platinium taq dna polymerase Thermo Fisher Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher platinum taq dna polymerase
    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 <t>Taq</t> <t>DNA</t> 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.
    Platinum Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 26513 article reviews
    Price from $9.99 to $1999.99
    platinum taq dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher platinum taq dna polymerase high fidelity
    RT-PCR analysis of the T HESCs cell line. (A) For the detection of the JAZF1/SUZ12 chimeric transcript, 1 μl cDNA was used as a template in PCR amplification with the primer combinations: human-JAZF1-284/Fusion-541R, Rhesus-JAZF1-284/Fusion-541R, human-JAZF1-286F/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R, the enzyme Platinum <t>Taq</t> <t>DNA</t> Polymerase High Fidelity and a touchdown PCR cycling protocol. Using the human-JAZF1-284/Fusion-541R and human-JAZF1-286F/Fusion-541R, JAZF1/SUZ12 chimeric transcripts were amplified in an endometrial stromal sarcoma carrying the t(7;17) chromosomal aberration [t(7;17)-ESS], whereas no JAZF1/SUZ12 chimeric transcripts were amplified in the T HESCs cell line. The Rhesus-JAZF1-284/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R did not generate any PCR products. (B) RT-PCR analysis showed that in the T HESCs cell line, both the normal JAZF1 and SUZ12 genes are expressed. The primer set for JAZF1 amplified a cDNA fragment that corresponds to the entire open reading of JAZF1 . The amplified cDNA fragment of SUZ12 contained part of exon 1, exons 2–6 and part of exon 7. M, 100-bp DNA ladder. Blank, no RNA in the cDNA synthesis.
    Platinum Taq Dna Polymerase High Fidelity, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6813 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq dna polymerase high fidelity/product/Thermo Fisher
    Average 99 stars, based on 6813 article reviews
    Price from $9.99 to $1999.99
    platinum taq dna polymerase high fidelity - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher taq polymerase
    RT-PCR analysis of the T HESCs cell line. (A) For the detection of the JAZF1/SUZ12 chimeric transcript, 1 μl cDNA was used as a template in PCR amplification with the primer combinations: human-JAZF1-284/Fusion-541R, Rhesus-JAZF1-284/Fusion-541R, human-JAZF1-286F/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R, the enzyme Platinum <t>Taq</t> <t>DNA</t> Polymerase High Fidelity and a touchdown PCR cycling protocol. Using the human-JAZF1-284/Fusion-541R and human-JAZF1-286F/Fusion-541R, JAZF1/SUZ12 chimeric transcripts were amplified in an endometrial stromal sarcoma carrying the t(7;17) chromosomal aberration [t(7;17)-ESS], whereas no JAZF1/SUZ12 chimeric transcripts were amplified in the T HESCs cell line. The Rhesus-JAZF1-284/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R did not generate any PCR products. (B) RT-PCR analysis showed that in the T HESCs cell line, both the normal JAZF1 and SUZ12 genes are expressed. The primer set for JAZF1 amplified a cDNA fragment that corresponds to the entire open reading of JAZF1 . The amplified cDNA fragment of SUZ12 contained part of exon 1, exons 2–6 and part of exon 7. M, 100-bp DNA ladder. Blank, no RNA in the cDNA synthesis.
    Taq Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 33062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase/product/Thermo Fisher
    Average 96 stars, based on 33062 article reviews
    Price from $9.99 to $1999.99
    taq polymerase - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    93
    Thermo Fisher platinum taq kit
    One-step <t>RT-PCR</t> for identification of contaminants in Kit I and Platinum <t>Taq</t> . (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.
    Platinum Taq Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq kit/product/Thermo Fisher
    Average 93 stars, based on 1515 article reviews
    Price from $9.99 to $1999.99
    platinum taq kit - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    92
    Thermo Fisher pcr
    One-step <t>RT-PCR</t> for identification of contaminants in Kit I and Platinum <t>Taq</t> . (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.
    Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 111176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr/product/Thermo Fisher
    Average 92 stars, based on 111176 article reviews
    Price from $9.99 to $1999.99
    pcr - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    89
    Thermo Fisher superscript iii rt platinum taq mix
    One-step <t>RT-PCR</t> for identification of contaminants in Kit I and Platinum <t>Taq</t> . (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.
    Superscript Iii Rt Platinum Taq Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii rt platinum taq mix/product/Thermo Fisher
    Average 89 stars, based on 463 article reviews
    Price from $9.99 to $1999.99
    superscript iii rt platinum taq mix - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    99
    Thermo Fisher platinum taq buffer
    One-step <t>RT-PCR</t> for identification of contaminants in Kit I and Platinum <t>Taq</t> . (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.
    Platinum Taq Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq buffer/product/Thermo Fisher
    Average 99 stars, based on 357 article reviews
    Price from $9.99 to $1999.99
    platinum taq buffer - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    88
    Thermo Fisher platinum taq high fidelity enzyme
    One-step <t>RT-PCR</t> for identification of contaminants in Kit I and Platinum <t>Taq</t> . (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.
    Platinum Taq High Fidelity Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq high fidelity enzyme/product/Thermo Fisher
    Average 88 stars, based on 138 article reviews
    Price from $9.99 to $1999.99
    platinum taq high fidelity enzyme - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    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.

