5 ng (lower panel) template DNA in the presence of 1 U and 4 U Taq DNA polymerase, respectively.
Figure Legend Snippet: Successful PCR Amplification of Larger Fragments by Overcoming PCR Inhibition. PCR-amplified DNA fragments of different sizes within the PITX2 gene locus using template DNA from FFPE tissue. The PCR was carried out using 1 µg (upper and middle panel) and 5 ng (lower panel) template DNA in the presence of 1 U and 4 U Taq DNA polymerase, respectively.

Techniques Used: Polymerase Chain Reaction, Amplification, Inhibition, Formalin-fixed Paraffin-Embedded

Degradation of DNA from FFPE Tissues and its Effect on PCR Amplification of Amplicons with Different Sizes. Analysis of DNA integrity of genomic and bisulfite-converted DNA from unfixed and FFPE tissues by means of (A) agarose gel electrophoresis and (B) end-point PCR using PCR fragments of different sizes within the PITX2 gene locus. (C) qPCR results applying increasing amounts (2.5–3,840 ng) of genomic template DNA from unfixed and (D) from FFPE tissue. Shown are the mean values (± standard deviations) from triplicate measurements. Each PCR was performed with 1 U Taq polymerase. DNA from unfixed specimens is considered high molecular weight (HMW) DNA.
Figure Legend Snippet: Degradation of DNA from FFPE Tissues and its Effect on PCR Amplification of Amplicons with Different Sizes. Analysis of DNA integrity of genomic and bisulfite-converted DNA from unfixed and FFPE tissues by means of (A) agarose gel electrophoresis and (B) end-point PCR using PCR fragments of different sizes within the PITX2 gene locus. (C) qPCR results applying increasing amounts (2.5–3,840 ng) of genomic template DNA from unfixed and (D) from FFPE tissue. Shown are the mean values (± standard deviations) from triplicate measurements. Each PCR was performed with 1 U Taq polymerase. DNA from unfixed specimens is considered high molecular weight (HMW) DNA.

Techniques Used: Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Molecular Weight

Transferability of the Findings to a qPCR Targeting an Alternative Genomic Locus. qPCR applying a 200-bp PCR fragment within the ACTB gene locus. Amplification with (A) HMW DNA from unfixed tissue and (B) DNA from FFPE tissue (2.5 ng to 3,840 ng) in the presence of 1 U and 4 U Taq DNA polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure Legend Snippet: Transferability of the Findings to a qPCR Targeting an Alternative Genomic Locus. qPCR applying a 200-bp PCR fragment within the ACTB gene locus. Amplification with (A) HMW DNA from unfixed tissue and (B) DNA from FFPE tissue (2.5 ng to 3,840 ng) in the presence of 1 U and 4 U Taq DNA polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Formalin-fixed Paraffin-Embedded

PCR Inhibition by Bisulfite-Converted Template DNA from FFPE Tissues. (A) qPCR–amplification of different amounts (10–3,840 ng) of bisulfite-converted template DNA from FFPE tissue using a 129-bp PCR fragment within the ACTB gene locus. 1 U and 4 U Taq polymerase were used for qPCR. Shown are the mean values (± standard deviations) from triplicate measurements. (B) PCR amplification of the specific PCR product was confirmed by agarose gel electrophoresis using 1 U (upper panel) and 4 U (lower panel) Taq polymerase.
Figure Legend Snippet: PCR Inhibition by Bisulfite-Converted Template DNA from FFPE Tissues. (A) qPCR–amplification of different amounts (10–3,840 ng) of bisulfite-converted template DNA from FFPE tissue using a 129-bp PCR fragment within the ACTB gene locus. 1 U and 4 U Taq polymerase were used for qPCR. Shown are the mean values (± standard deviations) from triplicate measurements. (B) PCR amplification of the specific PCR product was confirmed by agarose gel electrophoresis using 1 U (upper panel) and 4 U (lower panel) Taq polymerase.

Techniques Used: Polymerase Chain Reaction, Inhibition, Formalin-fixed Paraffin-Embedded, Real-time Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

PCR Inhibition by Template DNA from FFPE Tissues with Regard to PCR Conditions (dNTP Concentration and Thermal Cycling Profile). Shown are qPCR results applying a 200-bp fragment within the PITX2 gene locus using template DNA from FFPE tissue (80-1,920 ng) in the presence of 1 U Taq polymerase. (A) dNTP concentrations and (B) annealing and elongation times were varied. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure Legend Snippet: PCR Inhibition by Template DNA from FFPE Tissues with Regard to PCR Conditions (dNTP Concentration and Thermal Cycling Profile). Shown are qPCR results applying a 200-bp fragment within the PITX2 gene locus using template DNA from FFPE tissue (80-1,920 ng) in the presence of 1 U Taq polymerase. (A) dNTP concentrations and (B) annealing and elongation times were varied. Shown are the mean values (± standard deviations) from triplicate measurements.

