Structured Review

Thermo Fisher uracil dna glycosylase
Uracil Dna Glycosylase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/uracil dna glycosylase/product/Thermo Fisher
Average 99 stars, based on 16 article reviews
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uracil dna glycosylase - by Bioz Stars, 2020-02
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Amplification:

Article Title: A Missense Mutation in CASK Causes FG Syndrome in an Italian Family
Article Snippet: GAPDH was amplified as housekeeping gene for data normalization. .. In order to prevent PCR carry-over contamination, Uracil N-glycosylase was used (Amperase UNG, Applied Biosystems).

Article Title: Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia ▿
Article Snippet: .. Briefly, 4 μl of DNA (in 10 mM Tris HCl, 0.5 mM EDTA, pH 9.0) was amplified in duplicate in 25-μl volumes with 1 U HotStarTaq Plus DNA polymerase (QIAGEN) using final concentrations of 416 nM (each) primer, 256 nM probe labeled with 6-carboxyfluorescein and a black hole quencher (Biosearch Techonologies), 6 mM MgCl2 , 0.2 mM deoxynucleoside triphosphates (including dUTP at a dUTP/dTTP ratio of 1:9), and 0.25 U uracil DNA glycosylase (Invitrogen). .. Nonacetylated bovine serum albumin at a final concentration of 400 ng/μl was added to bind PCR inhibitors such as humic acids ( ).

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: .. A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA). .. The fragmented cDNA was labeled by terminal deoxynucleotidyltransferase using the DNA labeling reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA), which was covalently linked to biotin.

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: The temperature settings during the PCR amplification were 45 s at 98 C followed by 15 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C and a final 5 min extension at 72°C. .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR.

Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
Article Snippet: Each individual reaction contained 5 μl 2X TaqMan universal PCR master mix with uracil-N-glycosylase (Applied Biosystems), 1 μl 200 nmol/L fluorogenic probe, 1 μl primer ratio, 0.65 ng human blood DNA, and up to 3 μl HPV DNA or sterile HPLC-grade water in non-template controls. .. The amplification profile was initiated by a 2-minute incubation at 50°C, followed by a 10-minute incubation at 95°C, and a two-step amplification of 15 seconds at 95°C and 60 seconds at 59.3°C or 54.1°C for 45 cycles.

Article Title: Molecular Study of Microsporidiosis Due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ †
Article Snippet: Paragraph title: DNA extraction and PCR amplification. ... PCR was performed with a 25-μl volume containing 6 mM MgCl2 , 1× PCR Rxn buffer (Invitrogen), a 200 μM concentration of each deoxynucleoside triphosphate, 0.2 μM FEI1, 0.2 μM REI1, 0.4 μM S.INT, 0.5 U of uracil DNA glycosylase (Invitrogen), 1× Exo Mix IPC and 0.5× IPC DNA (Applied Biosystems), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 5 μl DNA extract.

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction. .. After initial incubations of 2 min at 50 °C and 10 min at 95 °C to activate uracil- N -glycosylase and Taq Gold, respectively, 40 cycles of amplification were performed as follows: 30 s at 95 °C, 30 s at 60 °C and 45 s at 72 °C.

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: The double-stranded cDNA was subsequently used as a template and amplified by T7 RNA polymerase producing many copies of antisense complementary RNA. .. The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix).

Article Title: Comprehensive Cancer Panel Sequencing Defines Genetic Diversity and Changes in the Mutational Characteristics of Pancreatic Cancer Patients Receiving Neoadjuvant Treatment
Article Snippet: .. To prevent sequencing artifacts, DNA samples were treated with uracil-DNA glycosylase prior to amplification., Targeted panel sequencing was performed with the Ion AmpliSeq Comprehensive Cancer Panel covering 409 genes (Ion Torrent, Life Technologies, Carlsbad, CA, USA). .. Libraries were prepared for sequencing according to the manufacturer’s instructions, and the quality of the libraries was determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Synthesized:

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: In the second cycle of cDNA synthesis, random hexamers are used to prime reverse transcription of the complementary RNA from the first cycle to produce ssDNA in the sense orientation. cDNA was synthesized using the GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix). .. The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix).

Quantitative RT-PCR:

Article Title: A Missense Mutation in CASK Causes FG Syndrome in an Italian Family
Article Snippet: Paragraph title: RT-PCR and Real-Time RT-PCR ... In order to prevent PCR carry-over contamination, Uracil N-glycosylase was used (Amperase UNG, Applied Biosystems).

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Paragraph title: Quantitative RT-PCR ... Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction.