    Journal: BMC Molecular Biology

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

    doi: 10.1186/1471-2199-10-113

    Figure Lengend 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.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: 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.

    Journal: BMC Molecular Biology

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

    doi: 10.1186/1471-2199-10-113

    Figure Lengend 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.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: 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.

    Journal: BMC Molecular Biology

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

    doi: 10.1186/1471-2199-10-113

    Figure Lengend 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.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: 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.

    Journal: BMC Molecular Biology

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

    doi: 10.1186/1471-2199-10-113

    Figure Lengend 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.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: 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.

    Journal: BMC Molecular Biology

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

    doi: 10.1186/1471-2199-10-113

    Figure Lengend 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.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

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

    Detection of the beta-lactamase gene in commercial Taq polymerase. Four dilutions of Amplitaq DNA polymerase were tested with primers for the beta-lactamase gene in the presence of 10 3 pUC19 plasmids (labeled 1000 pUC19 genomes) or H 2 O.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Detection of the beta-lactamase gene in commercial Taq polymerase. Four dilutions of Amplitaq DNA polymerase were tested with primers for the beta-lactamase gene in the presence of 10 3 pUC19 plasmids (labeled 1000 pUC19 genomes) or H 2 O.

    Article Snippet: Taq polymerases The following DNA polymerases from commercial vendors designed for qPCR were used in the experiments reported here: Amplitaq Gold (ABI, CA; Roche lot # J02913); Platinum Taq (Invitrogen, CA; cat # 10966–026; lot 1169610); Platinum HiFi Taq (Invitrogen, CA; cat# 11304–011; lot# 1267490); HotStar Taq (Qiagen, CA; Mat # 1007837; lot # 124125007); JumpStart Taq (Sigma, MO; cat # D-6558; lot # 71K9029).

    Techniques: Labeling

    Reportable range for T. cruzi and triatomine intestine unit quantification by real-time qPCR. Multiplex Taq Man qPCR assays were carried out with serially diluted DNA extracted from reconstituted triatomine intestine samples containing T. cruzi epimastigotes, ranging from 10 5 to 0.5 T. cruzi equivalents ( a ) and 5 to 0.002 triatomine intestine unit equivalents ( b ). The slope, R 2 and amplification efficiency (Eff) are indicated in the chart

    Journal: Parasites & Vectors

    Article Title: Development of conventional and real-time multiplex PCR-based assays for estimation of natural infection rates and Trypanosoma cruzi load in triatomine vectors

    doi: 10.1186/s13071-017-2343-x

    Figure Lengend Snippet: Reportable range for T. cruzi and triatomine intestine unit quantification by real-time qPCR. Multiplex Taq Man qPCR assays were carried out with serially diluted DNA extracted from reconstituted triatomine intestine samples containing T. cruzi epimastigotes, ranging from 10 5 to 0.5 T. cruzi equivalents ( a ) and 5 to 0.002 triatomine intestine unit equivalents ( b ). The slope, R 2 and amplification efficiency (Eff) are indicated in the chart

    Article Snippet: Multiplex conventional PCR (cPCR) Conventional PCR assays were carried out in a final volume of 50 μl, containing: 5 μl DNA (20–25 ng), 5 μl 10× Taq Platinum buffer, 0.2 mM dNTPs, 4.5 mM MgCl2 , 1.25 U Taq Platinum DNA polymerase (Life Technologies, Carlsbad, CA, USA), 200 nM 121/122 primers (T. cruzi kDNA) [ , ] and 100 nM P2B/P6R primers (triatomine 12S rRNA gene).