Techniques Used: Polymerase Chain Reaction, Inhibition, Formalin-fixed Paraffin-Embedded, Concentration Assay, Real-time Polymerase Chain Reaction

9) Product Images from "A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype"

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

Journal: PLoS ONE

doi: 10.1371/journal.pone.0114944

A comparison of potential enhancers on the PCR of α/β-tryptase gDNA using DY682-labeled (left panels) or digoxigenin-labeled (right panels) 3′-primer. The effects of ethylene glycol (0.5 to 2.5 M), betaine (0.5 to 2.5 M), glycerol (0.5 to 2.5 M), DMSO (0.5 to 10%) or no additive was examined on PCRs performed with gDNA amounts of 0.3-0.5 ng and Taq DNA polymerase of 0.75 U per PCR with ethylene glycol or betaine, or 1.2–2.0 ng of gDNA and 1.25 U of Taq DNA polymerase per PCR with glycerol or DMSO. Representative images of the 1017-28 bp amplimers from three independent experiments are shown. Band intensity data is stored in S3 Table .
Figure Legend Snippet: A comparison of potential enhancers on the PCR of α/β-tryptase gDNA using DY682-labeled (left panels) or digoxigenin-labeled (right panels) 3′-primer. The effects of ethylene glycol (0.5 to 2.5 M), betaine (0.5 to 2.5 M), glycerol (0.5 to 2.5 M), DMSO (0.5 to 10%) or no additive was examined on PCRs performed with gDNA amounts of 0.3-0.5 ng and Taq DNA polymerase of 0.75 U per PCR with ethylene glycol or betaine, or 1.2–2.0 ng of gDNA and 1.25 U of Taq DNA polymerase per PCR with glycerol or DMSO. Representative images of the 1017-28 bp amplimers from three independent experiments are shown. Band intensity data is stored in S3 Table .

Techniques Used: Polymerase Chain Reaction, Labeling

10) Product Images from "An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications"

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013042

Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.
Figure Legend Snippet: Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.

Techniques Used: Incubation, Real-time Polymerase Chain Reaction

Related Articles

Clone Assay:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche, Mannheim, Germany). .. For each region, at least 10 clones were sequenced using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied BioSystems, Foster City, CA) and ABI3100 autosequencer (Applied Biosystems).

Amplification:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: Paragraph title: PCR Amplification system ... Using the home-made qPCR mix, various Taq polymerases were tested for contamination with exogenous DNA: AmpliTaq Gold and AmpliTaq 360 DNA Polymerase (Applied Biosystems, CA, USA), FastStart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), HotStarTaq® DNA polymerase (Qiagen, Düsseldorf, Germany), GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI, USA).

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas
Article Snippet: Amplifications were carried out with FastStart Taq DNA Polymerase (Roche, Basel, Switzerland) with the following primers: CpG I: 5’-GTAGTAGTAGAAGAGAAGTAGATGA-3’ (sense) and 5’- ACCCAAATTATCCAACCAAAAACTC-3’ (antisense); CpG II: 5’-GGGTGAGGGTTTIGTTGGGA-3’ (sense) and 5’-CCCTCCCCTCIACTAACTTC-3’ (antisense). .. The amplified products were digested with the Taq α I restriction enzyme (New England Biolabs, Beverly, MA, USA) and subjected to 2% agarose gel electrophoresis and ethidium bromide staining.

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: Sequencing of sodium bisulfite-treated genomic DNA The sodium bisulfite-treated DNA (200–500 ng) was used as a template for PCR amplification of eight HTLV-I provirus regions. .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche, Mannheim, Germany).

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: To distinguish 5' and 3'-LTR, we used primers specific for gag and R region of LTR for amplification of 5'-LTR, and primers for pX region and U3 region were used for amplification of 3'-LTR. .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C.

Article Title: De Novo Human T-Cell Leukemia Virus Type 1 Infection of Human Lymphocytes in NOD-SCID, Common ?-Chain Knockout Mice ▿
Article Snippet: For a combined bisulfite restriction analysis (COBRA) , different regions of the HTLV-1 provirus were amplified from sodium bisulfite-treated genomic DNA ( ). .. The nested PCRs were performed using FastStart Taq DNA polymerase (Roche, Mannheim, Germany) under the following conditions: 95°C for 5 min; 40 cycles of 30 s at 95°C, 30 s at each annealing temperature, and 30 s at 72°C; and 2 min at 72°C for a final extension.