Real-time Polymerase Chain Reaction:

Article Title: Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia ▿
Article Snippet: Paragraph title: Real-time PCR. ... Briefly, 4 μl of DNA (in 10 mM Tris HCl, 0.5 mM EDTA, pH 9.0) was amplified in duplicate in 25-μl volumes with 1 U HotStarTaq Plus DNA polymerase (QIAGEN) using final concentrations of 416 nM (each) primer, 256 nM probe labeled with 6-carboxyfluorescein and a black hole quencher (Biosearch Techonologies), 6 mM MgCl2 , 0.2 mM deoxynucleoside triphosphates (including dUTP at a dUTP/dTTP ratio of 1:9), and 0.25 U uracil DNA glycosylase (Invitrogen).

Article Title: Transmission Probabilities of Mouse Parvovirus 1 to Sentinel Mice Chronically Exposed to Serial Dilutions of Contaminated Bedding
Article Snippet: Paragraph title: qPCR. ... Each 20-μl reaction consisted of 1× TaqMan buffer (50 mM KCl, 10 μM EDTA, 10 mM Tris-HCl [pH 8.3], and 60 nM passive reference dye); 5.5 mM MgCl2 ; 200 μM (each) dATP, dCTP, and dGTP; 400 μM dUTP; 300 nM primers; 125 nM probe; 0.2 U uracil-N-glycosylase (AmpErase, PE Applied Biosystems, Foster City, CA); 0.5 U Taq polymerase (AmpliTaq Gold, PE Applied Biosystems, Foster City, CA); and 2 μl template DNA (approximately 50 ng DNA).

Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
Article Snippet: Paragraph title: Real-Time PCR ... Each individual reaction contained 5 μl 2X TaqMan universal PCR master mix with uracil-N-glycosylase (Applied Biosystems), 1 μl 200 nmol/L fluorogenic probe, 1 μl primer ratio, 0.65 ng human blood DNA, and up to 3 μl HPV DNA or sterile HPLC-grade water in non-template controls.

Microarray:

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA). .. The mixed hybridization buffer was loaded into a microarray, and then the both septa were covered by round labels to prevent leaks and evaporation.

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix). .. Microarray data analyses were performed with R (version 3.0.0) using packages from Bioconductor.

Incubation:

Article Title: Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia ▿
Article Snippet: Briefly, 4 μl of DNA (in 10 mM Tris HCl, 0.5 mM EDTA, pH 9.0) was amplified in duplicate in 25-μl volumes with 1 U HotStarTaq Plus DNA polymerase (QIAGEN) using final concentrations of 416 nM (each) primer, 256 nM probe labeled with 6-carboxyfluorescein and a black hole quencher (Biosearch Techonologies), 6 mM MgCl2 , 0.2 mM deoxynucleoside triphosphates (including dUTP at a dUTP/dTTP ratio of 1:9), and 0.25 U uracil DNA glycosylase (Invitrogen). .. PCR conditions were as follows: an initial uracil DNA glycosylase incubation step at 37°C for 10 min followed by 94°C for 10 min and then 40 cycles of 94°C for 15 s and 60°C for 1 min. Nontemplate controls were added to each run and were always negative, i.e., no amplification was detected.

Article Title: Transmission Probabilities of Mouse Parvovirus 1 to Sentinel Mice Chronically Exposed to Serial Dilutions of Contaminated Bedding
Article Snippet: Each 20-μl reaction consisted of 1× TaqMan buffer (50 mM KCl, 10 μM EDTA, 10 mM Tris-HCl [pH 8.3], and 60 nM passive reference dye); 5.5 mM MgCl2 ; 200 μM (each) dATP, dCTP, and dGTP; 400 μM dUTP; 300 nM primers; 125 nM probe; 0.2 U uracil-N-glycosylase (AmpErase, PE Applied Biosystems, Foster City, CA); 0.5 U Taq polymerase (AmpliTaq Gold, PE Applied Biosystems, Foster City, CA); and 2 μl template DNA (approximately 50 ng DNA). .. Thermal cycling conditions consisted of 50 °C for 2 min for incubation of uracil-N-glycosylase, polymerase activation at 95 °C for 10 min, and then 45 cycles of 95 °C for 15 s followed by 60 °C for 1 min.

Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
Article Snippet: Each individual reaction contained 5 μl 2X TaqMan universal PCR master mix with uracil-N-glycosylase (Applied Biosystems), 1 μl 200 nmol/L fluorogenic probe, 1 μl primer ratio, 0.65 ng human blood DNA, and up to 3 μl HPV DNA or sterile HPLC-grade water in non-template controls. .. The amplification profile was initiated by a 2-minute incubation at 50°C, followed by a 10-minute incubation at 95°C, and a two-step amplification of 15 seconds at 95°C and 60 seconds at 59.3°C or 54.1°C for 45 cycles.

