expanded high fidelity pcr  (Roche)


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    Structured Review

    Roche expanded high fidelity pcr
    Expanded High Fidelity Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expanded high fidelity pcr/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    expanded high fidelity pcr - by Bioz Stars, 2021-07
    86/100 stars

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    Related Articles

    Transgenic Assay:

    Article Title: The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]
    Article Snippet: .. To generate transgenic plants expressing GUS protein under the regulation of MYC3 and MYC4 promoter regions, 2028 and 1549 bp, respectively, upstream of ATG (including the first 30 nucleotides of the coding sequence of each gene) were amplified with Expand High Fidelity polymerase (Roche) using appropriate primers (see online). .. PCR products were cloned into pENTR/D-TOPO (Invitrogen) and sequence verified.

    Expressing:

    Article Title: The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]
    Article Snippet: .. To generate transgenic plants expressing GUS protein under the regulation of MYC3 and MYC4 promoter regions, 2028 and 1549 bp, respectively, upstream of ATG (including the first 30 nucleotides of the coding sequence of each gene) were amplified with Expand High Fidelity polymerase (Roche) using appropriate primers (see online). .. PCR products were cloned into pENTR/D-TOPO (Invitrogen) and sequence verified.

    Sequencing:

    Article Title: The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]
    Article Snippet: .. To generate transgenic plants expressing GUS protein under the regulation of MYC3 and MYC4 promoter regions, 2028 and 1549 bp, respectively, upstream of ATG (including the first 30 nucleotides of the coding sequence of each gene) were amplified with Expand High Fidelity polymerase (Roche) using appropriate primers (see online). .. PCR products were cloned into pENTR/D-TOPO (Invitrogen) and sequence verified.

    Amplification:

    Article Title: The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]
    Article Snippet: .. To generate transgenic plants expressing GUS protein under the regulation of MYC3 and MYC4 promoter regions, 2028 and 1549 bp, respectively, upstream of ATG (including the first 30 nucleotides of the coding sequence of each gene) were amplified with Expand High Fidelity polymerase (Roche) using appropriate primers (see online). .. PCR products were cloned into pENTR/D-TOPO (Invitrogen) and sequence verified.

    Article Title: Generation and characterization of influenza A viruses with altered polymerase fidelity
    Article Snippet: Mutational frequency determination by TOPO cloning RNA (20–130 ng) extracted using RNeasy Mini Kit (Qiagen) were used for reverse transcription using Transcriptor High Fidelity cDNA Synthesis Kit (Roche), using random hexamer for cDNA synthesis. .. Amplification of HA gene was performed by using 10 μL of cDNA in 100 μL reaction using Expand High Fidelity PCR System (Roche) with forward and reverse primers: Wuhan95 (H3N2): 5′-GGTTTTCGCTCAAAAACTTCC-3′ and 5′-GTTTCCCGTTGATTTGGTTG-3′; VN04 (H5N1) 5′-ACCATGCAAACAACTCGACA-3′ and 5′-GCTATTTCTGAGCCCAGTCG-3′. .. Gel purified PCR producet (24–33 ng) was cloned into pCR™ 4-TOPO vector using TOPO® TA Cloning Kit (Life Technologies).

    Article Title: Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses
    Article Snippet: The sequence logo was generated using the web-based program WebLogo ( ). .. The cloning of EBV BILF1, Pan troglodytes lymphocryptovirus 1 (PtroLCV1) BILF1, PpygLCV1 BILF1, Symphalangus syndactylus lymphocryptovirus 1 (SsynLCV1) BILF1, and CalHV3 BILF1 was done after amplification of the ORF with standard PCR techniques using Expand high-fidelity polymerase (Roche, Switzerland). .. The ORF was inserted into the vectors pCMV-HA and pCMV-c-myc (TaKaRa Bio Europe, France) by cohesive end ligation and transformed into Escherichia coli .