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: Combined bisulfite restriction analysis (COBRA) For COBRA, eight different regions of HTLV-I provirus were amplified with sodium bisulfite treated genomic DNAs using each primer sets as shown in Table . .. The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension.

Real-time Polymerase Chain Reaction:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: .. Using the home-made qPCR mix, various Taq polymerases were tested for contamination with exogenous DNA: AmpliTaq Gold and AmpliTaq 360 DNA Polymerase (Applied Biosystems, CA, USA), FastStart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), HotStarTaq® DNA polymerase (Qiagen, Düsseldorf, Germany), GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI, USA). .. Contamination of PCR reagents with foreign DNA was tested using primers amplifying the B. taurus and the human mitochondrial D-loop.

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: .. The effect on PCR performance of irradiation of the qPCR reaction buffer, of the FastStart Taq DNA polymerase and of the PCR primers was assayed by performing qPCR experiments on non-irradiated phage λ DNA with PCR mixes prepared with separately irradiated buffer components and reagents and compared with a control reaction performed with non-irradiated reagents. ..

Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition
Article Snippet: .. Endpoint PCR and qPCR Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 . .. Cycling conditions were at 95°C for 15 min and 40 cycles at 95°C/30 s, 54°C/30 s, 72°C/30 s. qPCR was performed using 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA).

Formalin-fixed Paraffin-Embedded:

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas
Article Snippet: Evaluation of DACH1 promoter methylation status in colorectal cancers Using the QIAamp DNA FFPE Tissue kit (Qiagen, no. 56404), we extracted DNA from 18 of the 80 cancers described above. .. Amplifications were carried out with FastStart Taq DNA Polymerase (Roche, Basel, Switzerland) with the following primers: CpG I: 5’-GTAGTAGTAGAAGAGAAGTAGATGA-3’ (sense) and 5’- ACCCAAATTATCCAACCAAAAACTC-3’ (antisense); CpG II: 5’-GGGTGAGGGTTTIGTTGGGA-3’ (sense) and 5’-CCCTCCCCTCIACTAACTTC-3’ (antisense).

Activity Assay:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: The tests were carried out in the storage buffer of the FastStart® Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), supplemented with 10 mM MgCl2 , 1 mM CaCl2 and 1 mM DTT that are required by the dsDNases to be active or to be efficiently inactivated. .. When we compared the activity of the two dsDNAses, we found both enzymes to be 80 times less active in 50% Taq storage buffer and 300–400 times less active in the 90% supplemented storage buffer than in the recommended conditions of usage (see ).

Expressing:

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas
Article Snippet: DACH1 expression in these cancers was marked and ubiquitous in 6, patchy in 6, and completely lost in 6 (see examples in the Results section), and each of these 3 groups included 3 tumors that were MMR-proficient and 3 that were MMR-deficient. .. Amplifications were carried out with FastStart Taq DNA Polymerase (Roche, Basel, Switzerland) with the following primers: CpG I: 5’-GTAGTAGTAGAAGAGAAGTAGATGA-3’ (sense) and 5’- ACCCAAATTATCCAACCAAAAACTC-3’ (antisense); CpG II: 5’-GGGTGAGGGTTTIGTTGGGA-3’ (sense) and 5’-CCCTCCCCTCIACTAACTTC-3’ (antisense).

Immunoprecipitation:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: The chromatin solutions were immunoprecipitated with anti-acetyl-Histone H3 or anti-acetyl-Histone H4 (Upstate Biotechnology), or normal rabbit IgG, overnight at 4°C, and the immunoprecipitates were then collected with 50% protein A and G-Sepharose slurry preabsorbed with 0.1 mg/ml sonicated salmon sperm DNA. .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C.

Cell Culture:

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype
Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated. .. Human mast cell leukemia cells (HMC)-1,obtained from a patient with mast cell leukemia and provided by Dr. G. Gleich and Dr. J. Butterfield (Mayo Clinic, Rochester, MN) , known to contain only βI and βIII variants of the β-tryptase gene; and Mono Mac-6 cells , a cell line established from a patient with monoblastic leukemia and containing both α- and β- tryptase genes , were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, penicillin and streptomycin at 37°C/5% CO2 .

Irradiation:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: .. The effect on PCR performance of irradiation of the qPCR reaction buffer, of the FastStart Taq DNA polymerase and of the PCR primers was assayed by performing qPCR experiments on non-irradiated phage λ DNA with PCR mixes prepared with separately irradiated buffer components and reagents and compared with a control reaction performed with non-irradiated reagents. ..

Polymerase Chain Reaction:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: Paragraph title: PCR Amplification system ... Using the home-made qPCR mix, various Taq polymerases were tested for contamination with exogenous DNA: AmpliTaq Gold and AmpliTaq 360 DNA Polymerase (Applied Biosystems, CA, USA), FastStart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), HotStarTaq® DNA polymerase (Qiagen, Düsseldorf, Germany), GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI, USA).

