taq dna polymerase  (Thermo Fisher)


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    Name:
    Taq DNA Polymerase
    Description:
    Thermo Scientific Taq DNA Polymerase is a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus The enzyme catalyzes 5 →3 synthesis of DNA has no detectable 3 →5 exonuclease proofreading activity and possesses low 5 →3 exonuclease activity In addition Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity which frequently results in the addition of extra adenines at the 3 end of PCR products Recombinant Taq DNA Polymerase is the ideal tool for standard PCR of templates 5 kb or shorter Highlights• Thermostable half life is more than 40 min at 95°C• Generates PCR products with 3 dA overhangs• Supplied with two buffers 10X Taq Buffer with KCl and 10X Taq Buffer with NH4 2SO4 The latter allows for PCR at wide range of magnesium concentrations and decreases unspecific priming• Incorporates modified nucleotides e g biotin digoxigenin fluorescently labeled nucleotides Applications• Routine PCR amplification of DNA fragments up to 5 kb• High throughput PCR• DNA labelingNote• The error rate of Taq DNA Polymerase in PCR is 2 2 x 10 5 errors per nt per cycle Accordingly the accuracy of PCR is 4 5 x 104 Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs • The 10X Taq Buffer without Detergent is recommended for microarray experiments
    Catalog Number:
    ep0401
    Price:
    None
    Applications:
    PCR|PCR & Real-Time PCR|Routine PCR
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher taq dna polymerase
    A mobility shift assay for TaqS <t>DNA</t> polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- <t>Taq</t> S DNA polymerase, respectively.
    Thermo Scientific Taq DNA Polymerase is a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus The enzyme catalyzes 5 →3 synthesis of DNA has no detectable 3 →5 exonuclease proofreading activity and possesses low 5 →3 exonuclease activity In addition Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity which frequently results in the addition of extra adenines at the 3 end of PCR products Recombinant Taq DNA Polymerase is the ideal tool for standard PCR of templates 5 kb or shorter Highlights• Thermostable half life is more than 40 min at 95°C• Generates PCR products with 3 dA overhangs• Supplied with two buffers 10X Taq Buffer with KCl and 10X Taq Buffer with NH4 2SO4 The latter allows for PCR at wide range of magnesium concentrations and decreases unspecific priming• Incorporates modified nucleotides e g biotin digoxigenin fluorescently labeled nucleotides Applications• Routine PCR amplification of DNA fragments up to 5 kb• High throughput PCR• DNA labelingNote• The error rate of Taq DNA Polymerase in PCR is 2 2 x 10 5 errors per nt per cycle Accordingly the accuracy of PCR is 4 5 x 104 Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs • The 10X Taq Buffer without Detergent is recommended for microarray experiments
    https://www.bioz.com/result/taq dna polymerase/product/Thermo Fisher
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    Images

    1) Product Images from "Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization"

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184162

    A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.
    Figure Legend Snippet: A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Techniques Used: Mobility Shift, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).
    Figure Legend Snippet: Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.
    Figure Legend Snippet: Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Techniques Used: Activity Assay

    2) Product Images from "Genetic Diversity of Pasteurella dagmatis as Assessed by Analysis of the 16S rRNA and rpoB Gene Sequences"

    Article Title: Genetic Diversity of Pasteurella dagmatis as Assessed by Analysis of the 16S rRNA and rpoB Gene Sequences

    Journal: Current Microbiology

    doi: 10.1007/s00284-011-9949-6

    Agarose gel electrophoresis of a 476-bp fragment of the P . dagmatis 16S rRNA gene. Lanes 1 – 8 represent amplicons obtained from DNA of canine isolates, lanes 9 – 16 from DNA of feline isolates, and lanes 17 – 18 from DNA of the isolate from a tiger. For each isolate, intact amplicons ( lanes with odd numbers ) and Taq I-digested ones ( lanes with even numbers ) were run. M size marker (GeneRuler ™ 100 bp DNA Ladder [Fermentas])
    Figure Legend Snippet: Agarose gel electrophoresis of a 476-bp fragment of the P . dagmatis 16S rRNA gene. Lanes 1 – 8 represent amplicons obtained from DNA of canine isolates, lanes 9 – 16 from DNA of feline isolates, and lanes 17 – 18 from DNA of the isolate from a tiger. For each isolate, intact amplicons ( lanes with odd numbers ) and Taq I-digested ones ( lanes with even numbers ) were run. M size marker (GeneRuler ™ 100 bp DNA Ladder [Fermentas])

    Techniques Used: Agarose Gel Electrophoresis, Marker

    3) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    Journal: Nucleic Acids Research

    doi:

    Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
    Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

    Techniques Used: Sequencing

    Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
    Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

    Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

    Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
    Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

    Techniques Used: Incubation

    The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
    Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

    Techniques Used: Activity Assay, Labeling, Concentration Assay

    4) Product Images from "Targeted sequencing library preparation by genomic DNA circularization"

    Article Title: Targeted sequencing library preparation by genomic DNA circularization

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-122

    Targeted sequencing library preparation method . (a) Overview of the assay. (b) Specific preparation steps: (1) genomic DNA is digested using Mse I restriction endonuclease. (2) Then, genomic DNA fragments are circularized using thermostable DNA ligase and Taq DNA polymerase for 5' editing. Pool of oligonucleotides targeting 5' and 3' ends of the DNA fragments and vector oligonucleotide are used for targeted DNA capture. (3) After circularization, regular Illumina sequencing library can be prepared by PCR. (4) PCR amplified library fragments are similar to regular Illumina library constructs and anneal to immobilized primers on the flow cell. (5) Additionally, circular constructs can be directly sequenced as the adapted genomic DNA circles incorporate all DNA components required for library immobilization and sequencing. (c) Molecular structures of vector oligonucleotide and capture oligonucleotides.
    Figure Legend Snippet: Targeted sequencing library preparation method . (a) Overview of the assay. (b) Specific preparation steps: (1) genomic DNA is digested using Mse I restriction endonuclease. (2) Then, genomic DNA fragments are circularized using thermostable DNA ligase and Taq DNA polymerase for 5' editing. Pool of oligonucleotides targeting 5' and 3' ends of the DNA fragments and vector oligonucleotide are used for targeted DNA capture. (3) After circularization, regular Illumina sequencing library can be prepared by PCR. (4) PCR amplified library fragments are similar to regular Illumina library constructs and anneal to immobilized primers on the flow cell. (5) Additionally, circular constructs can be directly sequenced as the adapted genomic DNA circles incorporate all DNA components required for library immobilization and sequencing. (c) Molecular structures of vector oligonucleotide and capture oligonucleotides.

    Techniques Used: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Flow Cytometry

    5) Product Images from "Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish"

    Article Title: Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007754

    Conditional mutagenesis pipeline. Upon deciding which exon to flox, we recommend sequencing the target sites to identify polymorphisms compared to reference genome. Next, sgRNAs should be designed and tested by either direct sequencing of PCR fragments, T7 endonuclease assay or loss of a restriction enzyme site on bulk DNA from pooled embryos. Once active sgRNAs have been identified, experiments integrating the first loxP site should be performed. In the absence of conclusive data that certain HDR template performs significantly better than others (such experiments are not practical at the only level that matters–germline transmission), we recommend using the design we successfully used to integrate loxP into fleer , aldh1a2 and tcf21 : antisense to PAM, with 49-base 5’ homology arm and 21-base 3’ homology arm, with 3-nucleotide spacers flanking loxP site. As injected embryos are being raised, we then recommend to optimize nested PCR screening conditions DNA from pools of injected embryos. We found “plain” Taq polymerases (NEB #M0270, Thermofisher Scientific 2x PCR Master Mix Cat# AB-0575/DC and #EP0402, or similar) to be most suitable for nested PCR. In contrast, high-performance mixes such as Platinum Taq (Thermofisher #10966026) or Kapa 2G Fast ReadyMix + dye (Kapa Biosystems- KM5101) yield very high background and may only be used for the second (nested) reaction. It is also very helpful if primers for one end of the nested PCR are anchored within an exon. We recommend generating a deletion allele in parallel with integration of the first loxP site. Once highly active sgRNAs are identified, we recommend injecting a pair of sgRNAs flanking the exon to be floxed in order to confirm that removal of selected exon will yield an overt phenotype. We have been able to very efficiently delete exon 8 of aldh1a2 using sgRNAs spaced just over 450 base pairs, but larger deletions are certainly feasible too (1, 2). An additional benefit of a deletion allele is that it can be crossed to Cre drivers of interest, eliminating the need to back-cross floxed allele to obtain homozygotes. Screening for germline transmission should be performed by nested PCR on pools of embryos obtained from incross. Positive crosses should be analyzed by performing short flanking PCR (ideally under 400 base pairs) on DNA from individual embryos. Bands corresponding to loxP-containing allele should be extracted from gel and sequenced to ensure presence of intact loxP site. Siblings of screened embryos should be raised to adulthood and loxP-positive F1s should be identified by flanking PCR as well. Two strategies can be used for integration of the second loxP site. If speed is the main priority, loxP-positive F1s can be in-crossed and second sgRNA/HDR oligonucleotide can be injected. The main drawback of this strategy that there is only 50% likelihood that the second loxP site will integrate into a chromosome already containing the first loxP. It is therefore necessary to genotype adults for presence of the first loxP site before out-crossing. Even though we successfully used this strategy to engineer a floxed allele of tbx20 , we consider it impractical and would generally recommend to first generate adults homozygous for the first loxP site, incross them and then inject the second sgRNA/HDR oligonucleotide.
    Figure Legend Snippet: Conditional mutagenesis pipeline. Upon deciding which exon to flox, we recommend sequencing the target sites to identify polymorphisms compared to reference genome. Next, sgRNAs should be designed and tested by either direct sequencing of PCR fragments, T7 endonuclease assay or loss of a restriction enzyme site on bulk DNA from pooled embryos. Once active sgRNAs have been identified, experiments integrating the first loxP site should be performed. In the absence of conclusive data that certain HDR template performs significantly better than others (such experiments are not practical at the only level that matters–germline transmission), we recommend using the design we successfully used to integrate loxP into fleer , aldh1a2 and tcf21 : antisense to PAM, with 49-base 5’ homology arm and 21-base 3’ homology arm, with 3-nucleotide spacers flanking loxP site. As injected embryos are being raised, we then recommend to optimize nested PCR screening conditions DNA from pools of injected embryos. We found “plain” Taq polymerases (NEB #M0270, Thermofisher Scientific 2x PCR Master Mix Cat# AB-0575/DC and #EP0402, or similar) to be most suitable for nested PCR. In contrast, high-performance mixes such as Platinum Taq (Thermofisher #10966026) or Kapa 2G Fast ReadyMix + dye (Kapa Biosystems- KM5101) yield very high background and may only be used for the second (nested) reaction. It is also very helpful if primers for one end of the nested PCR are anchored within an exon. We recommend generating a deletion allele in parallel with integration of the first loxP site. Once highly active sgRNAs are identified, we recommend injecting a pair of sgRNAs flanking the exon to be floxed in order to confirm that removal of selected exon will yield an overt phenotype. We have been able to very efficiently delete exon 8 of aldh1a2 using sgRNAs spaced just over 450 base pairs, but larger deletions are certainly feasible too (1, 2). An additional benefit of a deletion allele is that it can be crossed to Cre drivers of interest, eliminating the need to back-cross floxed allele to obtain homozygotes. Screening for germline transmission should be performed by nested PCR on pools of embryos obtained from incross. Positive crosses should be analyzed by performing short flanking PCR (ideally under 400 base pairs) on DNA from individual embryos. Bands corresponding to loxP-containing allele should be extracted from gel and sequenced to ensure presence of intact loxP site. Siblings of screened embryos should be raised to adulthood and loxP-positive F1s should be identified by flanking PCR as well. Two strategies can be used for integration of the second loxP site. If speed is the main priority, loxP-positive F1s can be in-crossed and second sgRNA/HDR oligonucleotide can be injected. The main drawback of this strategy that there is only 50% likelihood that the second loxP site will integrate into a chromosome already containing the first loxP. It is therefore necessary to genotype adults for presence of the first loxP site before out-crossing. Even though we successfully used this strategy to engineer a floxed allele of tbx20 , we consider it impractical and would generally recommend to first generate adults homozygous for the first loxP site, incross them and then inject the second sgRNA/HDR oligonucleotide.