    Article Title: Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants
    Article Snippet: .. Amplification of HIV-1 gp120 sequences was performed by a double-nested PCR using the Expand High Fidelity PCR System (Roche). .. Primers for first round PCR were FW_5′-TAGCAATAGTTGTGTGGAC-3′ and Rev_5′-ATTGTTCTGCTGTTGCACTA-3′ and for second round PCR FW_5′-AGAAAGAGCAGAAGACAGTGGC-3′ and Rev_5′-TACCGTCAGCGTTATTGACG-3′.

    Article Title: Reduced Genetic Diversity in Lymphoid and Central Nervous System Tissues and Selection-Induced Tissue-Specific Compartmentalization of Neuropathogenic SIVsmmFGb during Acute Infection
    Article Snippet: DNA was pelleted by 15,000-rpm centrifugation for 30 min at 4°C, washed three times with 70% ethanol, air dried, and resuspended in ddH2 O. DNA concentration was quantified with a UV spectrophotometer and samples were frozen at −20°C. .. The nef, env , and int genes were amplified from proviral DNA by nested PCR, using an Expand High Fidelity PCR system kit (Roche, Indianapolis, IN), according to the manufacturer's protocol. .. After some difficulty in performing PCR amplification of viral genes from the CNS tissues of macaques PQo1 and PQq1, the primers in were analyzed and found to contain hairpins and dimers.

    Article Title: Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq
    Article Snippet: Northern blotting was performed with the Roche DIG system. .. DNA probes for 5S rRNA and SPA0079 were amplified with PCR using primers described in Table S2 and PCR DIG Probe Synthesis Kit (#11636090910, Roche). .. The reaction was performed with a total volume of 50 μl containing 0.2 μM of forward primer, 0.2 μM of reverse primer, 200 μM of dATP, dCTP, and dGTP, 130 μM of dTTP, 70 μM of DIG-11-dUTP, 1x PCR buffer, and 2.625 units of Enzyme mix, Expand High Fidelity (#1732641, Roche).

    Article Title: Differential expression of lysosome-associated protein transmembrane-4 beta (LAPTM4B) in granulosa cells of ovarian follicles and in other bovine tissues
    Article Snippet: Overexpression of bovine LAPTM4B in mammalian cells To confirm the detection of the two forms of LAPTM4B protein with our antibodies, overexpression experiments in mammalian cells were performed. .. The LAPTM4B open reading frame was amplified by PCR using the Expand High Fidelity polymerase (Roche Molecular Biochemicals) with specific LAPTM4B primers. .. The PCR fragment was purified and cloned into pQE-TriSystem His-Strep2 (Qiagen).

    Polymerase Chain Reaction:

    Article Title: Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models
    Article Snippet: Primer pairs encompassing the full-length phox2b gene were designed and used to amplify phox2b in the MO-injected embryos with the Superscript First strand synthesis kit (Invitrogen). .. 50 pg of 1st strand cDNA was used to amplify phox2b using the Expand High Fidelity Plus PCR system (Roche). .. Western blotting Immunoblotting was performed according to standard methods.

    Article Title: Generation and characterization of influenza A viruses with altered polymerase fidelity
    Article Snippet: Mutational frequency determination by TOPO cloning RNA (20–130 ng) extracted using RNeasy Mini Kit (Qiagen) were used for reverse transcription using Transcriptor High Fidelity cDNA Synthesis Kit (Roche), using random hexamer for cDNA synthesis. .. Amplification of HA gene was performed by using 10 μL of cDNA in 100 μL reaction using Expand High Fidelity PCR System (Roche) with forward and reverse primers: Wuhan95 (H3N2): 5′-GGTTTTCGCTCAAAAACTTCC-3′ and 5′-GTTTCCCGTTGATTTGGTTG-3′; VN04 (H5N1) 5′-ACCATGCAAACAACTCGACA-3′ and 5′-GCTATTTCTGAGCCCAGTCG-3′. .. Gel purified PCR producet (24–33 ng) was cloned into pCR™ 4-TOPO vector using TOPO® TA Cloning Kit (Life Technologies).