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche, Mannheim, Germany). .. The PCR primer pairs and annealing temperatures are shown in Table .

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: .. The effect on PCR performance of irradiation of the qPCR reaction buffer, of the FastStart Taq DNA polymerase and of the PCR primers was assayed by performing qPCR experiments on non-irradiated phage λ DNA with PCR mixes prepared with separately irradiated buffer components and reagents and compared with a control reaction performed with non-irradiated reagents. ..

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C. .. The PCR products were electrophoresed in an agarose gel and the results were analyzed using ATTO Densitograph Ver.

Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition
Article Snippet: .. Endpoint PCR and qPCR Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 . .. Cycling conditions were at 95°C for 15 min and 40 cycles at 95°C/30 s, 54°C/30 s, 72°C/30 s. qPCR was performed using 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA).

Article Title: De Novo Human T-Cell Leukemia Virus Type 1 Infection of Human Lymphocytes in NOD-SCID, Common ?-Chain Knockout Mice ▿
Article Snippet: The nested PCRs were performed using FastStart Taq DNA polymerase (Roche, Mannheim, Germany) under the following conditions: 95°C for 5 min; 40 cycles of 30 s at 95°C, 30 s at each annealing temperature, and 30 s at 72°C; and 2 min at 72°C for a final extension. .. The PCR products were digested for at least 4 h with TaqI restriction enzyme, which resulted in a single recognition site within each product.

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: This latter property makes these nucleases potentially superior to DNAse I because at these temperatures, it is likely that Hot-Start Taq polymerases are not prematurely activated and other proteins such as BSA are not denatured. dsDNase has been used to minimize contamination of buffer, dNTPs and primers prior to PCR, but not of the Taq DNA polymerase . .. The tests were carried out in the storage buffer of the FastStart® Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), supplemented with 10 mM MgCl2 , 1 mM CaCl2 and 1 mM DTT that are required by the dsDNases to be active or to be efficiently inactivated.

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: DNA methylation in neighboring regions of HTLV-I integration sites The integration sites of HTLV-I provirus has been determined by inverse long PCR, and DNA methylation of genomic DNAs neighboring integration sites was determined in both ATL cells and PBMCs. .. The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension.

Article Title: Comparison of Microsatellite Length Polymorphism and Multilocus Sequence Typing for DNA-Based Typing of Candida albicans ▿
Article Snippet: .. PCRs were carried out in a 50-μl reaction volume containing about 1 μl of extracted DNA, 5 μl of 10× PCR buffer, a 2.5 mM concentration of MgCl2 , a 0.200 mM concentration of each deoxynucleotide triphosphate, 10 pmol of each primer, and 1.25 U of FastStart Taq DNA polymerase (Roche Applied Science). .. The PCR conditions consisted of an initial denaturation step of 6 min at 95°C, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 min, with a final extension step of 5 min at 72°C.

Sonication:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: The chromatin solutions were immunoprecipitated with anti-acetyl-Histone H3 or anti-acetyl-Histone H4 (Upstate Biotechnology), or normal rabbit IgG, overnight at 4°C, and the immunoprecipitates were then collected with 50% protein A and G-Sepharose slurry preabsorbed with 0.1 mg/ml sonicated salmon sperm DNA. .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C.

Affinity Purification:

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype
Article Snippet: .. Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated. .. Human mast cell leukemia cells (HMC)-1,obtained from a patient with mast cell leukemia and provided by Dr. G. Gleich and Dr. J. Butterfield (Mayo Clinic, Rochester, MN) , known to contain only βI and βIII variants of the β-tryptase gene; and Mono Mac-6 cells , a cell line established from a patient with monoblastic leukemia and containing both α- and β- tryptase genes , were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, penicillin and streptomycin at 37°C/5% CO2 .

Recombinant:

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype
Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated. .. Primary human skin mast cells (SMCs), obtained from discarded surgical skin of 24 subjects (10 African Americans, 14 Caucasian or Latino), who ranged in age from 21 to 60 years, with written informed consent as approved by the Virginia Commonwealth University Internal Review Board, were cultured in serum-free X-Vivo-15 medium (Lonza Inc., Allendale, NJ) containing recombinant human SCF (100 ng/ml)(provided by Swedish Orphan Biovitrum, Stockholm, Sweden) as described .

