taq dna polymerase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher taq dna polymerase
    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 <t>DNA</t> and DNA isolated from precipitated chromatin were amplified by PCR with <t>Taq</t> 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.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) 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

    2) 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

    3) 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

    4) 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

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

    6) 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

    7) 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

    8) 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

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

    10) 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

    11) 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

    12) 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

    13) Product Images from "Examining the Effects of Netropsin on the Curvature of DNA A-Tracts Using Electrophoresis"

    Article Title: Examining the Effects of Netropsin on the Curvature of DNA A-Tracts Using Electrophoresis

    Journal: Molecules

    doi: 10.3390/molecules26195871

    Schematic of 98-bp DNA fragments. ( A ) Sequences of the DNA constructs studied here with A 6 -tracts shown in bold red. Possible unpaired 3′ deoxyadenosine residues from non-template-dependent terminal transferase activity of Taq polymerase are not shown. The first four sequence names (samples 0 to 4i) indicate the number of phased A 6 -tracts in the sample. Sample 2i/o contains two pairs of in-phase A 6 -tracts; the two pairs of phased A 6 -tracts are out-of-phase with each other. Sample 4o has four A 6 -tracts separated by 16 residues from each other. ( B ) Depiction of the shapes of the DNA fragments with A-tracts shown in red. Molecular models were rendered with PyMOL.
    Figure Legend Snippet: Schematic of 98-bp DNA fragments. ( A ) Sequences of the DNA constructs studied here with A 6 -tracts shown in bold red. Possible unpaired 3′ deoxyadenosine residues from non-template-dependent terminal transferase activity of Taq polymerase are not shown. The first four sequence names (samples 0 to 4i) indicate the number of phased A 6 -tracts in the sample. Sample 2i/o contains two pairs of in-phase A 6 -tracts; the two pairs of phased A 6 -tracts are out-of-phase with each other. Sample 4o has four A 6 -tracts separated by 16 residues from each other. ( B ) Depiction of the shapes of the DNA fragments with A-tracts shown in red. Molecular models were rendered with PyMOL.

    Techniques Used: Construct, Activity Assay, Sequencing

    14) Product Images from "Tandem blocking of PCR extension to form a single-stranded overhang for facile, visual, and ultrasensitive gene detection †"

    Article Title: Tandem blocking of PCR extension to form a single-stranded overhang for facile, visual, and ultrasensitive gene detection †

    Journal: RSC Advances

    doi: 10.1039/c8ra01471j

    Outline of the visual detection of designated gene proposed in this paper. In the “indenting PCR” (a), PCR products having an overhang are prepared by placing multiple “molecular blockers” (blue circles) in tandem in one of the primers. In (b), these PCR products are treated with AuNps, labelled with the probe oligonucleotide, in the presence of appropriate concentration of Mg 2+ . The overhang having complementary sequence, if any, should bind to the probe DNA on the AuNps and prevent the particles from aggregation (the left in (b)). Otherwise, the salt-induced aggregation promptly occurs (the right). Thus, the presence or the absence of the designated gene in the original specimen is differentiated simply in terms of the color of the solution (either red or blue).
    Figure Legend Snippet: Outline of the visual detection of designated gene proposed in this paper. In the “indenting PCR” (a), PCR products having an overhang are prepared by placing multiple “molecular blockers” (blue circles) in tandem in one of the primers. In (b), these PCR products are treated with AuNps, labelled with the probe oligonucleotide, in the presence of appropriate concentration of Mg 2+ . The overhang having complementary sequence, if any, should bind to the probe DNA on the AuNps and prevent the particles from aggregation (the left in (b)). Otherwise, the salt-induced aggregation promptly occurs (the right). Thus, the presence or the absence of the designated gene in the original specimen is differentiated simply in terms of the color of the solution (either red or blue).