    Article Title: Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses
    Article Snippet: The sequence logo was generated using the web-based program WebLogo ( ). .. The cloning of EBV BILF1, Pan troglodytes lymphocryptovirus 1 (PtroLCV1) BILF1, PpygLCV1 BILF1, Symphalangus syndactylus lymphocryptovirus 1 (SsynLCV1) BILF1, and CalHV3 BILF1 was done after amplification of the ORF with standard PCR techniques using Expand high-fidelity polymerase (Roche, Switzerland). .. The ORF was inserted into the vectors pCMV-HA and pCMV-c-myc (TaKaRa Bio Europe, France) by cohesive end ligation and transformed into Escherichia coli .

    Article Title: Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants
    Article Snippet: .. Amplification of HIV-1 gp120 sequences was performed by a double-nested PCR using the Expand High Fidelity PCR System (Roche). .. Primers for first round PCR were FW_5′-TAGCAATAGTTGTGTGGAC-3′ and Rev_5′-ATTGTTCTGCTGTTGCACTA-3′ and for second round PCR FW_5′-AGAAAGAGCAGAAGACAGTGGC-3′ and Rev_5′-TACCGTCAGCGTTATTGACG-3′.

    Article Title: Reduced Genetic Diversity in Lymphoid and Central Nervous System Tissues and Selection-Induced Tissue-Specific Compartmentalization of Neuropathogenic SIVsmmFGb during Acute Infection
    Article Snippet: DNA was pelleted by 15,000-rpm centrifugation for 30 min at 4°C, washed three times with 70% ethanol, air dried, and resuspended in ddH2 O. DNA concentration was quantified with a UV spectrophotometer and samples were frozen at −20°C. .. The nef, env , and int genes were amplified from proviral DNA by nested PCR, using an Expand High Fidelity PCR system kit (Roche, Indianapolis, IN), according to the manufacturer's protocol. .. After some difficulty in performing PCR amplification of viral genes from the CNS tissues of macaques PQo1 and PQq1, the primers in were analyzed and found to contain hairpins and dimers.

    Article Title: Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq
    Article Snippet: Northern blotting was performed with the Roche DIG system. .. DNA probes for 5S rRNA and SPA0079 were amplified with PCR using primers described in Table S2 and PCR DIG Probe Synthesis Kit (#11636090910, Roche). .. The reaction was performed with a total volume of 50 μl containing 0.2 μM of forward primer, 0.2 μM of reverse primer, 200 μM of dATP, dCTP, and dGTP, 130 μM of dTTP, 70 μM of DIG-11-dUTP, 1x PCR buffer, and 2.625 units of Enzyme mix, Expand High Fidelity (#1732641, Roche).

    Article Title: Differential expression of lysosome-associated protein transmembrane-4 beta (LAPTM4B) in granulosa cells of ovarian follicles and in other bovine tissues
    Article Snippet: Overexpression of bovine LAPTM4B in mammalian cells To confirm the detection of the two forms of LAPTM4B protein with our antibodies, overexpression experiments in mammalian cells were performed. .. The LAPTM4B open reading frame was amplified by PCR using the Expand High Fidelity polymerase (Roche Molecular Biochemicals) with specific LAPTM4B primers. .. The PCR fragment was purified and cloned into pQE-TriSystem His-Strep2 (Qiagen).

    Clone Assay:

    Article Title: Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses
    Article Snippet: The sequence logo was generated using the web-based program WebLogo ( ). .. The cloning of EBV BILF1, Pan troglodytes lymphocryptovirus 1 (PtroLCV1) BILF1, PpygLCV1 BILF1, Symphalangus syndactylus lymphocryptovirus 1 (SsynLCV1) BILF1, and CalHV3 BILF1 was done after amplification of the ORF with standard PCR techniques using Expand high-fidelity polymerase (Roche, Switzerland). .. The ORF was inserted into the vectors pCMV-HA and pCMV-c-myc (TaKaRa Bio Europe, France) by cohesive end ligation and transformed into Escherichia coli .