Combined Bisulfite Restriction Analysis Assay:

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas
Article Snippet: Sodium bisulfite conversion of genomic DNA was performed as previously described [ ], and the resulting DNA was subjected to combined bisulfite restriction analysis (COBRA) to determine the methylation status of two CpG islands located respectively upstream the transcription start site (CpG I) and in the first intron (CpG II) of the DACH1 gene. .. Amplifications were carried out with FastStart Taq DNA Polymerase (Roche, Basel, Switzerland) with the following primers: CpG I: 5’-GTAGTAGTAGAAGAGAAGTAGATGA-3’ (sense) and 5’- ACCCAAATTATCCAACCAAAAACTC-3’ (antisense); CpG II: 5’-GGGTGAGGGTTTIGTTGGGA-3’ (sense) and 5’-CCCTCCCCTCIACTAACTTC-3’ (antisense).

Article Title: De Novo Human T-Cell Leukemia Virus Type 1 Infection of Human Lymphocytes in NOD-SCID, Common ?-Chain Knockout Mice ▿
Article Snippet: Paragraph title: COBRA. ... The nested PCRs were performed using FastStart Taq DNA polymerase (Roche, Mannheim, Germany) under the following conditions: 95°C for 5 min; 40 cycles of 30 s at 95°C, 30 s at each annealing temperature, and 30 s at 72°C; and 2 min at 72°C for a final extension.

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: Paragraph title: Combined bisulfite restriction analysis (COBRA) ... The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension.

Methylation:

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas
Article Snippet: Paragraph title: Evaluation of DACH1 promoter methylation status in colorectal cancers ... Amplifications were carried out with FastStart Taq DNA Polymerase (Roche, Basel, Switzerland) with the following primers: CpG I: 5’-GTAGTAGTAGAAGAGAAGTAGATGA-3’ (sense) and 5’- ACCCAAATTATCCAACCAAAAACTC-3’ (antisense); CpG II: 5’-GGGTGAGGGTTTIGTTGGGA-3’ (sense) and 5’-CCCTCCCCTCIACTAACTTC-3’ (antisense).

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension. .. When CpG site within amplified region was methylated, it was resistant to sodium bisulfite treatment, resulting in digestion by these enzymes.

Size-exclusion Chromatography:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C. .. The PCR products were electrophoresed in an agarose gel and the results were analyzed using ATTO Densitograph Ver.

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension. .. RT-PCR Total RNA was isolated from PBMCs or lymph node cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) and RT-PCR was performed using RNA LA PCR Kit (AMV) Ver.

Purification:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche, Mannheim, Germany). .. The amplified PCR products were purified and subcloned into pGEM-T Easy vectors (Promega).

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype
Article Snippet: .. Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated. .. Human mast cell leukemia cells (HMC)-1,obtained from a patient with mast cell leukemia and provided by Dr. G. Gleich and Dr. J. Butterfield (Mayo Clinic, Rochester, MN) , known to contain only βI and βIII variants of the β-tryptase gene; and Mono Mac-6 cells , a cell line established from a patient with monoblastic leukemia and containing both α- and β- tryptase genes , were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, penicillin and streptomycin at 37°C/5% CO2 .

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: The resulting purified DNAs were subjected to PCR reactions using primer sets specific for 5'-LTR, env , pX and 3'-LTR. .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C.

Article Title: Comparison of Microsatellite Length Polymorphism and Multilocus Sequence Typing for DNA-Based Typing of Candida albicans ▿
Article Snippet: PCRs were carried out in a 50-μl reaction volume containing about 1 μl of extracted DNA, 5 μl of 10× PCR buffer, a 2.5 mM concentration of MgCl2 , a 0.200 mM concentration of each deoxynucleotide triphosphate, 10 pmol of each primer, and 1.25 U of FastStart Taq DNA polymerase (Roche Applied Science). .. Sequencing reactions were prepared using the ABI PRISM Big Dye Terminator v3.1 cycle sequencing kit (PE Applied Biosystems) and were analyzed, after a purification step by Sephadex columns, on an ABI PRISM 3130xl genetic analyzer (PE Applied Biosystems).

Sequencing:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: Paragraph title: Sequencing of sodium bisulfite-treated genomic DNA ... The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche, Mannheim, Germany).

Article Title: Comparison of Microsatellite Length Polymorphism and Multilocus Sequence Typing for DNA-Based Typing of Candida albicans ▿
Article Snippet: PCRs were carried out in a 50-μl reaction volume containing about 1 μl of extracted DNA, 5 μl of 10× PCR buffer, a 2.5 mM concentration of MgCl2 , a 0.200 mM concentration of each deoxynucleotide triphosphate, 10 pmol of each primer, and 1.25 U of FastStart Taq DNA polymerase (Roche Applied Science). .. Sequencing reactions were prepared using the ABI PRISM Big Dye Terminator v3.1 cycle sequencing kit (PE Applied Biosystems) and were analyzed, after a purification step by Sephadex columns, on an ABI PRISM 3130xl genetic analyzer (PE Applied Biosystems).