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Sequencing

    15) 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

    16) 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

    17) 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

    18) 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

    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: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

    20) 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

    21) 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

    22) Product Images from "Naturally Extended CT ? AG Repeats Increase H-DNA Structures and Promoter Activity in the Smooth Muscle Myosin Light Chain Kinase Gene ▿"

    Article Title: Naturally Extended CT ? AG Repeats Increase H-DNA Structures and Promoter Activity in the Smooth Muscle Myosin Light Chain Kinase Gene ▿

    Journal:

    doi: 10.1128/MCB.00960-07

    Expanded H-DNA at the SHR promoter. (A) Plasmids were probed with CAA and used to reveal chemically susceptible bases by primer extension using a Stoffel fragment of Taq polymerase. Primer extension products from the modified (lanes 5 to 7) DNA were separated
    Figure Legend Snippet: Expanded H-DNA at the SHR promoter. (A) Plasmids were probed with CAA and used to reveal chemically susceptible bases by primer extension using a Stoffel fragment of Taq polymerase. Primer extension products from the modified (lanes 5 to 7) DNA were separated

    Techniques Used: Cellular Antioxidant Activity Assay, Modification

    23) 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

    24) 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

    25) 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

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

    28) 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

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

    30) 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

    31) 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

    32) 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

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

    34) Product Images from "Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development"

    Article Title: Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063234

    Consensus sequence for CNBP DNA-binding site. ( A ) A consensus CNBP binding sequence was predicted by applying the MEME program to the 85 and 11 unique sequences obtained from M. musculus and D. rerio libraries, respectively. The logo shown was generated using Weblogo. The 14-nucleotide consensus sequence used for probes design is shown below the logo. Asterisks indicate the six more conserved guanine residues. ( B ) EMSAs were performed using labeled ssDNA probes (C14, C14-mut, C14-comp and dsC14) and increasing concentrations of recombinant CNBP (0.015, 0.050, 0.15, 0.50, 1.50 and 5 µM). Free and shifted probes are indicated by arrows at the left of the gels. K d for C14 is indicated below the gel.
    Figure Legend Snippet: Consensus sequence for CNBP DNA-binding site. ( A ) A consensus CNBP binding sequence was predicted by applying the MEME program to the 85 and 11 unique sequences obtained from M. musculus and D. rerio libraries, respectively. The logo shown was generated using Weblogo. The 14-nucleotide consensus sequence used for probes design is shown below the logo. Asterisks indicate the six more conserved guanine residues. ( B ) EMSAs were performed using labeled ssDNA probes (C14, C14-mut, C14-comp and dsC14) and increasing concentrations of recombinant CNBP (0.015, 0.050, 0.15, 0.50, 1.50 and 5 µM). Free and shifted probes are indicated by arrows at the left of the gels. K d for C14 is indicated below the gel.

    Techniques Used: Sequencing, Binding Assay, Generated, Labeling, Recombinant

    35) 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

    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 "One enzyme reverse transcription qPCR using Taq DNA polymerase"

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    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 in duplicate experiments. 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 in duplicate experiments. 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

    38) 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

    39) 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

    40) 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

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  • 94
    Thermo Fisher b16 cells
    Cell number changes of bone marrow cells on day 6 after culture with GM-CSF and CM from non-irradiated or 24 h after 24 Gy γ-irradiated <t>B16</t> cells. The cell number counted results of the adherent cells ( A ) and loosely/non-adherent cells ( B ). Mean ± SE, n = 3. *, p
    B16 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/b16 cells/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
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    94
    Thermo Fisher invitrogen taq
    E7 PCR using <t>Invitrogen</t> <t>Taq.</t> Yellow box highlights the double banding in positive samples showing an unexpected fragment.
    Invitrogen Taq, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taq dna polymerase
    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 <t>DNA</t> and DNA isolated from precipitated chromatin were amplified by PCR with <t>Taq</t> 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.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell number changes of bone marrow cells on day 6 after culture with GM-CSF and CM from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells. The cell number counted results of the adherent cells ( A ) and loosely/non-adherent cells ( B ). Mean ± SE, n = 3. *, p