    Techniques: Real-time Polymerase Chain Reaction, Multiplex Assay, Amplification

    MNN2 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 23 colonies for each condition. Dark grey: Petri dishes cultures, light grey: liquid cultures. B ) Average of the 3 assays presented in A (standard deviation=19.55). C ) PCR product visualized on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications have been carried out with the Platinium Taq.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: MNN2 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 23 colonies for each condition. Dark grey: Petri dishes cultures, light grey: liquid cultures. B ) Average of the 3 assays presented in A (standard deviation=19.55). C ) PCR product visualized on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications have been carried out with the Platinium Taq.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification, Standard Deviation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Efficiency of MNN5 gene amplification from wild type and modified strains. A ) Percentage of colonies picked from Petri dishes that amplified MNN5. The experiment was carried out on 47 colonies for each strain. Dark grey: BY4742; light grey: YiMMOgène. Amplification was carried out using the DreamTaq or the Platinium Taq. B ). PCR product visualisation, after amplification with the Platinium Taq, on a 1% agarose gel stained with SYBR safe. Left panel (1): BY4742, right panel (2): YiMMOgène, ML: Molecular ladder.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: Efficiency of MNN5 gene amplification from wild type and modified strains. A ) Percentage of colonies picked from Petri dishes that amplified MNN5. The experiment was carried out on 47 colonies for each strain. Dark grey: BY4742; light grey: YiMMOgène. Amplification was carried out using the DreamTaq or the Platinium Taq. B ). PCR product visualisation, after amplification with the Platinium Taq, on a 1% agarose gel stained with SYBR safe. Left panel (1): BY4742, right panel (2): YiMMOgène, ML: Molecular ladder.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Efficiency of MNN5 gene amplification from liquid culture of modified strain. A ) Percentage of MNN5 amplification from 47 colonies picked on Petri dishes and 47 colonies picked from liquid cultures in micro-plates. B ) Growth curve in micro-plate (YPD medium), average of two colonies of YiMMOgène. The OD 600 was measured in a micro-plate reader. ♦: OD 600 after 6 hour culture (early exponential phase). ●: OD 600 after 17 hour culture (late exponential phase). ▄: OD 600 after 24 hour culture (early stationary phase). C ) Percentage amplification of MNN5 from 47 YiMMOgène after different culture times in micro-plate. All amplifications have been carried out with the Platinium Taq.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: Efficiency of MNN5 gene amplification from liquid culture of modified strain. A ) Percentage of MNN5 amplification from 47 colonies picked on Petri dishes and 47 colonies picked from liquid cultures in micro-plates. B ) Growth curve in micro-plate (YPD medium), average of two colonies of YiMMOgène. The OD 600 was measured in a micro-plate reader. ♦: OD 600 after 6 hour culture (early exponential phase). ●: OD 600 after 17 hour culture (late exponential phase). ▄: OD 600 after 24 hour culture (early stationary phase). C ) Percentage amplification of MNN5 from 47 YiMMOgène after different culture times in micro-plate. All amplifications have been carried out with the Platinium Taq.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification

    MNN5 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 47 colonies for each condition. Dark grey: Petri dishes cultures, light grey: micro-plate cultures. B ) Average of the 3 assays presented in A (standard deviation =22.07). C) PCR product visualised on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications were carried out using Platinium Taq.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: MNN5 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 47 colonies for each condition. Dark grey: Petri dishes cultures, light grey: micro-plate cultures. B ) Average of the 3 assays presented in A (standard deviation =22.07). C) PCR product visualised on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications were carried out using Platinium Taq.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification, Standard Deviation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    RT-PCR analysis of the T HESCs cell line. (A) For the detection of the JAZF1/SUZ12 chimeric transcript, 1 μl cDNA was used as a template in PCR amplification with the primer combinations: human-JAZF1-284/Fusion-541R, Rhesus-JAZF1-284/Fusion-541R, human-JAZF1-286F/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R, the enzyme Platinum Taq DNA Polymerase High Fidelity and a touchdown PCR cycling protocol. Using the human-JAZF1-284/Fusion-541R and human-JAZF1-286F/Fusion-541R, JAZF1/SUZ12 chimeric transcripts were amplified in an endometrial stromal sarcoma carrying the t(7;17) chromosomal aberration [t(7;17)-ESS], whereas no JAZF1/SUZ12 chimeric transcripts were amplified in the T HESCs cell line. The Rhesus-JAZF1-284/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R did not generate any PCR products. (B) RT-PCR analysis showed that in the T HESCs cell line, both the normal JAZF1 and SUZ12 genes are expressed. The primer set for JAZF1 amplified a cDNA fragment that corresponds to the entire open reading of JAZF1 . The amplified cDNA fragment of SUZ12 contained part of exon 1, exons 2–6 and part of exon 7. M, 100-bp DNA ladder. Blank, no RNA in the cDNA synthesis.