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Transmission Assay, Injection, Nested PCR

    6) Product Images from "A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)"

    Article Title: A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)

    Journal: BMC Genetics

    doi: 10.1186/s12863-015-0284-y

    Differential peak morphologies of androgen receptor alleles resulting from DNA dilution and reagent use. Lynx positive control DNA sample amplified with Invitrogen Taq DNA Polymerase and diluted to 1:10 ( a ), 1:20 ( b ), and 1:50 ( c ) ratios with deionized water. Lynx positive control DNA sample amplified with Invitrogen Platinum Taq DNA Polymerase (no dilution necessary) ( d )
    Figure Legend Snippet: Differential peak morphologies of androgen receptor alleles resulting from DNA dilution and reagent use. Lynx positive control DNA sample amplified with Invitrogen Taq DNA Polymerase and diluted to 1:10 ( a ), 1:20 ( b ), and 1:50 ( c ) ratios with deionized water. Lynx positive control DNA sample amplified with Invitrogen Platinum Taq DNA Polymerase (no dilution necessary) ( d )

    Techniques Used: Positive Control, Amplification

    7) Product Images from "An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV"

    Article Title: An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

    Journal: Retrovirology

    doi: 10.1186/1742-4690-7-110

    One-step RT-PCR for identification of contaminants in Kit I and Platinum Taq . (A-C) One-step RT-PCR for identification of a contaminated component in Kit I. The experiments were conducted in two independent laboratories, IVR and JRC. In IVR, nucleic acids were extracted from 50 μl of the enzyme mix of the RT-PCR Kit I using an RNA purification column (QIAamp viral RNA mini kit [Cat. no. 52904] [QIAGEN]) and the presence of polytropic endogenous MLV was examined by using the RT-PCR Kit T (A) and Kit P (B). In JRC, nucleic acids were extracted from 75 μl of the enzyme mix of RT-PCR Kit I using an RNA/DNA purification column (PureLink™ Viral RNA/DNA Kit [Cat. no. 12280-050] [Invitrogen]), and the presence of polytropic endogenous MLV was examined using Kit Q (C). Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR conditions for Kit T and Kit P were the same as in Figure 1B. The RT-PCR conditions for Kit Q were as follows: reverse transcription at 50°C for 30 minutes; activation at 95°C for 15 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. Lanes 1 and 5, DW; lanes 2 and 6, column-purified carrier RNA (carrier); lanes 3 and 7, column-purified nucleic acids from enzyme mix (enzyme) of the Kit I; lanes 4 and 8, 1 μl buffer of the Kit I plus 4 μl DW (buffer). (D) One-step RT-PCR for the detection of MLV RNA in Platinum Taq. Nucleic acids were extracted from 50 μl of the Platinum Taq using an RNA purification column (QIAamp viral RNA mini kit [QIAGEN]) and the presence of MLV RNA was examined by using the RT-PCR Kit P. Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR condition was the same as in Figure 1B with the exception of the PCR cycles (60 cycles instead of 45 cycles). Abbreviation; DW: distilled water. M: DNA size marker.
    Figure Legend Snippet: One-step RT-PCR for identification of contaminants in Kit I and Platinum Taq . (A-C) One-step RT-PCR for identification of a contaminated component in Kit I. The experiments were conducted in two independent laboratories, IVR and JRC. In IVR, nucleic acids were extracted from 50 μl of the enzyme mix of the RT-PCR Kit I using an RNA purification column (QIAamp viral RNA mini kit [Cat. no. 52904] [QIAGEN]) and the presence of polytropic endogenous MLV was examined by using the RT-PCR Kit T (A) and Kit P (B). In JRC, nucleic acids were extracted from 75 μl of the enzyme mix of RT-PCR Kit I using an RNA/DNA purification column (PureLink™ Viral RNA/DNA Kit [Cat. no. 12280-050] [Invitrogen]), and the presence of polytropic endogenous MLV was examined using Kit Q (C). Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR conditions for Kit T and Kit P were the same as in Figure 1B. The RT-PCR conditions for Kit Q were as follows: reverse transcription at 50°C for 30 minutes; activation at 95°C for 15 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. Lanes 1 and 5, DW; lanes 2 and 6, column-purified carrier RNA (carrier); lanes 3 and 7, column-purified nucleic acids from enzyme mix (enzyme) of the Kit I; lanes 4 and 8, 1 μl buffer of the Kit I plus 4 μl DW (buffer). (D) One-step RT-PCR for the detection of MLV RNA in Platinum Taq. Nucleic acids were extracted from 50 μl of the Platinum Taq using an RNA purification column (QIAamp viral RNA mini kit [QIAGEN]) and the presence of MLV RNA was examined by using the RT-PCR Kit P. Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR condition was the same as in Figure 1B with the exception of the PCR cycles (60 cycles instead of 45 cycles). Abbreviation; DW: distilled water. M: DNA size marker.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Purification, DNA Purification, Activation Assay, Polymerase Chain Reaction, Marker

    8) Product Images from "Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection"

    Article Title: Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection

    Journal: RNA

    doi: 10.1261/rna.066753.118

    Effect of various ribonucleotide substitutions on iLock probe RNA detection assay with PBCV-1 ligase. Recognition of the invader structure and structure-specific endonucleolytic activity of Taq DNA polymerase can vary for different RNA substitutions. ( A ) Targeting let-7a with iLock probe. Ribonucleotides were introduced: at a terminal 3′ base (3); base in the 5′ arm, that an invading 3′ arm competes with for target binding (displaced base, 3D); base in the 5′ arm, that becomes a 5′-phosphorylated donor after iLock probe activation (3D5); in the flap sequence (3DF/DF). ( B ) iLocks were modified according to A , except nonchimeric iLock (DNA). The total number of RCPs for each probe is shown on y -axis. ( C ) PAGE of three selected iLock probes (DNA, 3, 3D) after activation and ligation, without (first three lanes) and with Taq DNA polymerase (last three lanes). Nonactivated iLock probe (79) is shortened upon activation by 14 nt (65) and ligated (seen as high molecular weight band at the top of the gel). (22) let-7a miRNA.
    Figure Legend Snippet: Effect of various ribonucleotide substitutions on iLock probe RNA detection assay with PBCV-1 ligase. Recognition of the invader structure and structure-specific endonucleolytic activity of Taq DNA polymerase can vary for different RNA substitutions. ( A ) Targeting let-7a with iLock probe. Ribonucleotides were introduced: at a terminal 3′ base (3); base in the 5′ arm, that an invading 3′ arm competes with for target binding (displaced base, 3D); base in the 5′ arm, that becomes a 5′-phosphorylated donor after iLock probe activation (3D5); in the flap sequence (3DF/DF). ( B ) iLocks were modified according to A , except nonchimeric iLock (DNA). The total number of RCPs for each probe is shown on y -axis. ( C ) PAGE of three selected iLock probes (DNA, 3, 3D) after activation and ligation, without (first three lanes) and with Taq DNA polymerase (last three lanes). Nonactivated iLock probe (79) is shortened upon activation by 14 nt (65) and ligated (seen as high molecular weight band at the top of the gel). (22) let-7a miRNA.

    Techniques Used: RNA Detection, Activity Assay, Binding Assay, Activation Assay, Sequencing, Modification, Polyacrylamide Gel Electrophoresis, Ligation, Molecular Weight

    9) Product Images from "Data of self-made Taq DNA polymerase prepared for screening purposes"

    Article Title: Data of self-made Taq DNA polymerase prepared for screening purposes

    Journal: Data in Brief

    doi: 10.1016/j.dib.2017.03.004

    Comparative PCR analyses of different samples with the self-made enzyme and the commercial ones. PCR analyses of the same DNA fragment as a part of a plasmid (A) and bacterial chromosome (B). The reactions contained (1) 2.5 u and (2) 5 u of Taq DNA polymerase supplied by Evrogen; (3) 0.5 μl, (4) 1 μl and (5) 2 μl of the self-made enzyme. Lane M contains the DNA ladder (New England Biolabs, #N3239S): 0.5, 1, 1.5, 2, 3 (more intense fragment), 4, 5, 6, 8, 10, 15, 20, 48.5 kbp. A common master mix was prepared for all samples. (C) PCR analyses of two cassettes incorporated in a binary plasmid DNA (3, 6) and in genomic DNA of transgenic chrysanthemum plant (2, 5). Lane M contains the DNA ladder (Thermo Fisher Scientific, #SM0321): 100, 200, 300, 400, 500 (more intense fragment), 600, 700, 800, 900, 1000 (more intense fragment), 1200, 1500, 2000, 3000 bp. On the left from lane M there are the reactions which contained 1 u of Thermo Fisher Scientific Taq DNA polymerase (#EP0402). On the right from the lane M there are the reactions which contained 1 μl of the self-made enzyme. Lanes (1 and 4) contains the control reactions without addition of DNA. A common master mix was prepared for all samples.
    Figure Legend Snippet: Comparative PCR analyses of different samples with the self-made enzyme and the commercial ones. PCR analyses of the same DNA fragment as a part of a plasmid (A) and bacterial chromosome (B). The reactions contained (1) 2.5 u and (2) 5 u of Taq DNA polymerase supplied by Evrogen; (3) 0.5 μl, (4) 1 μl and (5) 2 μl of the self-made enzyme. Lane M contains the DNA ladder (New England Biolabs, #N3239S): 0.5, 1, 1.5, 2, 3 (more intense fragment), 4, 5, 6, 8, 10, 15, 20, 48.5 kbp. A common master mix was prepared for all samples. (C) PCR analyses of two cassettes incorporated in a binary plasmid DNA (3, 6) and in genomic DNA of transgenic chrysanthemum plant (2, 5). Lane M contains the DNA ladder (Thermo Fisher Scientific, #SM0321): 100, 200, 300, 400, 500 (more intense fragment), 600, 700, 800, 900, 1000 (more intense fragment), 1200, 1500, 2000, 3000 bp. On the left from lane M there are the reactions which contained 1 u of Thermo Fisher Scientific Taq DNA polymerase (#EP0402). On the right from the lane M there are the reactions which contained 1 μl of the self-made enzyme. Lanes (1 and 4) contains the control reactions without addition of DNA. A common master mix was prepared for all samples.