    Journal: Medicines

    Article Title: A Combination of GM-CSF and Released Factors from Gamma-Irradiated Tumor Cells Enhances the Differentiation of Macrophages from Bone Marrow Cells and Their Antigen-Presenting Function and Polarization to Type 1

    doi: 10.3390/medicines8070035

    Figure Lengend Snippet: Cell number changes of bone marrow cells on day 6 after culture with GM-CSF and CM from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells. The cell number counted results of the adherent cells ( A ) and loosely/non-adherent cells ( B ). Mean ± SE, n = 3. *, p

    Article Snippet: B16 cells were cultured in Dulbecco’s modified Eagle’s medium (12800-017, Gibco, Life Technologies Corp., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, 10270, Gibco, Life Technologies Corp., Carlsbad, CA, USA) and 1% penicillin and streptomycin (P/S, Gibco, Life Technologies Corp., Carlsbad, CA, USA).

    Techniques: Irradiation

    In vitro culture system for bone marrow cell differentiation in the presence or absence of GM-CSF and CM from non-irradiated or 24 Gy γ-irradiated B16 cells. ( A ) schema of the in vitro culture. ( B ) The adherent and loosely/non-adherent cells were recovered after culture for 6 days in the presence or absence of GM-CSF plus CM obtained from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells, stained with a mouse macrophage marker F4/80 antibody and analyzed by flow cytometry. ( C ) F4/80 positive cell percentages in adherent cells and loosely/non-adherent cells. ( D ) Real-time PCR analysis of Csf2 gene e xpression in B16 cells 24 h after γ-irradiation. #, undetected; Mean ± SE, n = 3.

    Journal: Medicines

    Article Title: A Combination of GM-CSF and Released Factors from Gamma-Irradiated Tumor Cells Enhances the Differentiation of Macrophages from Bone Marrow Cells and Their Antigen-Presenting Function and Polarization to Type 1

    doi: 10.3390/medicines8070035

    Figure Lengend Snippet: In vitro culture system for bone marrow cell differentiation in the presence or absence of GM-CSF and CM from non-irradiated or 24 Gy γ-irradiated B16 cells. ( A ) schema of the in vitro culture. ( B ) The adherent and loosely/non-adherent cells were recovered after culture for 6 days in the presence or absence of GM-CSF plus CM obtained from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells, stained with a mouse macrophage marker F4/80 antibody and analyzed by flow cytometry. ( C ) F4/80 positive cell percentages in adherent cells and loosely/non-adherent cells. ( D ) Real-time PCR analysis of Csf2 gene e xpression in B16 cells 24 h after γ-irradiation. #, undetected; Mean ± SE, n = 3.

    Article Snippet: B16 cells were cultured in Dulbecco’s modified Eagle’s medium (12800-017, Gibco, Life Technologies Corp., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, 10270, Gibco, Life Technologies Corp., Carlsbad, CA, USA) and 1% penicillin and streptomycin (P/S, Gibco, Life Technologies Corp., Carlsbad, CA, USA).

    Techniques: In Vitro, Cell Differentiation, Irradiation, Staining, Marker, Flow Cytometry, Real-time Polymerase Chain Reaction

    Real-time PCR analysis of the expression of type 1 macrophage markers and cytokine genes expression profiles in adherent cell fractions. ( A ) mRNA expression level of macrophage type 1 markers, Il6 , Il1b , and Tnfa . Bone marrow cells on day 6 after culture with different concentrations of GM-CSF and CM obtained from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells. Mean ± SE, n = 3. *, p