Nested PCR:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension. .. RT-PCR Total RNA was isolated from PBMCs or lymph node cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) and RT-PCR was performed using RNA LA PCR Kit (AMV) Ver.

Chromatin Immunoprecipitation:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) assay ... The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C.

Software:

Article Title: Comparison of Microsatellite Length Polymorphism and Multilocus Sequence Typing for DNA-Based Typing of Candida albicans ▿
Article Snippet: PCRs were carried out in a 50-μl reaction volume containing about 1 μl of extracted DNA, 5 μl of 10× PCR buffer, a 2.5 mM concentration of MgCl2 , a 0.200 mM concentration of each deoxynucleotide triphosphate, 10 pmol of each primer, and 1.25 U of FastStart Taq DNA polymerase (Roche Applied Science). .. The data were stored and analyzed with SeqScape software (Applied Biosystems).

SYBR Green Assay:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: We either used commercial Master mixes for qPCR, i.e., PLATINUM® Quantitative PCR SUPERMIX-UDG (Invitrogen, St. Louis, MO, USA) and FastStart DNA MasterPLUS SYBR Green mix (Roche Applied Science, Mannheim, Germany), or a home-made qPCR mix prepared according to Lutfalla and Uze . .. Using the home-made qPCR mix, various Taq polymerases were tested for contamination with exogenous DNA: AmpliTaq Gold and AmpliTaq 360 DNA Polymerase (Applied Biosystems, CA, USA), FastStart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), HotStarTaq® DNA polymerase (Qiagen, Düsseldorf, Germany), GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI, USA).

Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition
Article Snippet: Endpoint PCR and qPCR Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 . .. 20-µL reactions were conducted in triplicates, containing 0.4 µM of each primer (0.15 µM ACTB bisulfite primer, ), 0.3 µM locus-specific hydrolysis probe , 0.25 mM dNTPs, 1 x PCR buffer [35 mM Tris (pH8.4), 50 mM KCl, 6 mM MgCl2 ], 0.125 x SYBR Green® I (Lonza, Rockland, ME, USA) and varying amounts of FastStart Taq DNA polymerase.

Agarose Gel Electrophoresis:

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas
Article Snippet: Amplifications were carried out with FastStart Taq DNA Polymerase (Roche, Basel, Switzerland) with the following primers: CpG I: 5’-GTAGTAGTAGAAGAGAAGTAGATGA-3’ (sense) and 5’- ACCCAAATTATCCAACCAAAAACTC-3’ (antisense); CpG II: 5’-GGGTGAGGGTTTIGTTGGGA-3’ (sense) and 5’-CCCTCCCCTCIACTAACTTC-3’ (antisense). .. The amplified products were digested with the Taq α I restriction enzyme (New England Biolabs, Beverly, MA, USA) and subjected to 2% agarose gel electrophoresis and ethidium bromide staining.

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C, 35 or 37 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C. .. The PCR products were electrophoresed in an agarose gel and the results were analyzed using ATTO Densitograph Ver.

Preserving:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: To decontaminate Taq DNA polymerase, we chose to use conditions that minimize changes in the Taq storage buffer composition in order to enable long term preservation of the enzyme at −20°C after decontamination in batch. .. The tests were carried out in the storage buffer of the FastStart® Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), supplemented with 10 mM MgCl2 , 1 mM CaCl2 and 1 mM DTT that are required by the dsDNases to be active or to be efficiently inactivated.

DNA Methylation Assay:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: Paragraph title: DNA methylation in neighboring regions of HTLV-I integration sites ... The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension.

Concentration Assay:

Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications
Article Snippet: To test the efficiency of the dsDNases as a decontaminating agent of Taq polymerases, primers and dNTPs and to find the minimal enzyme concentration ensuring most efficient decontamination, we evaluated the degradation of λ DNA using dilutions of dsDNAses in different buffer conditions. .. The tests were carried out in the storage buffer of the FastStart® Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), supplemented with 10 mM MgCl2 , 1 mM CaCl2 and 1 mM DTT that are required by the dsDNases to be active or to be efficiently inactivated.

Article Title: Comparison of Microsatellite Length Polymorphism and Multilocus Sequence Typing for DNA-Based Typing of Candida albicans ▿
Article Snippet: .. PCRs were carried out in a 50-μl reaction volume containing about 1 μl of extracted DNA, 5 μl of 10× PCR buffer, a 2.5 mM concentration of MgCl2 , a 0.200 mM concentration of each deoxynucleotide triphosphate, 10 pmol of each primer, and 1.25 U of FastStart Taq DNA polymerase (Roche Applied Science). .. The PCR conditions consisted of an initial denaturation step of 6 min at 95°C, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 min, with a final extension step of 5 min at 72°C.