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Transgenic Assay

    Analysis of Taq DNA polymerase activity in the enzyme stock fractions: (1–3) the PCR reactions with 0.5 µl, 1 µl and 2 µl of the working enzyme solution, (4–6) the PCR reaction with 0.5 µl, 1 µl and 2 µl of the AS suspension supernatant.
    Figure Legend Snippet: Analysis of Taq DNA polymerase activity in the enzyme stock fractions: (1–3) the PCR reactions with 0.5 µl, 1 µl and 2 µl of the working enzyme solution, (4–6) the PCR reaction with 0.5 µl, 1 µl and 2 µl of the AS suspension supernatant.

    Techniques Used: Activity Assay, Polymerase Chain Reaction

    10) Product Images from "Demonstration of a potent RET transcriptional inhibitor for the treatment of medullary thyroid carcinoma based on an ellipticine derivative"

    Article Title: Demonstration of a potent RET transcriptional inhibitor for the treatment of medullary thyroid carcinoma based on an ellipticine derivative

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2017.3994

    Taq DNA polymerase assay and DMS footprinting to validate the stabilization of RET G-quadruplex by NSC311153. (A) DNA polymerase stop assay at increasing concentrations of NSC311153. Lanes A, G, T and C represent the di-deoxy sequencing reactions with the same template, which serve as the marker to locate the exact stop site. Lane P represents the position of the free primer on the gel. (B) DMS footprinting on the RET-WT G-quadruplex forming sequence in the absence and presence of NSC311153 (5 equivalents) following 0.2% DMS treatment (lanes C and D, respectively). Purine and pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes AG and TC, respectively). (C) Schematic models for the parallel G-quadruplexes formed by RET-WT sequence in the absence and presence of NSC311153.
    Figure Legend Snippet: Taq DNA polymerase assay and DMS footprinting to validate the stabilization of RET G-quadruplex by NSC311153. (A) DNA polymerase stop assay at increasing concentrations of NSC311153. Lanes A, G, T and C represent the di-deoxy sequencing reactions with the same template, which serve as the marker to locate the exact stop site. Lane P represents the position of the free primer on the gel. (B) DMS footprinting on the RET-WT G-quadruplex forming sequence in the absence and presence of NSC311153 (5 equivalents) following 0.2% DMS treatment (lanes C and D, respectively). Purine and pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes AG and TC, respectively). (C) Schematic models for the parallel G-quadruplexes formed by RET-WT sequence in the absence and presence of NSC311153.

    Techniques Used: Footprinting, Sequencing, Marker, Activated Clotting Time Assay

    11) Product Images from "Rapid Detection of rpoB Gene Mutations in Rifampin-Resistant Mycobacterium tuberculosis Isolates in Shanghai by Using the Amplification Refractory Mutation System"

    Article Title: Rapid Detection of rpoB Gene Mutations in Rifampin-Resistant Mycobacterium tuberculosis Isolates in Shanghai by Using the Amplification Refractory Mutation System

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.3.993-997.2003

    Comparison of DNA sequences of rpoB genes in Rif s and Rif r M. tuberculosis isolates. The mutated nucleotides of Rif r isolates are boldfaced. ARMS primers used in this study are shown above and below the sequences. The line with the arrow shows that PCR can be performed well, whereas the line with the dot shows that the ARMS primer is refractory to extension by Taq DNA polymerase. Underlined letters indicate the nucleotide alterations introduced to enhance the 3′ mismatch effect. Codon numbers are assigned on the basis of alignment of the translated E. coli rpoB sequence with a portion of the translated M. tuberculosis sequence and are not the positions of the actual M. tuberculosis rpoB codons.
    Figure Legend Snippet: Comparison of DNA sequences of rpoB genes in Rif s and Rif r M. tuberculosis isolates. The mutated nucleotides of Rif r isolates are boldfaced. ARMS primers used in this study are shown above and below the sequences. The line with the arrow shows that PCR can be performed well, whereas the line with the dot shows that the ARMS primer is refractory to extension by Taq DNA polymerase. Underlined letters indicate the nucleotide alterations introduced to enhance the 3′ mismatch effect. Codon numbers are assigned on the basis of alignment of the translated E. coli rpoB sequence with a portion of the translated M. tuberculosis sequence and are not the positions of the actual M. tuberculosis rpoB codons.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    12) Product Images from "A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)"

    Article Title: A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)

    Journal: BMC Genetics

    doi: 10.1186/s12863-015-0284-y

    Differential peak morphologies of androgen receptor alleles resulting from DNA dilution and reagent use. Lynx positive control DNA sample amplified with Invitrogen Taq DNA Polymerase and diluted to 1:10 ( a ), 1:20 ( b ), and 1:50 ( c ) ratios with deionized water. Lynx positive control DNA sample amplified with Invitrogen Platinum Taq DNA Polymerase (no dilution necessary) ( d )
    Figure Legend Snippet: Differential peak morphologies of androgen receptor alleles resulting from DNA dilution and reagent use. Lynx positive control DNA sample amplified with Invitrogen Taq DNA Polymerase and diluted to 1:10 ( a ), 1:20 ( b ), and 1:50 ( c ) ratios with deionized water. Lynx positive control DNA sample amplified with Invitrogen Platinum Taq DNA Polymerase (no dilution necessary) ( d )

    Techniques Used: Positive Control, Amplification

    13) Product Images from "Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis"

    Article Title: Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070942

    Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.
    Figure Legend Snippet: Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Serial Dilution, Plasmid Preparation, Negative Control

    14) Product Images from "Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein"

    Article Title: Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    A) Gel agarose visualization of PCR products amplified by internal primers after amplification of cDNA as template. M (DNA marker); 1–3 (interest fragment amplified in optimized Tm=58° C ); 4 (negative control, human genomic DNA); B) PCR amplification results of the PTZ57R-PR vector containing protease ORF (301 bp ). Lanes 1 and 2 (DNA marker); lanes 3–6 (amplified by Taq DNA polymerase); lanes 7–8 (amplified by pfu DNA polymerase); lane 9 (negative control).
    Figure Legend Snippet: A) Gel agarose visualization of PCR products amplified by internal primers after amplification of cDNA as template. M (DNA marker); 1–3 (interest fragment amplified in optimized Tm=58° C ); 4 (negative control, human genomic DNA); B) PCR amplification results of the PTZ57R-PR vector containing protease ORF (301 bp ). Lanes 1 and 2 (DNA marker); lanes 3–6 (amplified by Taq DNA polymerase); lanes 7–8 (amplified by pfu DNA polymerase); lane 9 (negative control).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Negative Control, Plasmid Preparation

    15) Product Images from "rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira"

    Article Title: rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira

    Journal: Journal of Clinical Microbiology

    doi:

    Specific PCR amplification of the spirochetal rpoB gene. The PCR assay was performed by using Taq polymerase with an annealing temperature of 52°C. Lanes: 1, molecular mass markers (DNA molecular weight marker VI; Boehringer); 2, Borrelia burgdorferi ; 3, Borrelia recurrentis ; 4, Treponema pallidum ; 5, Leptospira biflexa serovar patoc; 6, Leptospira interrogans , serovar australis; 7, Leptospira interrogans , serovar icterohaemmorragiae; 8, Escherichia coli ; 9, Staphylococcus aureus ; 10, Streptococcus salivarius ; 11, Pseudomonas aeruginosa ; 12, negative control without DNA. (A) rpoB gene primers LTB 1730F and LTB 2900R. (B) rpoB gene primers LTB 1730F and LTB 3700R. (C) 16S rRNA gene primers FD1 and RP2.
    Figure Legend Snippet: Specific PCR amplification of the spirochetal rpoB gene. The PCR assay was performed by using Taq polymerase with an annealing temperature of 52°C. Lanes: 1, molecular mass markers (DNA molecular weight marker VI; Boehringer); 2, Borrelia burgdorferi ; 3, Borrelia recurrentis ; 4, Treponema pallidum ; 5, Leptospira biflexa serovar patoc; 6, Leptospira interrogans , serovar australis; 7, Leptospira interrogans , serovar icterohaemmorragiae; 8, Escherichia coli ; 9, Staphylococcus aureus ; 10, Streptococcus salivarius ; 11, Pseudomonas aeruginosa ; 12, negative control without DNA. (A) rpoB gene primers LTB 1730F and LTB 2900R. (B) rpoB gene primers LTB 1730F and LTB 3700R. (C) 16S rRNA gene primers FD1 and RP2.

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Negative Control

    16) Product Images from "Functional Characterization of the Stringent Response Regulatory Gene dksA of Vibrio cholerae and Its Role in Modulation of Virulence Phenotypes"

    Article Title: Functional Characterization of the Stringent Response Regulatory Gene dksA of Vibrio cholerae and Its Role in Modulation of Virulence Phenotypes

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00518-12

    (A) Growth complementation of the Δ dksA Ec strain with a functional dksA Vc gene in M9M medium. E. coli strains used are as follows: Wt, CF1648; ΔdksA, CF9240; ΔdksA+pDksAvc, CF9240(pDDKW1); ΔdksA+pDksAec, CF9240(pJK537); ΔdksA+pEmpty, CF9240(pDrive). Error bars indicate standard deviations. (B) Genomic arrangement of the dksA gene (gray arrow), including its flanking genetic determinants (VC0590 to VC0600), in V. cholerae . The direction of each arrow indicates the direction of transcription of a gene. VC0598 and VC0599 are two small hypothetical ORFs (white arrows). The insertion location of the kanamycin resistance gene ( kan ) cassette (small filled triangle) and its direction of transcription are also shown. (C) RT-PCR analysis to show that the deletion of dksA Vc did not hamper transcription of downstream genes. V. cholerae strains used are the Wt (C6709) and the Δ dksA Vc mutant (C-DksA1). Lanes: M, pBluescript II KS(+) plasmid DNA digested with HaeIII, used as markers; sizes (in kb) of the DNA fragments are given in the left margin; R, RT with Taq DNA polymerase; N, only Taq DNA polymerase (used as a negative control).
    Figure Legend Snippet: (A) Growth complementation of the Δ dksA Ec strain with a functional dksA Vc gene in M9M medium. E. coli strains used are as follows: Wt, CF1648; ΔdksA, CF9240; ΔdksA+pDksAvc, CF9240(pDDKW1); ΔdksA+pDksAec, CF9240(pJK537); ΔdksA+pEmpty, CF9240(pDrive). Error bars indicate standard deviations. (B) Genomic arrangement of the dksA gene (gray arrow), including its flanking genetic determinants (VC0590 to VC0600), in V. cholerae . The direction of each arrow indicates the direction of transcription of a gene. VC0598 and VC0599 are two small hypothetical ORFs (white arrows). The insertion location of the kanamycin resistance gene ( kan ) cassette (small filled triangle) and its direction of transcription are also shown. (C) RT-PCR analysis to show that the deletion of dksA Vc did not hamper transcription of downstream genes. V. cholerae strains used are the Wt (C6709) and the Δ dksA Vc mutant (C-DksA1). Lanes: M, pBluescript II KS(+) plasmid DNA digested with HaeIII, used as markers; sizes (in kb) of the DNA fragments are given in the left margin; R, RT with Taq DNA polymerase; N, only Taq DNA polymerase (used as a negative control).