    Journal: Oncology Letters

    Article Title: Absence of the JAZF1/SUZ12 chimeric transcript in the immortalized non-neoplastic endometrial stromal cell line T HESCs

    doi: 10.3892/ol.2010.185

    Figure Lengend Snippet: RT-PCR analysis of the T HESCs cell line. (A) For the detection of the JAZF1/SUZ12 chimeric transcript, 1 μl cDNA was used as a template in PCR amplification with the primer combinations: human-JAZF1-284/Fusion-541R, Rhesus-JAZF1-284/Fusion-541R, human-JAZF1-286F/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R, the enzyme Platinum Taq DNA Polymerase High Fidelity and a touchdown PCR cycling protocol. Using the human-JAZF1-284/Fusion-541R and human-JAZF1-286F/Fusion-541R, JAZF1/SUZ12 chimeric transcripts were amplified in an endometrial stromal sarcoma carrying the t(7;17) chromosomal aberration [t(7;17)-ESS], whereas no JAZF1/SUZ12 chimeric transcripts were amplified in the T HESCs cell line. The Rhesus-JAZF1-284/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R did not generate any PCR products. (B) RT-PCR analysis showed that in the T HESCs cell line, both the normal JAZF1 and SUZ12 genes are expressed. The primer set for JAZF1 amplified a cDNA fragment that corresponds to the entire open reading of JAZF1 . The amplified cDNA fragment of SUZ12 contained part of exon 1, exons 2–6 and part of exon 7. M, 100-bp DNA ladder. Blank, no RNA in the cDNA synthesis.

    Article Snippet: For the detection of the JAZF1/SUZ12 fusion transcript, PCR was performed with Platinum Taq DNA Polymerase High Fidelity (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Touchdown PCR

    Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P

    Journal: Genes to cells : devoted to molecular & cellular mechanisms

    Article Title: Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

    doi: 10.1111/j.1365-2443.2010.01483.x

    Figure Lengend Snippet: Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P

    Article Snippet: PCR was carried out in a 50-μL reaction mixture containing 5 μL 10× high-fidelity PCR buffer, 1 μL dNTP (10 μM each), 2 μL MgSO4 (50 μM), 4 μL primers (5 μM each), 1 μL template cDNA and 1 μL Platinum® Taq High Fidelity DNA Polymerase (5 U) (Invitrogen).

    Techniques: Recombinant, Plasmid Preparation, Transfection

    Smad4 mutagenesis. (A) Schematic of mutations introduced into wild-type HA-tagged murine Smad4; DBD, DNA-binding domain; NES, nuclear export signal. Briefly, WT HA-Smad4 was used as a template for PCR reactions using Platinum High Fidelity Taq polymerase

    Journal: Journal of Cell Science

    Article Title: Postnatal ablation of osteoblast Smad4 enhances proliferative responses to canonical Wnt signaling through interactions with β-catenin

    doi: 10.1242/jcs.132233

    Figure Lengend Snippet: Smad4 mutagenesis. (A) Schematic of mutations introduced into wild-type HA-tagged murine Smad4; DBD, DNA-binding domain; NES, nuclear export signal. Briefly, WT HA-Smad4 was used as a template for PCR reactions using Platinum High Fidelity Taq polymerase

    Article Snippet: Briefly, WT HA-Smad4 was used as a template for PCR reaction using Platinum High Fidelity Taq polymerase (Invitrogen) and the following primers: (ΔDBD F) 5′-phospho-CATGTGATCTATGCCCGTC-3′ and (ΔDBD R) 5′-phospho-TCCATCCAATGTTCTCTGTAT-3′; (ΔNES F) 5′-phospho-AGTAATGCTCCAAGTATGTTA-3′ and (ΔNES R) 5′-phospho-GACAACCCGCTCATAGTG-3′; (ΔMH2 F) 5′-phospho-TGCTGGATTGAGATTCACCT-3′ and (ΔMH2 R) 5′-phospho-AGGATGATTGGAAATGGGAG-3′; (ΔMH1 F) 5′-phospho-TCACCTGGAATTGATCTCTC-3′ and (ΔMH1 R) 5′-phospho-GCTCAGACAGGCATCGTT-3′; (ΔLinker F) 5′-phospho-CATCCTGCTCCTGAGTAC-3′ and (ΔLinker R) 5′-phospho-CTGCAGTGTTAATCCTGA G-3′; (R100T F) 5′-phospho-ACGTGGCCTGATCTACACAAGAATG-3′ and (R100T R) 5′-phospho-CGTCCACAGACGGGCATAGATCAC-3′.

    Techniques: Mutagenesis, Binding Assay, Polymerase Chain Reaction

    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: We used the following RT-PCR kits which were purchased in Japan: SuperScript® III One-Step RT-PCR System with the Platinum® Taq High Fidelity Kit (Cat. no. 12574-030) (Invitrogen, Carlsbad, CA, USA) (abbreviated as Kit I); AccessQuick™RT-PCR Sysytem (Cat. no. A1701) (Promega, Madison, WI, USA) (abbreviated as Kit P); One Step RT-PCR Kit (Cat. no. PRO24A) (TaKaRa, Ohtsu, Shiga, Japan) (Abbreviated as Kit T); One Step RT-PCR Kit (Cat. no. 210210) (QIAGEN GmbH, Hilden, Germany) (Abbreviated as Kit Q).

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