    Nested PCR:

    Article Title: Reduced Genetic Diversity in Lymphoid and Central Nervous System Tissues and Selection-Induced Tissue-Specific Compartmentalization of Neuropathogenic SIVsmmFGb during Acute Infection
    Article Snippet: DNA was pelleted by 15,000-rpm centrifugation for 30 min at 4°C, washed three times with 70% ethanol, air dried, and resuspended in ddH2 O. DNA concentration was quantified with a UV spectrophotometer and samples were frozen at −20°C. .. The nef, env , and int genes were amplified from proviral DNA by nested PCR, using an Expand High Fidelity PCR system kit (Roche, Indianapolis, IN), according to the manufacturer's protocol. .. After some difficulty in performing PCR amplification of viral genes from the CNS tissues of macaques PQo1 and PQq1, the primers in were analyzed and found to contain hairpins and dimers.

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    Roche expand high fidelity plus pcr system
    <t>Phox2b</t> deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real <t>time-PCR</t> analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P
    Expand High Fidelity Plus Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand high fidelity plus pcr system/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    expand high fidelity plus pcr system - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    86
    Roche expand high fidelity pcr system
    Replication kinetics of wild-type and PB1-V43I viruses under competition or suboptimal temperatures (a) One-step growth kinetics of wild-type and PB1-V43I H3N2 and H5N1 viruses using MOI 1–2 TCID 50 per cell in MDCK cells. Viral supernatants were collected every 2h post-infection and viral titres (mean ± SD log 10 TCID 50 per mL) from triplicates were shown. The replication kinetics has been repeated three times for the H3N2 wild-type and PB1-V43I viruses and once for the H5N1 wild-type and PB1-V43I viruses. (b) Competitive replication of wild-type and PB1-V43I mutant viruses in vitro . Wild-type and PB1-V43I viruses were premixed at different ratios prior to infection of MDCK cells. To determine the actual ratio in both the premixed (inoculum) and the viral supernatant after incubation for 2 days (progeny virus), clonal sequencing was performed to determine the ratio between <t>Wuhan95</t> wild-type and PB1-V43I viruses. (c) As an alternative, in a separate experiment, instead of clonal sequencing, plaque assay was performed for the inoculums and passage-one viral supernatant after incubation for 2 days. Then, 32 clones were picked for each of the three inoculums and three corresponding passage-one viral cultures for viral RNA isolation and <t>RT-PCR</t> of the PB1 gene region that can distinguish wild-type virus from its V43I mutant counterpart. The actual ratio in the inoculums and the viral supernatant was shown in the same fashion. (d) Mean plaque sizes formed by the Wuhan95 wild-type and PB1-V43I viruses incubated at different temperatures. Wild-type or PB1-V43I mutant viruses were used to infect MDCK monolayers in triplicates and incubated under 0.5% agar overlay for 48 hours at 33°C, 37°C, or 39°C. The experiments were repeated independently twice and one representative result was shown. (e) Picture taken for the one representative plaque assay experiment performed for wild-type and V43I mutant viruses at the three temperatures. (f) Growth curve of wild-type and V43I mutant Wuhan95 viruses performed at 37°C, (g) 33°C, and (h) 39°C. For (f) to (h), viral titres (mean±SD log 10 TCID 50 per mL) from quadruplicated wells were shown. Three independent experiments were performed, with one representative experiment being displayed. P-values were based on Two-way ANOVA test with bonferroni post-tests. N.S., not statistically significant.
    Expand High Fidelity Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand high fidelity pcr system/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    expand high fidelity pcr system - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

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    Phox2b deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real time-PCR analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P

    Journal: PLoS Genetics

    Article Title: Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models

    doi: 10.1371/journal.pgen.1003533

    Figure Lengend Snippet: Phox2b deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real time-PCR analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P

    Article Snippet: 50 pg of 1st strand cDNA was used to amplify phox2b using the Expand High Fidelity Plus PCR system (Roche).