    Techniques Used: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Plasmid Preparation, Negative Control

    17) Product Images from "The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance"

    Article Title: The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance

    Journal: TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik

    doi: 10.1007/s00122-007-0667-1

    Patterns of amplified DNA of resistant parent cv. Rywal ( lanes 1, 3, 5 and 7) and susceptible parent Accent ( lanes 2, 4, 6 and 8 ), indicating the ISSR marker UBC895 1200 ( lane 1 ), the STS marker SC895 1139 ( lane 3 ), the SCAR marker GP41 443 ( lane 5 ) and the COSII marker C2_At3g16840 after digestion with Taq I ( lanes 7 and 8 ). Arrows point to the marker products that were scored. Lanes M contain the 250-bp ( upper ) and 100-bp ( lower ) DNA ladders as molecular size markers
    Figure Legend Snippet: Patterns of amplified DNA of resistant parent cv. Rywal ( lanes 1, 3, 5 and 7) and susceptible parent Accent ( lanes 2, 4, 6 and 8 ), indicating the ISSR marker UBC895 1200 ( lane 1 ), the STS marker SC895 1139 ( lane 3 ), the SCAR marker GP41 443 ( lane 5 ) and the COSII marker C2_At3g16840 after digestion with Taq I ( lanes 7 and 8 ). Arrows point to the marker products that were scored. Lanes M contain the 250-bp ( upper ) and 100-bp ( lower ) DNA ladders as molecular size markers

    Techniques Used: Amplification, Marker

    18) Product Images from "Large scale multiplex PCR improves pathogen detection by DNA microarrays"

    Article Title: Large scale multiplex PCR improves pathogen detection by DNA microarrays

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-1

    Large scale multiplex PCR with 800 primer pairs . Gel electrophoresis of PCR products obtained with high complexity 800-primer pair mix (Additional file 1 ) with a final concentration of 0.02 μM for each individual primer pair and using Taq polymerase (standard LSplex) (A) or using vent exo-polymerase (B and C). Efficiency of LSplex using primer mix with different individual primer concentrations (B). Optimized LSplex amplification of various DNA templates from Gram-negative, Gram-positive bacteria and Candida albicans (C). 100 ng genomic DNA from each indicated species served as template.
    Figure Legend Snippet: Large scale multiplex PCR with 800 primer pairs . Gel electrophoresis of PCR products obtained with high complexity 800-primer pair mix (Additional file 1 ) with a final concentration of 0.02 μM for each individual primer pair and using Taq polymerase (standard LSplex) (A) or using vent exo-polymerase (B and C). Efficiency of LSplex using primer mix with different individual primer concentrations (B). Optimized LSplex amplification of various DNA templates from Gram-negative, Gram-positive bacteria and Candida albicans (C). 100 ng genomic DNA from each indicated species served as template.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Concentration Assay, Amplification

    19) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.05.27.120238

    Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
    Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification

    SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.
    Figure Legend Snippet: SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Amplification

    TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.
    Figure Legend Snippet: TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

    Techniques Used: Quantitative RT-PCR, Amplification

    20) Product Images from "Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus"

    Article Title: Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

    Journal: Enzyme Research

    doi: 10.1155/2015/837842

    PCR reactions to amplify intermediate and long regions of DNA. Reactions were conducted as described under Section 2 . PCR reactions were allowed to proceed for 30 cycles. The same amount of primer and template was used in Tth pol III HE and Taq polymerase reactions. Primers used are shown in Table 3 . PCR product sizes are as indicated. (a) The template was pET Blue-2 plasmid (Novagen) and primers were designed to yield 1500 and 2500 bp products. (b) The template was Lambda DNA- Hind III Digest and primers were designed to yield a 4650 and 7500 bp product. (c) The template was Lambda DNA- Hind III Digest and primers were designed to yield a 12500 and 15000 bp product.
    Figure Legend Snippet: PCR reactions to amplify intermediate and long regions of DNA. Reactions were conducted as described under Section 2 . PCR reactions were allowed to proceed for 30 cycles. The same amount of primer and template was used in Tth pol III HE and Taq polymerase reactions. Primers used are shown in Table 3 . PCR product sizes are as indicated. (a) The template was pET Blue-2 plasmid (Novagen) and primers were designed to yield 1500 and 2500 bp products. (b) The template was Lambda DNA- Hind III Digest and primers were designed to yield a 4650 and 7500 bp product. (c) The template was Lambda DNA- Hind III Digest and primers were designed to yield a 12500 and 15000 bp product.

    Techniques Used: Polymerase Chain Reaction, Positron Emission Tomography, Plasmid Preparation, Lambda DNA Preparation

    PCR reactions for amplification of short regions of DNA. PCR reactions and agarose gel analysis were as described under Section 2 . PCR cycles consisted of 94°C/30 s, 55°C/1 min, and 72°C/2 min. Taq Pol indicates PCR reactions using Taq DNA polymerase (18038-018, Invitrogen) per manufacturer's instructions. Primers used are shown in Table 3 . (a) PCR reactions using primers designed to yield a 200 bp PCR product. (b) PCR reactions using primers designed to yield a 512 bp PCR product. The number of PCR cycles is indicated at bottom of the figures.
    Figure Legend Snippet: PCR reactions for amplification of short regions of DNA. PCR reactions and agarose gel analysis were as described under Section 2 . PCR cycles consisted of 94°C/30 s, 55°C/1 min, and 72°C/2 min. Taq Pol indicates PCR reactions using Taq DNA polymerase (18038-018, Invitrogen) per manufacturer's instructions. Primers used are shown in Table 3 . (a) PCR reactions using primers designed to yield a 200 bp PCR product. (b) PCR reactions using primers designed to yield a 512 bp PCR product. The number of PCR cycles is indicated at bottom of the figures.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    21) Product Images from "The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance"

    Article Title: The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance

    Journal: TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik

    doi: 10.1007/s00122-007-0667-1

    Patterns of amplified DNA of resistant parent cv. Rywal ( lanes 1, 3, 5 and 7) and susceptible parent Accent ( lanes 2, 4, 6 and 8 ), indicating the ISSR marker UBC895 1200 ( lane 1 ), the STS marker SC895 1139 ( lane 3 ), the SCAR marker GP41 443 ( lane 5 ) and the COSII marker C2_At3g16840 after digestion with Taq I ( lanes 7 and 8 ). Arrows point to the marker products that were scored. Lanes M contain the 250-bp ( upper ) and 100-bp ( lower ) DNA ladders as molecular size markers
    Figure Legend Snippet: Patterns of amplified DNA of resistant parent cv. Rywal ( lanes 1, 3, 5 and 7) and susceptible parent Accent ( lanes 2, 4, 6 and 8 ), indicating the ISSR marker UBC895 1200 ( lane 1 ), the STS marker SC895 1139 ( lane 3 ), the SCAR marker GP41 443 ( lane 5 ) and the COSII marker C2_At3g16840 after digestion with Taq I ( lanes 7 and 8 ). Arrows point to the marker products that were scored. Lanes M contain the 250-bp ( upper ) and 100-bp ( lower ) DNA ladders as molecular size markers

    Techniques Used: Amplification, Marker

    22) Product Images from "Selective control of primer usage in multiplex one-step reverse transcription PCR"

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-10-113

    Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.
    Figure Legend Snippet: Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Polymerase Chain Reaction

    One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.
    Figure Legend Snippet: One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.
    Figure Legend Snippet: Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Incubation

    Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.
    Figure Legend Snippet: Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Standard Deviation, Agarose Gel Electrophoresis

    Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.
    Figure Legend Snippet: Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    23) Product Images from "Mechanical properties of DNA-like polymers"

    Article Title: Mechanical properties of DNA-like polymers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt808

    Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
    Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

    Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

    24) Product Images from "Identification of Four Entamoeba histolytica Organellar DNA Polymerases of the Family B and Cellular Localization of the Ehodp1 Gene and EhODP1 Protein"

    Article Title: Identification of Four Entamoeba histolytica Organellar DNA Polymerases of the Family B and Cellular Localization of the Ehodp1 Gene and EhODP1 Protein

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2010/734898

    Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m
    Figure Legend Snippet: Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m

    Techniques Used: In Situ, Polymerase Chain Reaction, Staining, Microscopy, Amplification, Negative Control, Labeling

    25) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.05.27.120238

    Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
    Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification

    SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.
    Figure Legend Snippet: SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Amplification

    TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.
    Figure Legend Snippet: TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

    Techniques Used: Quantitative RT-PCR, Amplification

    26) Product Images from "Quantitative Detection of Hepadnavirus-Infected Lymphoid Cells by In Situ PCR Combined with Flow Cytometry: Implications for the Study of Occult Virus Persistence"

    Article Title: Quantitative Detection of Hepadnavirus-Infected Lymphoid Cells by In Situ PCR Combined with Flow Cytometry: Implications for the Study of Occult Virus Persistence

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.2.970-979.2003

    Sensitivity and specificity of WHV DNA detection by PCR with the oligonucleotide primer pair homologous to the virus core gene sequence containing a fluorescein-conjugated sense primer. Serial 10-fold dilutions of full-length, recombinant WHV DNA (rWHV DNA) were amplified with either PPCC-CCOV (A) or FITC-PPCC-CCOV (B) primer pairs under the same PCR conditions. In parallel, supernatants from WHV-naive PBMC or PBMC derived from a chronic WHV carrier obtained after in situ PCR with FITC-PPCC-CCOV or, as controls, FITC-PPCC in the absence of Taq DNA polymerase or in the absence of both FITC-PPCC and Taq DNA polymerase were analyzed (B). The amplified WHV DNA sequences were identified by Southern blot hybridization to 32 P-labeled recombinant WHV DNA as a probe. The results show that either standard PCR procedure employing labeled or unlabeled PPCC primer or in situ PCR method with the FITC-PPCC primer generate DNA fragments of identical molecular size (423 bp) and specificity and that the use of the FITC-PPCC primer does not modify the sensitivity of WHV core gene detection.
    Figure Legend Snippet: Sensitivity and specificity of WHV DNA detection by PCR with the oligonucleotide primer pair homologous to the virus core gene sequence containing a fluorescein-conjugated sense primer. Serial 10-fold dilutions of full-length, recombinant WHV DNA (rWHV DNA) were amplified with either PPCC-CCOV (A) or FITC-PPCC-CCOV (B) primer pairs under the same PCR conditions. In parallel, supernatants from WHV-naive PBMC or PBMC derived from a chronic WHV carrier obtained after in situ PCR with FITC-PPCC-CCOV or, as controls, FITC-PPCC in the absence of Taq DNA polymerase or in the absence of both FITC-PPCC and Taq DNA polymerase were analyzed (B). The amplified WHV DNA sequences were identified by Southern blot hybridization to 32 P-labeled recombinant WHV DNA as a probe. The results show that either standard PCR procedure employing labeled or unlabeled PPCC primer or in situ PCR method with the FITC-PPCC primer generate DNA fragments of identical molecular size (423 bp) and specificity and that the use of the FITC-PPCC primer does not modify the sensitivity of WHV core gene detection.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Recombinant, Amplification, Derivative Assay, In Situ, Southern Blot, Hybridization, Labeling

    Detection of WHV DNA in lymphoid cells from WHV-infected woodchucks and a healthy control by in situ PCR coupled with flow cytometry or with hybridization analysis of cell supernatants obtained after PCR amplification. (A) Representative flow cytometry results showing WHV DNA-positive populations of PBMC from an animal with progressive WHV surface antigen-positive chronic hepatitis (Evident infection) and from a woodchuck with silent WHV infection continuing for 12 months after resolution of acute hepatitis (Silent infection) and from a control, WHV-naive woodchuck (top row). The same cell samples treated under identical cycling conditions but in the absence of FITC-PPCC primer and Taq DNA polymerase (pol.) showing almost nonexisting background autofluorescence (middle row) or after omitting Taq DNA polymerase illustrating signals obtained due to hybridization of the FITC-labeled PCCC primer with WHV DNA in PBMC from chronic and residual WHV infections but not from a healthy animal (bottom row). (B) Dot blot hybridization analysis of supernatants collected after PBMC underwent in situ PCR, confirming the validity of positive flow cytometry signals and negative controls shown in panel A.
    Figure Legend Snippet: Detection of WHV DNA in lymphoid cells from WHV-infected woodchucks and a healthy control by in situ PCR coupled with flow cytometry or with hybridization analysis of cell supernatants obtained after PCR amplification. (A) Representative flow cytometry results showing WHV DNA-positive populations of PBMC from an animal with progressive WHV surface antigen-positive chronic hepatitis (Evident infection) and from a woodchuck with silent WHV infection continuing for 12 months after resolution of acute hepatitis (Silent infection) and from a control, WHV-naive woodchuck (top row). The same cell samples treated under identical cycling conditions but in the absence of FITC-PPCC primer and Taq DNA polymerase (pol.) showing almost nonexisting background autofluorescence (middle row) or after omitting Taq DNA polymerase illustrating signals obtained due to hybridization of the FITC-labeled PCCC primer with WHV DNA in PBMC from chronic and residual WHV infections but not from a healthy animal (bottom row). (B) Dot blot hybridization analysis of supernatants collected after PBMC underwent in situ PCR, confirming the validity of positive flow cytometry signals and negative controls shown in panel A.