    Journal: Medicines

    Article Title: A Combination of GM-CSF and Released Factors from Gamma-Irradiated Tumor Cells Enhances the Differentiation of Macrophages from Bone Marrow Cells and Their Antigen-Presenting Function and Polarization to Type 1

    doi: 10.3390/medicines8070035

    Figure Lengend Snippet: Real-time PCR analysis of the expression of type 1 macrophage markers and cytokine genes expression profiles in adherent cell fractions. ( A ) mRNA expression level of macrophage type 1 markers, Il6 , Il1b , and Tnfa . Bone marrow cells on day 6 after culture with different concentrations of GM-CSF and CM obtained from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells. Mean ± SE, n = 3. *, p

    Article Snippet: B16 cells were cultured in Dulbecco’s modified Eagle’s medium (12800-017, Gibco, Life Technologies Corp., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, 10270, Gibco, Life Technologies Corp., Carlsbad, CA, USA) and 1% penicillin and streptomycin (P/S, Gibco, Life Technologies Corp., Carlsbad, CA, USA).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Irradiation

    Real-time PCR analysis of the expression level of the genes involved in antigen presentation in the adherent and loosely/non-adherent cells. ( A ) H2-Ab1 mRNA expression level. ( B ) Ccr7 , Cd80 , Cd83 , Cd86 , and Il12b mRNA expression levels. ( C ) Pdl1 mRNA expression level in the adherent cells. Bone marrow cells on day 6 after culture with different concentrations of GM-CSF and CM obtained from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells. Mean ± SE, n = 3. *, p

    Journal: Medicines

    Article Title: A Combination of GM-CSF and Released Factors from Gamma-Irradiated Tumor Cells Enhances the Differentiation of Macrophages from Bone Marrow Cells and Their Antigen-Presenting Function and Polarization to Type 1

    doi: 10.3390/medicines8070035

    Figure Lengend Snippet: Real-time PCR analysis of the expression level of the genes involved in antigen presentation in the adherent and loosely/non-adherent cells. ( A ) H2-Ab1 mRNA expression level. ( B ) Ccr7 , Cd80 , Cd83 , Cd86 , and Il12b mRNA expression levels. ( C ) Pdl1 mRNA expression level in the adherent cells. Bone marrow cells on day 6 after culture with different concentrations of GM-CSF and CM obtained from non-irradiated or 24 h after 24 Gy γ-irradiated B16 cells. Mean ± SE, n = 3. *, p

    Article Snippet: B16 cells were cultured in Dulbecco’s modified Eagle’s medium (12800-017, Gibco, Life Technologies Corp., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, 10270, Gibco, Life Technologies Corp., Carlsbad, CA, USA) and 1% penicillin and streptomycin (P/S, Gibco, Life Technologies Corp., Carlsbad, CA, USA).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Irradiation

    E7 PCR using Invitrogen Taq. Yellow box highlights the double banding in positive samples showing an unexpected fragment.

    Journal: medRxiv

    Article Title: HPV 51: A candidate for type-replacement following vaccination?

    doi: 10.1101/2021.07.27.21261121

    Figure Lengend Snippet: E7 PCR using Invitrogen Taq. Yellow box highlights the double banding in positive samples showing an unexpected fragment.

    Article Snippet: During the course of this project, a PhD student saw improved specificity with the use of Hotstar Taq instead of Invitrogen Taq in E7 PCR ( and ).

    Techniques: Polymerase Chain Reaction

    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.

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

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

    doi: 10.1073/pnas.1019452108

    Figure Lengend 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.

    Article Snippet: ABCC1 ( ) and Hey1 ( ) were amplified by PCR with Taq DNA polymerase (Invitrogen).

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

    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.

    Journal: Retrovirology

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

    doi: 10.1186/1742-4690-7-110

    Figure Lengend 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.

    Article Snippet: In this study, we evaluated several one-step RT-PCR kits and a Taq DNA polymerase for the contamination of MLV-related genomes and found that the test kit and the Taq DNA polymerase from Invitrogen were contaminated with MLV-related genomes.

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