    Techniques: In Situ Hybridization, Labeling, Expressing, Injection, Real-time Polymerase Chain Reaction

    Replication kinetics of wild-type and PB1-V43I viruses under competition or suboptimal temperatures (a) One-step growth kinetics of wild-type and PB1-V43I H3N2 and H5N1 viruses using MOI 1–2 TCID 50 per cell in MDCK cells. Viral supernatants were collected every 2h post-infection and viral titres (mean ± SD log 10 TCID 50 per mL) from triplicates were shown. The replication kinetics has been repeated three times for the H3N2 wild-type and PB1-V43I viruses and once for the H5N1 wild-type and PB1-V43I viruses. (b) Competitive replication of wild-type and PB1-V43I mutant viruses in vitro . Wild-type and PB1-V43I viruses were premixed at different ratios prior to infection of MDCK cells. To determine the actual ratio in both the premixed (inoculum) and the viral supernatant after incubation for 2 days (progeny virus), clonal sequencing was performed to determine the ratio between Wuhan95 wild-type and PB1-V43I viruses. (c) As an alternative, in a separate experiment, instead of clonal sequencing, plaque assay was performed for the inoculums and passage-one viral supernatant after incubation for 2 days. Then, 32 clones were picked for each of the three inoculums and three corresponding passage-one viral cultures for viral RNA isolation and RT-PCR of the PB1 gene region that can distinguish wild-type virus from its V43I mutant counterpart. The actual ratio in the inoculums and the viral supernatant was shown in the same fashion. (d) Mean plaque sizes formed by the Wuhan95 wild-type and PB1-V43I viruses incubated at different temperatures. Wild-type or PB1-V43I mutant viruses were used to infect MDCK monolayers in triplicates and incubated under 0.5% agar overlay for 48 hours at 33°C, 37°C, or 39°C. The experiments were repeated independently twice and one representative result was shown. (e) Picture taken for the one representative plaque assay experiment performed for wild-type and V43I mutant viruses at the three temperatures. (f) Growth curve of wild-type and V43I mutant Wuhan95 viruses performed at 37°C, (g) 33°C, and (h) 39°C. For (f) to (h), viral titres (mean±SD log 10 TCID 50 per mL) from quadruplicated wells were shown. Three independent experiments were performed, with one representative experiment being displayed. P-values were based on Two-way ANOVA test with bonferroni post-tests. N.S., not statistically significant.

    Journal: Nature communications

    Article Title: Generation and characterization of influenza A viruses with altered polymerase fidelity

    doi: 10.1038/ncomms5794

    Figure Lengend Snippet: Replication kinetics of wild-type and PB1-V43I viruses under competition or suboptimal temperatures (a) One-step growth kinetics of wild-type and PB1-V43I H3N2 and H5N1 viruses using MOI 1–2 TCID 50 per cell in MDCK cells. Viral supernatants were collected every 2h post-infection and viral titres (mean ± SD log 10 TCID 50 per mL) from triplicates were shown. The replication kinetics has been repeated three times for the H3N2 wild-type and PB1-V43I viruses and once for the H5N1 wild-type and PB1-V43I viruses. (b) Competitive replication of wild-type and PB1-V43I mutant viruses in vitro . Wild-type and PB1-V43I viruses were premixed at different ratios prior to infection of MDCK cells. To determine the actual ratio in both the premixed (inoculum) and the viral supernatant after incubation for 2 days (progeny virus), clonal sequencing was performed to determine the ratio between Wuhan95 wild-type and PB1-V43I viruses. (c) As an alternative, in a separate experiment, instead of clonal sequencing, plaque assay was performed for the inoculums and passage-one viral supernatant after incubation for 2 days. Then, 32 clones were picked for each of the three inoculums and three corresponding passage-one viral cultures for viral RNA isolation and RT-PCR of the PB1 gene region that can distinguish wild-type virus from its V43I mutant counterpart. The actual ratio in the inoculums and the viral supernatant was shown in the same fashion. (d) Mean plaque sizes formed by the Wuhan95 wild-type and PB1-V43I viruses incubated at different temperatures. Wild-type or PB1-V43I mutant viruses were used to infect MDCK monolayers in triplicates and incubated under 0.5% agar overlay for 48 hours at 33°C, 37°C, or 39°C. The experiments were repeated independently twice and one representative result was shown. (e) Picture taken for the one representative plaque assay experiment performed for wild-type and V43I mutant viruses at the three temperatures. (f) Growth curve of wild-type and V43I mutant Wuhan95 viruses performed at 37°C, (g) 33°C, and (h) 39°C. For (f) to (h), viral titres (mean±SD log 10 TCID 50 per mL) from quadruplicated wells were shown. Three independent experiments were performed, with one representative experiment being displayed. P-values were based on Two-way ANOVA test with bonferroni post-tests. N.S., not statistically significant.