    Techniques Used: Infection, In Situ, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Hybridization, Amplification, Labeling, Dot Blot

    27) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

    Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-4-21

    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

    28) Product Images from "Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine"

    Article Title: Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1610-5

    Taq DNA polymerase stop assay showing the stabilization of the RET G-quadruplex with berberine and its structural analogues. a Structures of berberine and its analogues. b Sequence of the single-stranded template DNA annealed with the 5′-[ 32 P] labelled primer used in DNA polymerase stop assay. c DNA polymerase stop assay at increasing concentrations of berberine (0, 0.75, 1.5, 15 μg/ml) and its analogues (0, 0.75, 1.5,3, 15 μg/ml). Lanes A, G, T C represent the di-deoxy sequencing reactions with the same template, which serve as the marker to identify the exact stop site. Lane P represents the position of the free primer on the gel
    Figure Legend Snippet: Taq DNA polymerase stop assay showing the stabilization of the RET G-quadruplex with berberine and its structural analogues. a Structures of berberine and its analogues. b Sequence of the single-stranded template DNA annealed with the 5′-[ 32 P] labelled primer used in DNA polymerase stop assay. c DNA polymerase stop assay at increasing concentrations of berberine (0, 0.75, 1.5, 15 μg/ml) and its analogues (0, 0.75, 1.5,3, 15 μg/ml). Lanes A, G, T C represent the di-deoxy sequencing reactions with the same template, which serve as the marker to identify the exact stop site. Lane P represents the position of the free primer on the gel

    Techniques Used: Sequencing, Marker

    29) Product Images from "Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization"

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184162

    A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.
    Figure Legend Snippet: A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Techniques Used: Mobility Shift, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).
    Figure Legend Snippet: Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.
    Figure Legend Snippet: Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Techniques Used: Activity Assay

    30) Product Images from "Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection"

    Article Title: Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection

    Journal: RNA

    doi: 10.1261/rna.066753.118

    Effect of various ribonucleotide substitutions on iLock probe RNA detection assay with PBCV-1 ligase. Recognition of the invader structure and structure-specific endonucleolytic activity of Taq DNA polymerase can vary for different RNA substitutions. ( A ) Targeting let-7a with iLock probe. Ribonucleotides were introduced: at a terminal 3′ base (3); base in the 5′ arm, that an invading 3′ arm competes with for target binding (displaced base, 3D); base in the 5′ arm, that becomes a 5′-phosphorylated donor after iLock probe activation (3D5); in the flap sequence (3DF/DF). ( B ) iLocks were modified according to A , except nonchimeric iLock (DNA). The total number of RCPs for each probe is shown on y -axis. ( C ) PAGE of three selected iLock probes (DNA, 3, 3D) after activation and ligation, without (first three lanes) and with Taq DNA polymerase (last three lanes). Nonactivated iLock probe (79) is shortened upon activation by 14 nt (65) and ligated (seen as high molecular weight band at the top of the gel). (22) let-7a miRNA.
    Figure Legend Snippet: Effect of various ribonucleotide substitutions on iLock probe RNA detection assay with PBCV-1 ligase. Recognition of the invader structure and structure-specific endonucleolytic activity of Taq DNA polymerase can vary for different RNA substitutions. ( A ) Targeting let-7a with iLock probe. Ribonucleotides were introduced: at a terminal 3′ base (3); base in the 5′ arm, that an invading 3′ arm competes with for target binding (displaced base, 3D); base in the 5′ arm, that becomes a 5′-phosphorylated donor after iLock probe activation (3D5); in the flap sequence (3DF/DF). ( B ) iLocks were modified according to A , except nonchimeric iLock (DNA). The total number of RCPs for each probe is shown on y -axis. ( C ) PAGE of three selected iLock probes (DNA, 3, 3D) after activation and ligation, without (first three lanes) and with Taq DNA polymerase (last three lanes). Nonactivated iLock probe (79) is shortened upon activation by 14 nt (65) and ligated (seen as high molecular weight band at the top of the gel). (22) let-7a miRNA.

    Techniques Used: RNA Detection, Activity Assay, Binding Assay, Activation Assay, Sequencing, Modification, Polyacrylamide Gel Electrophoresis, Ligation, Molecular Weight

    31) Product Images from "Association of two SNPs in the coding region of the insulin-like growth factor 1 receptor (IGF1R) gene with growth-related traits in Angus cattle"

    Article Title: Association of two SNPs in the coding region of the insulin-like growth factor 1 receptor (IGF1R) gene with growth-related traits in Angus cattle

    Journal: Journal of Applied Genetics

    doi: 10.1007/s13353-013-0155-z

    Ethidium bromide-stained PCR products after digestion and gel electrophoresis. Lanes 1, 5, and 9: M , the DNA length marker pUC19/ Msp I. Lanes 2–4: the IGF1R /e12/ Msp I polymorphism. Lanes 6–8: the IGF1R /3′UTR/ Taq I polymorphism (the small 18-bp fragment is not visible in the gel)
    Figure Legend Snippet: Ethidium bromide-stained PCR products after digestion and gel electrophoresis. Lanes 1, 5, and 9: M , the DNA length marker pUC19/ Msp I. Lanes 2–4: the IGF1R /e12/ Msp I polymorphism. Lanes 6–8: the IGF1R /3′UTR/ Taq I polymorphism (the small 18-bp fragment is not visible in the gel)

    Techniques Used: Staining, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Marker

    32) Product Images from "Notch1 regulates the expression of the multidrug resistance gene ABCC1/MRP1 in cultured cancer cells"

    Article Title: Notch1 regulates the expression of the multidrug resistance gene ABCC1/MRP1 in cultured cancer cells

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

    doi: 10.1073/pnas.1019452108

    ChIP assay and EMSA reveal an interaction between the N1 IC -CBF1 protein and putative CBF1 binding elements. ( A ) ABCC1 p-411 promoter sequence containing four putative CBF1 binding sites. They are I, GTGGAGA (−376/−370); II, GTGGGCC (−168/−162); III, GTGGGGG (−141/−135); and IV, GTGGGGC (−40/−34). +1 is the transcription start site. ( B ) ChIP-PCR analysis. Input DNA and DNA isolated from precipitated chromatin were amplified by PCR with Taq polymerase and separated by 1.5% agarose gel. Lanes: M, marker; 1, Input (1% DNA); 2, IgG (negative control); 3, Notch1 antibody; and 4, CBF1 antibody. I, PCR product (171 bp) covers the first CBF1 element. II, ChIP-PCR product (221 bp) covers the second CBF1 element. III, ChIP-PCR product (72 bp) covers the third CBF1 element. ( C ) EMSA analysis. Lanes: 1–2, CBF1 probe without (1) and with (2) nuclear extract; 3, putative CBF1 probe with nuclear extract; 4–5, putative CBF1 and 50× (4) or 100× (5) molar excess cold putative CBF1 probe with nuclear extract; 6–7, putative CBF1 and 50× (6) or 100× (7) molar excess cold mutant CBF1 probe with nuclear extract; and 8–9, putative CBF1 and 50× (8) or 100× (9) molar excess cold CBF1 probe with nuclear extract.
    Figure Legend Snippet: ChIP assay and EMSA reveal an interaction between the N1 IC -CBF1 protein and putative CBF1 binding elements. ( A ) ABCC1 p-411 promoter sequence containing four putative CBF1 binding sites. They are I, GTGGAGA (−376/−370); II, GTGGGCC (−168/−162); III, GTGGGGG (−141/−135); and IV, GTGGGGC (−40/−34). +1 is the transcription start site. ( B ) ChIP-PCR analysis. Input DNA and DNA isolated from precipitated chromatin were amplified by PCR with Taq polymerase and separated by 1.5% agarose gel. Lanes: M, marker; 1, Input (1% DNA); 2, IgG (negative control); 3, Notch1 antibody; and 4, CBF1 antibody. I, PCR product (171 bp) covers the first CBF1 element. II, ChIP-PCR product (221 bp) covers the second CBF1 element. III, ChIP-PCR product (72 bp) covers the third CBF1 element. ( C ) EMSA analysis. Lanes: 1–2, CBF1 probe without (1) and with (2) nuclear extract; 3, putative CBF1 probe with nuclear extract; 4–5, putative CBF1 and 50× (4) or 100× (5) molar excess cold putative CBF1 probe with nuclear extract; 6–7, putative CBF1 and 50× (6) or 100× (7) molar excess cold mutant CBF1 probe with nuclear extract; and 8–9, putative CBF1 and 50× (8) or 100× (9) molar excess cold CBF1 probe with nuclear extract.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Sequencing, Polymerase Chain Reaction, Isolation, Amplification, Agarose Gel Electrophoresis, Marker, Negative Control, Mutagenesis

    33) Product Images from "Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage"

    Article Title: Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni058

    Synthesis of a functional chloramphenicol acetyltransferase gene with changed codon composition. The ratio r of ‘active clones’ to ‘analyzed clones’ as described in the text is shown for different gene synthesis methods with or without an EMC step. A significant increase of r can be observed only in the cases where EMC is combined with an exonuclease activity present in the reaction or in the later amplification reaction. Prolonged incubation with E.coli endonuclease V results in no detectable product after the amplification steps (ss, single-stranded synthesis, ds, double-stranded synthesis; VII, T4 endonuclease VII; V, E.coli endonuclease V; T, Taq DNA polymerase; and Vn, Vent DNA polymerase).
    Figure Legend Snippet: Synthesis of a functional chloramphenicol acetyltransferase gene with changed codon composition. The ratio r of ‘active clones’ to ‘analyzed clones’ as described in the text is shown for different gene synthesis methods with or without an EMC step. A significant increase of r can be observed only in the cases where EMC is combined with an exonuclease activity present in the reaction or in the later amplification reaction. Prolonged incubation with E.coli endonuclease V results in no detectable product after the amplification steps (ss, single-stranded synthesis, ds, double-stranded synthesis; VII, T4 endonuclease VII; V, E.coli endonuclease V; T, Taq DNA polymerase; and Vn, Vent DNA polymerase).