DNA Purification:

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype
Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated. .. Genomic DNA from SMCs, HMC-1, or Mono Mac-6 cells was purified using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI).

Staining:

Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas
Article Snippet: Amplifications were carried out with FastStart Taq DNA Polymerase (Roche, Basel, Switzerland) with the following primers: CpG I: 5’-GTAGTAGTAGAAGAGAAGTAGATGA-3’ (sense) and 5’- ACCCAAATTATCCAACCAAAAACTC-3’ (antisense); CpG II: 5’-GGGTGAGGGTTTIGTTGGGA-3’ (sense) and 5’-CCCTCCCCTCIACTAACTTC-3’ (antisense). .. The amplified products were digested with the Taq α I restriction enzyme (New England Biolabs, Beverly, MA, USA) and subjected to 2% agarose gel electrophoresis and ethidium bromide staining.

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    Roche pcr reactions
    PTPN11 mutations identified in MC participants. (A) Schematic of the exonic structure of PTPN11 (above) and the corresponding protein structure of SHP2 (below). The locations of mutations that were identified in MC are indicated with black lines. Predicted protein changes are indicated for the nonsense (blue) and frameshift (red) mutations, while the cDNA designation is indicated for the splice-site mutations (green). (B) Log2 values of the number of Illumina reads obtained per 50 bp window in participant S, divided by the average number of reads obtained in other participants whose <t>DNA</t> was captured simultaneously using the second capture array. Shown are all 50 bp windows spanning regions of PTPN11 targeted by the array, with the corresponding exonic structure of PTPN11 shown below. The red bar indicates a region spanning exon 7, in which the average log2 value is approximately −1, suggesting a heterozygous deletion. <t>PCR</t> amplification and sequencing of the breakpoint, using primers on either end of the deletion, indicate that 14,629 bp of sequence have been deleted and replaced with a single CA dinucleotide.
    Pcr Reactions, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 794 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche faststart taq dna polymerase
    Efficiency of hl-dsDNase treatment. 1.3 pg of λ <t>DNA</t> was incubated with various quantities of hl-dsDNase in 90% <t>Taq</t> DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.
    Faststart Taq Dna Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/faststart taq dna polymerase/product/Roche
    Average 99 stars, based on 488 article reviews
    Price from $9.99 to $1999.99
    faststart taq dna polymerase - by Bioz Stars, 2020-04
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    Image Search Results


    PTPN11 mutations identified in MC participants. (A) Schematic of the exonic structure of PTPN11 (above) and the corresponding protein structure of SHP2 (below). The locations of mutations that were identified in MC are indicated with black lines. Predicted protein changes are indicated for the nonsense (blue) and frameshift (red) mutations, while the cDNA designation is indicated for the splice-site mutations (green). (B) Log2 values of the number of Illumina reads obtained per 50 bp window in participant S, divided by the average number of reads obtained in other participants whose DNA was captured simultaneously using the second capture array. Shown are all 50 bp windows spanning regions of PTPN11 targeted by the array, with the corresponding exonic structure of PTPN11 shown below. The red bar indicates a region spanning exon 7, in which the average log2 value is approximately −1, suggesting a heterozygous deletion. PCR amplification and sequencing of the breakpoint, using primers on either end of the deletion, indicate that 14,629 bp of sequence have been deleted and replaced with a single CA dinucleotide.

    Journal: PLoS Genetics

    Article Title: Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome

    doi: 10.1371/journal.pgen.1002050

    Figure Lengend Snippet: PTPN11 mutations identified in MC participants. (A) Schematic of the exonic structure of PTPN11 (above) and the corresponding protein structure of SHP2 (below). The locations of mutations that were identified in MC are indicated with black lines. Predicted protein changes are indicated for the nonsense (blue) and frameshift (red) mutations, while the cDNA designation is indicated for the splice-site mutations (green). (B) Log2 values of the number of Illumina reads obtained per 50 bp window in participant S, divided by the average number of reads obtained in other participants whose DNA was captured simultaneously using the second capture array. Shown are all 50 bp windows spanning regions of PTPN11 targeted by the array, with the corresponding exonic structure of PTPN11 shown below. The red bar indicates a region spanning exon 7, in which the average log2 value is approximately −1, suggesting a heterozygous deletion. PCR amplification and sequencing of the breakpoint, using primers on either end of the deletion, indicate that 14,629 bp of sequence have been deleted and replaced with a single CA dinucleotide.