    Article Snippet: Amplification of HA gene was performed by using 10 μL of cDNA in 100 μL reaction using Expand High Fidelity PCR System (Roche) with forward and reverse primers: Wuhan95 (H3N2): 5′-GGTTTTCGCTCAAAAACTTCC-3′ and 5′-GTTTCCCGTTGATTTGGTTG-3′; VN04 (H5N1) 5′-ACCATGCAAACAACTCGACA-3′ and 5′-GCTATTTCTGAGCCCAGTCG-3′.

    Techniques: Infection, Mutagenesis, In Vitro, Incubation, Sequencing, Plaque Assay, Clone Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    A. Schematic illustration (top) and partial G-banded karyotype (bottom) illustrating the 7;12 translocation. B. RT-PCR detected ACTB-GLI1 fusion transcripts using ACTB 61F-868R and ACTB 18F-1246R primers, which amplified DNA products of 700 bp (lane 1) and 1119 bp (lane 2) respectively. M, 1 kb DNA ladder.

    Journal: Human pathology

    Article Title: Pericytoma with t(7;12) and ACTB-GLI1 Fusion Arising in Bone

    doi: 10.1016/j.humpath.2012.01.019

    Figure Lengend Snippet: A. Schematic illustration (top) and partial G-banded karyotype (bottom) illustrating the 7;12 translocation. B. RT-PCR detected ACTB-GLI1 fusion transcripts using ACTB 61F-868R and ACTB 18F-1246R primers, which amplified DNA products of 700 bp (lane 1) and 1119 bp (lane 2) respectively. M, 1 kb DNA ladder.

    Article Snippet: To detect the ACTB-GLI fusion transcript, PCR was performed using the Expand High Fidelity PCR System (Roche, Mannheim, Germany) with gene specific primer pairs ACTB61F-GLI868R and ACTB18F-GLI1246R (ACTB61F: 5’-CCGCCAGCTCAC CATGGATGATG, GLI 868R: 5’-GTGGCACACGAACTCCTTCCGCTC; ACTB 18 F: 5’-CACAGAGCCTC GCCTTTGCCGA, GLI 1246R: 5’-GCCGTTTGGTCACATGG GCGTC) [ ].

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

    Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Article Snippet: We then performed long-range PCR of the samples (approximately 100 ng input DNA) using either the Expand High Fidelity PCR System, dNTPack (Roche), or the Long Range PCR Kit (Qiagen) together with GAA-B-F (5′-AATGGATTTCCTGGCAGGACGC-3′) and GAA-B-R (5′-GCATTGGGCGATCTTGGCTTAA-3′) primers as previously described ( ).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

    Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Article Snippet: We then performed long-range PCR of the samples (approximately 100 ng input DNA) using either the Expand High Fidelity PCR System, dNTPack (Roche), or the Long Range PCR Kit (Qiagen) together with GAA-B-F (5′-AATGGATTTCCTGGCAGGACGC-3′) and GAA-B-R (5′-GCATTGGGCGATCTTGGCTTAA-3′) primers as previously described ( ).

    Techniques: Polymerase Chain Reaction, Marker, Mouse Assay