    Techniques Used: Functional Assay, Clone Assay, Activity Assay, Amplification, Incubation

    34) Product Images from "1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer"

    Article Title: 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-41

    The effect of blood inhibitors and enhancers on PCR, melting temperature, and polymerase activity . (A) DNA fragment of NP_001035158.1 gene (Fr. 7; 735 bp; 73.1% GC) was amplified in PCR mix II supplemented with the corresponding primers, 37.5 μM hemoglobin and 0.2 M trehalose alone (T), 1 M 1,2-propanediol alone (P) or their combination (PT). PCR mix supplemented with H 2 O instead of enhancers served as a control (C'). A typical experiment of four performed is shown. (B) Melting temperature of GC-rich dsDNA oligonucleotide primer No 7, reverse, and the anti-primer (72.2% GC; 1 μM final concentration) in the presence (+) or absence (-) of hemoglobin (37.5 μM) and various enhancers (as in A). Melting temperature was determined as in Figure 6, except that SGI was used at higher concentration (1.32 μM). (C) Enzymatic activity of Taq DNA polymerase in PCR mix II buffer supplemented with (+) or without (-) 10% blood and various enhancers (as in A). Samples supplemented with H 2 O instead of enhancers (C') served as controls. Data in B and C indicate means ± S.D. (n = 4). Asterisks indicate statistically significant differences (P
    Figure Legend Snippet: The effect of blood inhibitors and enhancers on PCR, melting temperature, and polymerase activity . (A) DNA fragment of NP_001035158.1 gene (Fr. 7; 735 bp; 73.1% GC) was amplified in PCR mix II supplemented with the corresponding primers, 37.5 μM hemoglobin and 0.2 M trehalose alone (T), 1 M 1,2-propanediol alone (P) or their combination (PT). PCR mix supplemented with H 2 O instead of enhancers served as a control (C'). A typical experiment of four performed is shown. (B) Melting temperature of GC-rich dsDNA oligonucleotide primer No 7, reverse, and the anti-primer (72.2% GC; 1 μM final concentration) in the presence (+) or absence (-) of hemoglobin (37.5 μM) and various enhancers (as in A). Melting temperature was determined as in Figure 6, except that SGI was used at higher concentration (1.32 μM). (C) Enzymatic activity of Taq DNA polymerase in PCR mix II buffer supplemented with (+) or without (-) 10% blood and various enhancers (as in A). Samples supplemented with H 2 O instead of enhancers (C') served as controls. Data in B and C indicate means ± S.D. (n = 4). Asterisks indicate statistically significant differences (P

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Amplification, Concentration Assay

    The effect of various DNA dyes and enhancers on ssDNA fluorescence and dsDNA melting temperature . (A) TNF-1 oligonucleotide (ssDNA, 45.5% GC; 1 μM final concentration) in PCR mix II (without dNTPs, Taq DNA polymerase and anti-Taq) was mixed with H 2 O (Control; Co) or enhancers [0.2 M trehalose (T; final concentration), 1 M 1,2-propanediol (P) or both 1 M 1,2-propanediol and 0.2 M trehalose (PT)] and various DNA dyes at final concentrations as indicated in Table 1. After heating at 95°C for 2 min the samples were cooled to 50°C and fluorescence was determined using Mastercycler ep realplex. (B) Oligonucleotide primer No 7, reverse (ssDNA; 72.2% GC; 1 μM final concentration) in PCR mix II was combined with various additives and DNA dyes, and fluorescence at 50°C was determined as in A. (C) Oligonucleotide mixture of TNF-1 and anti-TNF-1 (dsDNA, 45.5% GC; 1 μM final concentration) was prepared in mix II supplemented with various additives and DNA dyes. The samples were heated to 95°C for 2 min, then cooled to 30°C and temperature-dependent changes in fluorescence were obtained during heating from 30 to 95°C (0.2°C increments) in Mastercycler ep realplex. Melting temperatures were determined from the melting curves. (D) Oligonucleotide mixture of the primer No 7, reverse, and the anti-primer 7 (dsDNA, 72.2% GC; final concentration 1 μM) was combined in mix II with additives and DNA dyes and analyzed as in C. Means ± S.D. were calculated from 3 - 5 measurements.
    Figure Legend Snippet: The effect of various DNA dyes and enhancers on ssDNA fluorescence and dsDNA melting temperature . (A) TNF-1 oligonucleotide (ssDNA, 45.5% GC; 1 μM final concentration) in PCR mix II (without dNTPs, Taq DNA polymerase and anti-Taq) was mixed with H 2 O (Control; Co) or enhancers [0.2 M trehalose (T; final concentration), 1 M 1,2-propanediol (P) or both 1 M 1,2-propanediol and 0.2 M trehalose (PT)] and various DNA dyes at final concentrations as indicated in Table 1. After heating at 95°C for 2 min the samples were cooled to 50°C and fluorescence was determined using Mastercycler ep realplex. (B) Oligonucleotide primer No 7, reverse (ssDNA; 72.2% GC; 1 μM final concentration) in PCR mix II was combined with various additives and DNA dyes, and fluorescence at 50°C was determined as in A. (C) Oligonucleotide mixture of TNF-1 and anti-TNF-1 (dsDNA, 45.5% GC; 1 μM final concentration) was prepared in mix II supplemented with various additives and DNA dyes. The samples were heated to 95°C for 2 min, then cooled to 30°C and temperature-dependent changes in fluorescence were obtained during heating from 30 to 95°C (0.2°C increments) in Mastercycler ep realplex. Melting temperatures were determined from the melting curves. (D) Oligonucleotide mixture of the primer No 7, reverse, and the anti-primer 7 (dsDNA, 72.2% GC; final concentration 1 μM) was combined in mix II with additives and DNA dyes and analyzed as in C. Means ± S.D. were calculated from 3 - 5 measurements.

    Techniques Used: Fluorescence, Concentration Assay, Polymerase Chain Reaction

    35) Product Images from "Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization"

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184162

    A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.
    Figure Legend Snippet: A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Techniques Used: Mobility Shift, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).
    Figure Legend Snippet: Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.
    Figure Legend Snippet: Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Techniques Used: Activity Assay

    36) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.05.27.120238

    Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
    Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification

    SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.
    Figure Legend Snippet: SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Amplification

    TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.
    Figure Legend Snippet: TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

    Techniques Used: Quantitative RT-PCR, Amplification

    37) Product Images from "Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis"

    Article Title: Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070942

    Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.
    Figure Legend Snippet: Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Serial Dilution, Plasmid Preparation, Negative Control

    38) Product Images from "Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization"

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184162

    A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.
    Figure Legend Snippet: A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Techniques Used: Mobility Shift, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).
    Figure Legend Snippet: Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.
    Figure Legend Snippet: Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Techniques Used: Activity Assay

    39) Product Images from "Identification, technological and safety characterization of Lactobacillus sakei and Lactobacillus curvatus isolated from Argentinean anchovies (Engraulis anchoita)"

    Article Title: Identification, technological and safety characterization of Lactobacillus sakei and Lactobacillus curvatus isolated from Argentinean anchovies (Engraulis anchoita)

    Journal: SpringerPlus

    doi: 10.1186/2193-1801-2-257

    Electrophoresis of Hind III-digested PCR products of the multiplex reaction (A) and Taq I digestion patterns (B) of Lactobacillus isolates SACB 7 01, SACB 7 01a, SACB 7 05, SACB 7 08, SACB03a. Lactobacillus sakei CRL978 and Lactobacillus curvatus CRL1000 were used as type strains; 100-bp DNA ladder was used as molecular weight marker.
    Figure Legend Snippet: Electrophoresis of Hind III-digested PCR products of the multiplex reaction (A) and Taq I digestion patterns (B) of Lactobacillus isolates SACB 7 01, SACB 7 01a, SACB 7 05, SACB 7 08, SACB03a. Lactobacillus sakei CRL978 and Lactobacillus curvatus CRL1000 were used as type strains; 100-bp DNA ladder was used as molecular weight marker.

    Techniques Used: Electrophoresis, Polymerase Chain Reaction, Multiplex Assay, Molecular Weight, Marker

    40) Product Images from "Preferential Completion of Human Immunodeficiency Virus Type 1 Proviruses Initiated with tRNA3Lys rather than tRNA1,2Lys"

    Article Title: Preferential Completion of Human Immunodeficiency Virus Type 1 Proviruses Initiated with tRNA3Lys rather than tRNA1,2Lys

    Journal: Journal of Virology

    doi:

    Endogenous reverse transcription-PCR designed to specifically amplify the minus-strand strong-stop DNA elongated from tRNA 1 Lys , tRNA 2 Lys , and tRNA 3 Lys . (A) Endogenous reverse transcription reactions were carried out with HIV-1 produced from transfected 293T cells or infected SupT1 cells. PCR was performed with primer P1 (plus-strand sense DNA primer located in the R region) and primer P2 (Lys1,2,3 primer, corresponding to the common sequence [nt 8 to 26] shared by tRNA 1 Lys , tRNA 2 Lys , and tRNA 3 Lys ). Note that, at the beginning of the PCR, P1 primer can only bind to the minus-strand strong-stop DNA that extended from endogenous tRNA primers. Taq DNA polymerase extends the P1 primer by copying the sequences of the minus-strand DNA and the still-attached tRNA. P2 primer binds to the DNA sequence copied from the 5′ ends of the attached tRNA 1 Lys or tRNA 2 Lys and tRNA 3 Lys . (B) Secondary structures of tRNA 3 Lys and tRNA 1,2 Lys ). Note that there are only 2-nt differences between tRNA 1 Lys and tRNA 2 Lys . The 3′-terminal 18 nt in these tRNAs are highlighted in boldface.
    Figure Legend Snippet: Endogenous reverse transcription-PCR designed to specifically amplify the minus-strand strong-stop DNA elongated from tRNA 1 Lys , tRNA 2 Lys , and tRNA 3 Lys . (A) Endogenous reverse transcription reactions were carried out with HIV-1 produced from transfected 293T cells or infected SupT1 cells. PCR was performed with primer P1 (plus-strand sense DNA primer located in the R region) and primer P2 (Lys1,2,3 primer, corresponding to the common sequence [nt 8 to 26] shared by tRNA 1 Lys , tRNA 2 Lys , and tRNA 3 Lys ). Note that, at the beginning of the PCR, P1 primer can only bind to the minus-strand strong-stop DNA that extended from endogenous tRNA primers. Taq DNA polymerase extends the P1 primer by copying the sequences of the minus-strand DNA and the still-attached tRNA. P2 primer binds to the DNA sequence copied from the 5′ ends of the attached tRNA 1 Lys or tRNA 2 Lys and tRNA 3 Lys . (B) Secondary structures of tRNA 3 Lys and tRNA 1,2 Lys ). Note that there are only 2-nt differences between tRNA 1 Lys and tRNA 2 Lys . The 3′-terminal 18 nt in these tRNAs are highlighted in boldface.