    Article Snippet: Libraries used for hybridization to the first capture array were amplified according to two strategies: 3 µl amplified for 18 cycles in four 50 µl PCR reactions (Phusion High-Fidelity DNA polymerase, Finnzymes), or 2 µl amplified for 11 cycles in one 50 µl PCR reaction that was then purified and amplified for 17 cycles in ten 50 µl PCR reactions (FastStart Taq DNA polymerase, Roche).

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing

    Loss of the wild-type PTPN11 allele in the cartilage of two exostoses. (A) Electropherograms of PCR amplified template DNA that had been extracted from whole blood, a section of an exostosis, or the cartilage core of the same exostosis. Exostoses were available from patients A-IV-5 and A-IV-8. The site of the 5 bp deletion in exon 4 of PTPN11 in both patients is indicated with a box. Note that that the heights of the peaks corresponding to the mutant sequence are markedly reduced in amplimers from the cartilage-core compared to amplimers from blood or from a section that contains cartilage, bone and fibrous tissue. This is consistent with loss-of-heterozygosity in the cartilage component of the exostoses. (B) Electropherograms of PCR amplified template DNA extracted from blood from participants A-III-9, A-III-10 and A-IV-5, as well as DNA extracted from the cartilage core of the exostosis from participant A-IV-5 shown in (A). Blood DNA electropherograms indicate that participants A-III-9 and A-IV-10 are heterozygous at a position (asterisk) in intron 11 of PTPN11 . This is the site of a known common polymorphism (rs41279092). Exostosis cartilage DNA electropherograms have a reduced adenine peak height at this position. This suggests that the wild-type PTPN11 allele inherited from the unaffected parent (A-III-9), which carries an adenine at this position, has been lost in cells that contribute to formation of the exostosis' cartilage core.

    Journal: PLoS Genetics

    Article Title: Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome

    doi: 10.1371/journal.pgen.1002050

    Figure Lengend Snippet: Loss of the wild-type PTPN11 allele in the cartilage of two exostoses. (A) Electropherograms of PCR amplified template DNA that had been extracted from whole blood, a section of an exostosis, or the cartilage core of the same exostosis. Exostoses were available from patients A-IV-5 and A-IV-8. The site of the 5 bp deletion in exon 4 of PTPN11 in both patients is indicated with a box. Note that that the heights of the peaks corresponding to the mutant sequence are markedly reduced in amplimers from the cartilage-core compared to amplimers from blood or from a section that contains cartilage, bone and fibrous tissue. This is consistent with loss-of-heterozygosity in the cartilage component of the exostoses. (B) Electropherograms of PCR amplified template DNA extracted from blood from participants A-III-9, A-III-10 and A-IV-5, as well as DNA extracted from the cartilage core of the exostosis from participant A-IV-5 shown in (A). Blood DNA electropherograms indicate that participants A-III-9 and A-IV-10 are heterozygous at a position (asterisk) in intron 11 of PTPN11 . This is the site of a known common polymorphism (rs41279092). Exostosis cartilage DNA electropherograms have a reduced adenine peak height at this position. This suggests that the wild-type PTPN11 allele inherited from the unaffected parent (A-III-9), which carries an adenine at this position, has been lost in cells that contribute to formation of the exostosis' cartilage core.

    Article Snippet: Libraries used for hybridization to the first capture array were amplified according to two strategies: 3 µl amplified for 18 cycles in four 50 µl PCR reactions (Phusion High-Fidelity DNA polymerase, Finnzymes), or 2 µl amplified for 11 cycles in one 50 µl PCR reaction that was then purified and amplified for 17 cycles in ten 50 µl PCR reactions (FastStart Taq DNA polymerase, Roche).

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis, Sequencing

    Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.

    Journal: PLoS ONE

    Article Title: An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

    doi: 10.1371/journal.pone.0013042

    Figure Lengend Snippet: Efficiency of hl-dsDNase treatment. 1.3 pg of λ DNA was incubated with various quantities of hl-dsDNase in 90% Taq DNA polymerase storage buffer for 30 minutes at 25°C and qPCR quantification of a 73 bp fragment was performed. The average of the percentage of remaining DNA is plotted as a function of the quantities of hl-dsDNase.

    Article Snippet: The effect on PCR performance of irradiation of the qPCR reaction buffer, of the FastStart Taq DNA polymerase and of the PCR primers was assayed by performing qPCR experiments on non-irradiated phage λ DNA with PCR mixes prepared with separately irradiated buffer components and reagents and compared with a control reaction performed with non-irradiated reagents.

    Techniques: Incubation, Real-time Polymerase Chain Reaction

    DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

    Journal: Retrovirology

    Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

    doi: 10.1186/1742-4690-2-64

    Figure Lengend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

    Article Snippet: The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension.

    Techniques: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation