expand high fidelity polymerase chain reaction pcr system  (Roche)


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    Roche expand high fidelity polymerase chain reaction pcr system
    Expand High Fidelity Polymerase Chain Reaction 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
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    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: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare
    Article Snippet: We also obtained genomic DNA samples from cerebellum autopsy tissues from three FRDA patients (tissues were registered with the HTA under Brunel University Licensing number 12543) and five ear biopsies from previously described GAA repeat expansion-based Y47R and YG8sR FRDA mouse models ( ) (animal procedures were carried out in accordance with the UK Home Office “Animals (Scientific Procedures) Act 1986” and with approval from the Brunel University London Animal Welfare and Ethical Review Board). .. 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 ( ). .. The thermocycling conditions used were (i) Roche Kit: 94°C for 2 min; 10 cycles of 94°C for 10 s, 60°C for 30 s, 68°C for 45 s; 20 cycles of 94°C for 10 s, 60°C for 30 s, 68°C for 1 min with 20 s increments; and a final cycle of 68°C for 10 mins, or (ii) Qiagen Kit: 93°C for 3 min; 35 cycles of 93°C for 15 s, 62°C for 30 s, 68°C for 5 min, and a final cycle of 68°C for 10 min.

    Article Title: A novel eukaryotic factor for cytosolic Fe-S cluster assembly
    Article Snippet: .. To clone the CFD1 gene, yeast genomic DNA was prepared as previously described ( ) and used for amplification of CFD1 genes by the polymerase chain reaction (PCR) using the Expand High Fidelity PCR System (Roche), according to the manufacturer’s recommendation. .. The CFD1 gene plus 456 bp upstream and 168 bp downstream of the ORF was amplified using the following primer set: 3F, 5′-ATGGAAGAAAGAGCCGTGAA-3′ (5′ end); 3R, 5′-CACTTGAAGAACATGCTGCA-3′ (3′ end).

    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: Pericytoma with t(7;12) and ACTB-GLI1 Fusion Arising in Bone
    Article Snippet: The reaction was incubated at 42°C for 1 hr followed by 94°C for 5 min to arrest cDNA synthesis. .. 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) [ ]. .. 5 ul of diluted cDNA was used as template for PCR in 50 μ of assay containing 20 mM Tris-HCl, 50 mM KCL, 1.25 mM MgCl2, 0.4 mM dNTPs and 0.2 uM of each primer and 1 ul of High Fidelity Enzyme Mix.

    Amplification:

    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: A novel eukaryotic factor for cytosolic Fe-S cluster assembly
    Article Snippet: .. To clone the CFD1 gene, yeast genomic DNA was prepared as previously described ( ) and used for amplification of CFD1 genes by the polymerase chain reaction (PCR) using the Expand High Fidelity PCR System (Roche), according to the manufacturer’s recommendation. .. The CFD1 gene plus 456 bp upstream and 168 bp downstream of the ORF was amplified using the following primer set: 3F, 5′-ATGGAAGAAAGAGCCGTGAA-3′ (5′ end); 3R, 5′-CACTTGAAGAACATGCTGCA-3′ (3′ end).

    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′.

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    Roche expand high fidelity pcr system
    A. Schematic illustration (top) and partial G-banded karyotype (bottom) illustrating the 7;12 translocation. B. <t>RT-PCR</t> detected <t>ACTB-GLI1</t> 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.
    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
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    Roche high fidelity pcr kit
    Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction . ( A ) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. ( B ) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by <t>RT-PCR</t> using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the <t>DNA</t> molecular weight markers (lane M) is indicated in bp.
    High Fidelity Pcr Kit, 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
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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
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    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

    Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction . ( A ) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. ( B ) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by RT-PCR using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Journal: Virology Journal

    Article Title: Hepatitis A virus (HAV) packaging size limit

    doi: 10.1186/1743-422X-6-204

    Figure Lengend Snippet: Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction . ( A ) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. ( B ) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by RT-PCR using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Article Snippet: PCR-based DNA fragments were amplified using expand high fidelity PCR kit (Roche) in 25 cycles consisting of 95°C for 30 sec, 55°C for 1 min, and 72°C for 2-3 min. For overlap PCR, DNA fragments were denatured at 94°C and annealed at 45°C for 2 min in each step.

    Techniques: Construct, Transfection, In Vitro, Incubation, Selection, Microscopy, Recombinant, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight

    Rescue and stability of the HAV constructs containing the EMCV IRES at the 5'NTR . ( A ) IF analysis of FRhK4 cells transfected with in vitro RNA transcripts from pHAVvec9, pHAVvec9-Bsd, or pHAV-IRES or mock-transfected. Cells were fixed with acetone two weeks post-transfection, and stained with anti-HAV neutralizing monoclonal antibodies K2-4F2 and K3-4C8 and FITC-conjugated goat anti-mouse antibodies. Micrographs were taken with a Zeiss microscope at 400× magnification. ( B ) Analysis of the stability of HAV recombinants containing the EMCV IRES. RT-PCR analysis of genomic RNA extracted form HAV/7, HAV-IRES, HAVvec9-Bsd virions and amplified using primers corresponding to nts 484-507 and 1167-1194 of HAV. As negative control, T7 polymerase in vitro transcripts from pHAV-IRES were spiked into media, layered on top of a 40% sucrose cushion, and sedimented by ultracentrifugation. RNA extracted from the pellet was used for the RT-PCR analysis (Naked-RNA control). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Journal: Virology Journal

    Article Title: Hepatitis A virus (HAV) packaging size limit

    doi: 10.1186/1743-422X-6-204

    Figure Lengend Snippet: Rescue and stability of the HAV constructs containing the EMCV IRES at the 5'NTR . ( A ) IF analysis of FRhK4 cells transfected with in vitro RNA transcripts from pHAVvec9, pHAVvec9-Bsd, or pHAV-IRES or mock-transfected. Cells were fixed with acetone two weeks post-transfection, and stained with anti-HAV neutralizing monoclonal antibodies K2-4F2 and K3-4C8 and FITC-conjugated goat anti-mouse antibodies. Micrographs were taken with a Zeiss microscope at 400× magnification. ( B ) Analysis of the stability of HAV recombinants containing the EMCV IRES. RT-PCR analysis of genomic RNA extracted form HAV/7, HAV-IRES, HAVvec9-Bsd virions and amplified using primers corresponding to nts 484-507 and 1167-1194 of HAV. As negative control, T7 polymerase in vitro transcripts from pHAV-IRES were spiked into media, layered on top of a 40% sucrose cushion, and sedimented by ultracentrifugation. RNA extracted from the pellet was used for the RT-PCR analysis (Naked-RNA control). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Article Snippet: PCR-based DNA fragments were amplified using expand high fidelity PCR kit (Roche) in 25 cycles consisting of 95°C for 30 sec, 55°C for 1 min, and 72°C for 2-3 min. For overlap PCR, DNA fragments were denatured at 94°C and annealed at 45°C for 2 min in each step.

    Techniques: Construct, Transfection, In Vitro, Staining, Microscopy, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Molecular Weight

    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