    Techniques Used: Polymerase Chain Reaction, Produced, Transfection, Infection, Sequencing

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    Clone Assay:

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
    Article Snippet: .. Six histidine codons were introduced onto the N-terminus of the Taq DNA polymerase and Taq DNA pol/TBD gene and the recombinant genes were cloned in frame into the NdeI site of the pLEX vector (Invitrogen). .. 5′–3′ Exonuclease-deficient mutants which lack the first 235 amino acids of the N-terminal were likewise generated.

    Amplification:

    Article Title: Epigenetic Reactivation of RASSF1A by Phenethyl Isothiocyanate (PEITC) and promotion of apoptosis in LNCaP cells
    Article Snippet: .. Briefly, the DNA sequences were amplified by mixing bisulfite-converted DNA (500 ng) with primer MU379 (50 pmoles) and primer ML730 (50 pmoles) in reaction buffer (20 μl) containing dNTPs (200 μM each dNTP) using Platinum PCR Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). .. The reaction conditions consisted of 30 cycles of 95 °C for 1 min, 55 °C for 1 min and 74 °C for 2 min. A semi-nested PCR using the amplified products at a 1:50 dilution, the internal primer ML561 and primer MU379 was performed using similar PCR conditions as described above.

    Article Title: A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)
    Article Snippet: .. Amplification was conducted in a 10ul reaction containing deionized water (Invitrogen), 10X PCR Reaction Buffer (Invitrogen), 50 mM MgCl2 (Invitrogen), 100 mM dNTP solution (Invitrogen), 3 mg/mL BSA, 40uM forward and reverse primers (Integrated DNA Technologies) mentioned above (forward primers labeled with the fluorescent dye HEX), 0.0375U Invitrogen Taq DNA Polymerase, and 5 ng of DNA. .. The PCR reaction was run in a Bio-Rad DNA Engine Dyad and Dyad Disciple thermocycler under the following conditions: 94 °C for 15 min; followed by 29 cycles of 94 °C for 30 s, 52 °C for 1 min 30 s, and 72 °C for 1 min 30 s, and completed with a step of 60 °C for 45 min. Amplified samples were run on an 80 mL, 1.5 % agarose gel stained with ethidium bromide at 90 volts for 45 min, and visualized under ultraviolet light and to determine sex.

    Labeling:

    Article Title: A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)
    Article Snippet: .. Amplification was conducted in a 10ul reaction containing deionized water (Invitrogen), 10X PCR Reaction Buffer (Invitrogen), 50 mM MgCl2 (Invitrogen), 100 mM dNTP solution (Invitrogen), 3 mg/mL BSA, 40uM forward and reverse primers (Integrated DNA Technologies) mentioned above (forward primers labeled with the fluorescent dye HEX), 0.0375U Invitrogen Taq DNA Polymerase, and 5 ng of DNA. .. The PCR reaction was run in a Bio-Rad DNA Engine Dyad and Dyad Disciple thermocycler under the following conditions: 94 °C for 15 min; followed by 29 cycles of 94 °C for 30 s, 52 °C for 1 min 30 s, and 72 °C for 1 min 30 s, and completed with a step of 60 °C for 45 min. Amplified samples were run on an 80 mL, 1.5 % agarose gel stained with ethidium bromide at 90 volts for 45 min, and visualized under ultraviolet light and to determine sex.

    other:

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization
    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Polymerase Chain Reaction:

    Article Title: Epigenetic Reactivation of RASSF1A by Phenethyl Isothiocyanate (PEITC) and promotion of apoptosis in LNCaP cells
    Article Snippet: .. Briefly, the DNA sequences were amplified by mixing bisulfite-converted DNA (500 ng) with primer MU379 (50 pmoles) and primer ML730 (50 pmoles) in reaction buffer (20 μl) containing dNTPs (200 μM each dNTP) using Platinum PCR Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). .. The reaction conditions consisted of 30 cycles of 95 °C for 1 min, 55 °C for 1 min and 74 °C for 2 min. A semi-nested PCR using the amplified products at a 1:50 dilution, the internal primer ML561 and primer MU379 was performed using similar PCR conditions as described above.

    Article Title: Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish
    Article Snippet: .. All PCR reactions were performed using either NEB 2X Taq Master Mix (M0270L), Thermo Scientific Taq (EP0402 and 2x PCR Master Mix Cat# AB-0575/DC), or Kapa 2G Fast ReadyMix +dye (Kapa Biosystems-KM5101). .. CRISPR activity was assessed by either direct sequencing of PCR amplicons, T7 assay (NEB M0302S), Surveyor assay (IDT 706025), or RFLP.

    Article Title: A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)
    Article Snippet: .. Amplification was conducted in a 10ul reaction containing deionized water (Invitrogen), 10X PCR Reaction Buffer (Invitrogen), 50 mM MgCl2 (Invitrogen), 100 mM dNTP solution (Invitrogen), 3 mg/mL BSA, 40uM forward and reverse primers (Integrated DNA Technologies) mentioned above (forward primers labeled with the fluorescent dye HEX), 0.0375U Invitrogen Taq DNA Polymerase, and 5 ng of DNA. .. The PCR reaction was run in a Bio-Rad DNA Engine Dyad and Dyad Disciple thermocycler under the following conditions: 94 °C for 15 min; followed by 29 cycles of 94 °C for 30 s, 52 °C for 1 min 30 s, and 72 °C for 1 min 30 s, and completed with a step of 60 °C for 45 min. Amplified samples were run on an 80 mL, 1.5 % agarose gel stained with ethidium bromide at 90 volts for 45 min, and visualized under ultraviolet light and to determine sex.

    Article Title: Activation of alternative Jdp2 promoters and functional protein isoforms in T-cell lymphomas by retroviral insertion mutagenesis
    Article Snippet: .. To look for alternative splicing between published exons 1a through 1d, PCR reactions were done with forward primers Exon1a-154 (5′-tgggcaccgcgcctgcagcag-3′), Exon1b-148 (5′-ggaggagcgcgagcat-3′), Exon1c-70 (5′-gctctggctgggttaggagggaac-3′) or Exon1d-150 (5′-cagctgcctctctccatctt-3′) and reverse primer Intron2-87 (5′-tccttcgctcttcttcctcgtctagctt-3′) using 1/500 of the cDNA (corresponding to 4 ng of total RNA) per PCR reaction (Taq polymerase, Invitrogen). .. Each PCR reaction consisted of an initial melting step at 95°C for 5 min followed by 35 cycles of amplification, each consisting of a 30 s melting step at 95°C, annealing at 55–60°C for 30 s and elongation at 72°C for 45 s. A final elongation step at 72°C was done for 10 min. Quantitative real-time PCR (QRT-PCR) was done using Platinum SYBR Green QRT-PCR SuperMix UDG (Invitrogen) following the manufacturer's recommendations.

    Recombinant:

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
    Article Snippet: .. Six histidine codons were introduced onto the N-terminus of the Taq DNA polymerase and Taq DNA pol/TBD gene and the recombinant genes were cloned in frame into the NdeI site of the pLEX vector (Invitrogen). .. 5′–3′ Exonuclease-deficient mutants which lack the first 235 amino acids of the N-terminal were likewise generated.

    Plasmid Preparation:

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
    Article Snippet: .. Six histidine codons were introduced onto the N-terminus of the Taq DNA polymerase and Taq DNA pol/TBD gene and the recombinant genes were cloned in frame into the NdeI site of the pLEX vector (Invitrogen). .. 5′–3′ Exonuclease-deficient mutants which lack the first 235 amino acids of the N-terminal were likewise generated.

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  • 99
    Thermo Fisher taq dna polymerase
    A mobility shift assay for TaqS <t>DNA</t> polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- <t>Taq</t> S DNA polymerase, respectively.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher gene exp plin2 hs00605340 m1
    Localization of ATGL, <t>PLIN2</t> and PLIN3 in term placenta tissue. ( a ) ATGL was localized exclusively in the syncytiotrophoblast layer. ( b ) PLIN2 was detectable on the syncytiotrophoblast by a clear punctate staining. ( c ) PLIN3 was mainly localized in the syncytiotrophoblast. ( d ) Cytokeratin 7 was used as positive control for the syncytiotrophoblast layer. Negative control staining for rabbit IgG ( e ) and mouse IgG ( f ) was performed with equal IgG concentrations. Scale bar, 50 μm.
    Gene Exp Plin2 Hs00605340 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Mobility Shift, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Polymerase Chain Reaction, Amplification, Marker

    Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Activity Assay

    Conditional mutagenesis pipeline. Upon deciding which exon to flox, we recommend sequencing the target sites to identify polymorphisms compared to reference genome. Next, sgRNAs should be designed and tested by either direct sequencing of PCR fragments, T7 endonuclease assay or loss of a restriction enzyme site on bulk DNA from pooled embryos. Once active sgRNAs have been identified, experiments integrating the first loxP site should be performed. In the absence of conclusive data that certain HDR template performs significantly better than others (such experiments are not practical at the only level that matters–germline transmission), we recommend using the design we successfully used to integrate loxP into fleer , aldh1a2 and tcf21 : antisense to PAM, with 49-base 5’ homology arm and 21-base 3’ homology arm, with 3-nucleotide spacers flanking loxP site. As injected embryos are being raised, we then recommend to optimize nested PCR screening conditions DNA from pools of injected embryos. We found “plain” Taq polymerases (NEB #M0270, Thermofisher Scientific 2x PCR Master Mix Cat# AB-0575/DC and #EP0402, or similar) to be most suitable for nested PCR. In contrast, high-performance mixes such as Platinum Taq (Thermofisher #10966026) or Kapa 2G Fast ReadyMix + dye (Kapa Biosystems- KM5101) yield very high background and may only be used for the second (nested) reaction. It is also very helpful if primers for one end of the nested PCR are anchored within an exon. We recommend generating a deletion allele in parallel with integration of the first loxP site. Once highly active sgRNAs are identified, we recommend injecting a pair of sgRNAs flanking the exon to be floxed in order to confirm that removal of selected exon will yield an overt phenotype. We have been able to very efficiently delete exon 8 of aldh1a2 using sgRNAs spaced just over 450 base pairs, but larger deletions are certainly feasible too (1, 2). An additional benefit of a deletion allele is that it can be crossed to Cre drivers of interest, eliminating the need to back-cross floxed allele to obtain homozygotes. Screening for germline transmission should be performed by nested PCR on pools of embryos obtained from incross. Positive crosses should be analyzed by performing short flanking PCR (ideally under 400 base pairs) on DNA from individual embryos. Bands corresponding to loxP-containing allele should be extracted from gel and sequenced to ensure presence of intact loxP site. Siblings of screened embryos should be raised to adulthood and loxP-positive F1s should be identified by flanking PCR as well. Two strategies can be used for integration of the second loxP site. If speed is the main priority, loxP-positive F1s can be in-crossed and second sgRNA/HDR oligonucleotide can be injected. The main drawback of this strategy that there is only 50% likelihood that the second loxP site will integrate into a chromosome already containing the first loxP. It is therefore necessary to genotype adults for presence of the first loxP site before out-crossing. Even though we successfully used this strategy to engineer a floxed allele of tbx20 , we consider it impractical and would generally recommend to first generate adults homozygous for the first loxP site, incross them and then inject the second sgRNA/HDR oligonucleotide.

    Journal: PLoS Genetics

    Article Title: Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish

    doi: 10.1371/journal.pgen.1007754

    Figure Lengend Snippet: Conditional mutagenesis pipeline. Upon deciding which exon to flox, we recommend sequencing the target sites to identify polymorphisms compared to reference genome. Next, sgRNAs should be designed and tested by either direct sequencing of PCR fragments, T7 endonuclease assay or loss of a restriction enzyme site on bulk DNA from pooled embryos. Once active sgRNAs have been identified, experiments integrating the first loxP site should be performed. In the absence of conclusive data that certain HDR template performs significantly better than others (such experiments are not practical at the only level that matters–germline transmission), we recommend using the design we successfully used to integrate loxP into fleer , aldh1a2 and tcf21 : antisense to PAM, with 49-base 5’ homology arm and 21-base 3’ homology arm, with 3-nucleotide spacers flanking loxP site. As injected embryos are being raised, we then recommend to optimize nested PCR screening conditions DNA from pools of injected embryos. We found “plain” Taq polymerases (NEB #M0270, Thermofisher Scientific 2x PCR Master Mix Cat# AB-0575/DC and #EP0402, or similar) to be most suitable for nested PCR. In contrast, high-performance mixes such as Platinum Taq (Thermofisher #10966026) or Kapa 2G Fast ReadyMix + dye (Kapa Biosystems- KM5101) yield very high background and may only be used for the second (nested) reaction. It is also very helpful if primers for one end of the nested PCR are anchored within an exon. We recommend generating a deletion allele in parallel with integration of the first loxP site. Once highly active sgRNAs are identified, we recommend injecting a pair of sgRNAs flanking the exon to be floxed in order to confirm that removal of selected exon will yield an overt phenotype. We have been able to very efficiently delete exon 8 of aldh1a2 using sgRNAs spaced just over 450 base pairs, but larger deletions are certainly feasible too (1, 2). An additional benefit of a deletion allele is that it can be crossed to Cre drivers of interest, eliminating the need to back-cross floxed allele to obtain homozygotes. Screening for germline transmission should be performed by nested PCR on pools of embryos obtained from incross. Positive crosses should be analyzed by performing short flanking PCR (ideally under 400 base pairs) on DNA from individual embryos. Bands corresponding to loxP-containing allele should be extracted from gel and sequenced to ensure presence of intact loxP site. Siblings of screened embryos should be raised to adulthood and loxP-positive F1s should be identified by flanking PCR as well. Two strategies can be used for integration of the second loxP site. If speed is the main priority, loxP-positive F1s can be in-crossed and second sgRNA/HDR oligonucleotide can be injected. The main drawback of this strategy that there is only 50% likelihood that the second loxP site will integrate into a chromosome already containing the first loxP. It is therefore necessary to genotype adults for presence of the first loxP site before out-crossing. Even though we successfully used this strategy to engineer a floxed allele of tbx20 , we consider it impractical and would generally recommend to first generate adults homozygous for the first loxP site, incross them and then inject the second sgRNA/HDR oligonucleotide.

    Article Snippet: All PCR reactions were performed using either NEB 2X Taq Master Mix (M0270L), Thermo Scientific Taq (EP0402 and 2x PCR Master Mix Cat# AB-0575/DC), or Kapa 2G Fast ReadyMix +dye (Kapa Biosystems-KM5101).

    Techniques: Mutagenesis, Sequencing, Polymerase Chain Reaction, Transmission Assay, Injection, Nested PCR

    Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

    Journal: bioRxiv

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    doi: 10.1101/2020.05.27.120238

    Figure Lengend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

    Article Snippet: Both NEB and Thermo Fisher Taq DNA polymerases were also able to perform TaqMan RT-qPCR analysis of RNaseP armored RNA using the CDC assay ( ).

    Techniques: Quantitative RT-PCR, Amplification

    SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.

    Journal: bioRxiv

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    doi: 10.1101/2020.05.27.120238

    Figure Lengend Snippet: SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.

    Article Snippet: Both NEB and Thermo Fisher Taq DNA polymerases were also able to perform TaqMan RT-qPCR analysis of RNaseP armored RNA using the CDC assay ( ).

    Techniques: Quantitative RT-PCR, Amplification

    TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

    Journal: bioRxiv

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    doi: 10.1101/2020.05.27.120238

    Figure Lengend Snippet: TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

    Article Snippet: Both NEB and Thermo Fisher Taq DNA polymerases were also able to perform TaqMan RT-qPCR analysis of RNaseP armored RNA using the CDC assay ( ).

    Techniques: Quantitative RT-PCR, Amplification

    Localization of ATGL, PLIN2 and PLIN3 in term placenta tissue. ( a ) ATGL was localized exclusively in the syncytiotrophoblast layer. ( b ) PLIN2 was detectable on the syncytiotrophoblast by a clear punctate staining. ( c ) PLIN3 was mainly localized in the syncytiotrophoblast. ( d ) Cytokeratin 7 was used as positive control for the syncytiotrophoblast layer. Negative control staining for rabbit IgG ( e ) and mouse IgG ( f ) was performed with equal IgG concentrations. Scale bar, 50 μm.

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal obesity modulates intracellular lipid turnover in the human term placenta

    doi: 10.1038/ijo.2016.188

    Figure Lengend Snippet: Localization of ATGL, PLIN2 and PLIN3 in term placenta tissue. ( a ) ATGL was localized exclusively in the syncytiotrophoblast layer. ( b ) PLIN2 was detectable on the syncytiotrophoblast by a clear punctate staining. ( c ) PLIN3 was mainly localized in the syncytiotrophoblast. ( d ) Cytokeratin 7 was used as positive control for the syncytiotrophoblast layer. Negative control staining for rabbit IgG ( e ) and mouse IgG ( f ) was performed with equal IgG concentrations. Scale bar, 50 μm.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) gene expression assays and antibodies for protein analysis Gene expression assays were purchased from Applied Biosystems (Darmstadt, Germany) and included the following genes: adipose triglyceride lipase ATGL (PNPLA2, gene ID 57104, Taq man assay no. Hs00386101_m1), hormone sensitive lipase, HSL (LIPE, gene ID 3991, Taq man assay no. Hs00193510_m1), α/β hydrolase domain containing 5, CGI-58, (ABHD5, gene ID 51099, Taq man assay no. HS01104373_m1), perilipin 1 (PLIN1, gene ID 5346, Taq man assay no. Hs00160173_m1), perilipin 2, ADRP (PLIN2, gene ID 123, Taq man assay no. HS00605340_m1), perilipin 3, TIP47 (PLIN3, gene ID 10226, Taq man assay no. HS00998416_m1), perilipin 4 (PLIN4, gene ID 729359, Taq man assay no. Hs00287411_m1), perilipin 5, OXPAT (PLIN5, gene ID 440503, Taq man assay no. Hs00965990_m1), TATA box-binding protein (TBP, gene ID 6908, Taq man assay no. Hs00427620_m1).

    Techniques: Staining, Positive Control, Negative Control

    Placental CGI-58 is regulated by maternal pre-pregnancy obesity. Lower panel: representative western blots ( n =4 per group) of total placental tissue. ( a ) ATGL protein expression. ( b ) PLIN2 protein expression. ( c ) CGI-58 protein expression. All protein signals were quantitated by densitometry, normalized to β-actin as loading control and to one protein sample which was used on each blot to correct for inter blot variations. Sum rank test was performed, differences between the lean (BMI

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal obesity modulates intracellular lipid turnover in the human term placenta

    doi: 10.1038/ijo.2016.188

    Figure Lengend Snippet: Placental CGI-58 is regulated by maternal pre-pregnancy obesity. Lower panel: representative western blots ( n =4 per group) of total placental tissue. ( a ) ATGL protein expression. ( b ) PLIN2 protein expression. ( c ) CGI-58 protein expression. All protein signals were quantitated by densitometry, normalized to β-actin as loading control and to one protein sample which was used on each blot to correct for inter blot variations. Sum rank test was performed, differences between the lean (BMI

    Article Snippet: Quantitative real-time PCR (qRT-PCR) gene expression assays and antibodies for protein analysis Gene expression assays were purchased from Applied Biosystems (Darmstadt, Germany) and included the following genes: adipose triglyceride lipase ATGL (PNPLA2, gene ID 57104, Taq man assay no. Hs00386101_m1), hormone sensitive lipase, HSL (LIPE, gene ID 3991, Taq man assay no. Hs00193510_m1), α/β hydrolase domain containing 5, CGI-58, (ABHD5, gene ID 51099, Taq man assay no. HS01104373_m1), perilipin 1 (PLIN1, gene ID 5346, Taq man assay no. Hs00160173_m1), perilipin 2, ADRP (PLIN2, gene ID 123, Taq man assay no. HS00605340_m1), perilipin 3, TIP47 (PLIN3, gene ID 10226, Taq man assay no. HS00998416_m1), perilipin 4 (PLIN4, gene ID 729359, Taq man assay no. Hs00287411_m1), perilipin 5, OXPAT (PLIN5, gene ID 440503, Taq man assay no. Hs00965990_m1), TATA box-binding protein (TBP, gene ID 6908, Taq man assay no. Hs00427620_m1).

    Techniques: Western Blot, Expressing

    Association of placental CGI-58 with maternal metabolic parameters. Correlation analysis was performed between CGI-58 mRNA or protein expression in placenta tissue and maternal pre-pregnancy BMI ( a ) or maternal plasma insulin ( b ). PLIN2 mRNA and protein levels were correlated with ( c ) maternal pre-pregnancy BMI and maternal plasma insulin levels ( d ). Black circles (•) protein expression on thr open circles (○) mRNA expression. Spearman correlation was defined as significant if P -values were

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal obesity modulates intracellular lipid turnover in the human term placenta

    doi: 10.1038/ijo.2016.188

    Figure Lengend Snippet: Association of placental CGI-58 with maternal metabolic parameters. Correlation analysis was performed between CGI-58 mRNA or protein expression in placenta tissue and maternal pre-pregnancy BMI ( a ) or maternal plasma insulin ( b ). PLIN2 mRNA and protein levels were correlated with ( c ) maternal pre-pregnancy BMI and maternal plasma insulin levels ( d ). Black circles (•) protein expression on thr open circles (○) mRNA expression. Spearman correlation was defined as significant if P -values were

    Article Snippet: Quantitative real-time PCR (qRT-PCR) gene expression assays and antibodies for protein analysis Gene expression assays were purchased from Applied Biosystems (Darmstadt, Germany) and included the following genes: adipose triglyceride lipase ATGL (PNPLA2, gene ID 57104, Taq man assay no. Hs00386101_m1), hormone sensitive lipase, HSL (LIPE, gene ID 3991, Taq man assay no. Hs00193510_m1), α/β hydrolase domain containing 5, CGI-58, (ABHD5, gene ID 51099, Taq man assay no. HS01104373_m1), perilipin 1 (PLIN1, gene ID 5346, Taq man assay no. Hs00160173_m1), perilipin 2, ADRP (PLIN2, gene ID 123, Taq man assay no. HS00605340_m1), perilipin 3, TIP47 (PLIN3, gene ID 10226, Taq man assay no. HS00998416_m1), perilipin 4 (PLIN4, gene ID 729359, Taq man assay no. Hs00287411_m1), perilipin 5, OXPAT (PLIN5, gene ID 440503, Taq man assay no. Hs00965990_m1), TATA box-binding protein (TBP, gene ID 6908, Taq man assay no. Hs00427620_m1).

    Techniques: Expressing, IF-P