Article Title: Evolution of the Mycobacterial SigK Regulon ▿
Article Snippet: .. After incubation for 16 h at 15°C, 0.5 U of uracil DNA glycosylase and the corresponding 5× buffer (Gibco Life Technologies) were added and incubated for 20 min at 37°C. .. This solution served as template for PCR, using the AccuPrime Taq DNA high-fidelity polymerase (Invitrogen), following the manufacturer's instructions, using Uni70F-EcoRIR or Uni70R-EcoRIR primers.

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix). .. Hybridization was performed using 5 µg of biotinylated target, which was incubated with the GeneChip Human gene 2.0 array (Affymetrix) at 45°C for 16–20 h. After hybridization, non–specifically bound material was removed by washing and specifically bound target was detected using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix).

Formalin-fixed Paraffin-Embedded:

Article Title: Comprehensive Cancer Panel Sequencing Defines Genetic Diversity and Changes in the Mutational Characteristics of Pancreatic Cancer Patients Receiving Neoadjuvant Treatment
Article Snippet: Comprehensive cancer panel Genomic DNA of patients was extracted from blood samples using QIAamp blood DNA mini kits (Qiagen, Valencia, CA, USA) and from formalin-fixed paraffin embedded (FFPE) endoscopic ultrasound (EUS)-guided biopsy samples, and surgically resected tissue using QIAamp DNA FFPE tissue kits (Qiagen). .. To prevent sequencing artifacts, DNA samples were treated with uracil-DNA glycosylase prior to amplification., Targeted panel sequencing was performed with the Ion AmpliSeq Comprehensive Cancer Panel covering 409 genes (Ion Torrent, Life Technologies, Carlsbad, CA, USA).

Expressing:

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction. .. Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12, GAPD) expression was analyzed as above with an annealing temperature of 58 °C for primers fGAPD 101c (5′-CCTTCATTGACCTCAACTACAT-3′) and fGAPD 225n (5′-GAAGATGGTGATGGGCTTT-3′).

Hybridization:

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA). .. The resulting labeled cDNAs (5.2 µg) were dissolved in 160 μL of hybridization mix solution, then denatured at 99 °C for 5 min.

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix). .. Hybridization was performed using 5 µg of biotinylated target, which was incubated with the GeneChip Human gene 2.0 array (Affymetrix) at 45°C for 16–20 h. After hybridization, non–specifically bound material was removed by washing and specifically bound target was detected using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix).

High Performance Liquid Chromatography:

Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
Article Snippet: .. Each individual reaction contained 5 μl 2X TaqMan universal PCR master mix with uracil-N-glycosylase (Applied Biosystems), 1 μl 200 nmol/L fluorogenic probe, 1 μl primer ratio, 0.65 ng human blood DNA, and up to 3 μl HPV DNA or sterile HPLC-grade water in non-template controls. .. The amplification profile was initiated by a 2-minute incubation at 50°C, followed by a 10-minute incubation at 95°C, and a two-step amplification of 15 seconds at 95°C and 60 seconds at 59.3°C or 54.1°C for 45 cycles.

Sequencing:

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: Sequencing was performed on the Illumina Nextseq500 (75bp single end reads). .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR.

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Quantitative RT-PCR (qRT-PCR) assays for feline βgal mRNA were performed on an Applied Biosystems model 7700 sequence detector using primers 3a and 2b ( ). .. Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction.

Article Title: Evolution of the Mycobacterial SigK Regulon ▿
Article Snippet: Paragraph title: Determination of homologues and sequence analysis. ... After incubation for 16 h at 15°C, 0.5 U of uracil DNA glycosylase and the corresponding 5× buffer (Gibco Life Technologies) were added and incubated for 20 min at 37°C.

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: Using 100 ng of total RNA, double-stranded cDNA was synthesized with random hexamers tagged with a T7 promoter sequence. .. The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix).

Article Title: Comprehensive Cancer Panel Sequencing Defines Genetic Diversity and Changes in the Mutational Characteristics of Pancreatic Cancer Patients Receiving Neoadjuvant Treatment
Article Snippet: .. To prevent sequencing artifacts, DNA samples were treated with uracil-DNA glycosylase prior to amplification., Targeted panel sequencing was performed with the Ion AmpliSeq Comprehensive Cancer Panel covering 409 genes (Ion Torrent, Life Technologies, Carlsbad, CA, USA). .. Libraries were prepared for sequencing according to the manufacturer’s instructions, and the quality of the libraries was determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Ligation:

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR. .. A detailed protocol to generate in situ Hi-C libraries including sequence alignment and Aggregate Peak Analysis can be obtained from ( ).

Article Title: Evolution of the Mycobacterial SigK Regulon ▿
Article Snippet: The ligation step was performed in 20 μl, by adding 1 ng of digested genomic DNA, annealing primers (final concentration, 0.67 pmol/μl), 5× T4 DNA ligase buffer, and 1 U of T4 DNA ligase (Invitrogen). .. After incubation for 16 h at 15°C, 0.5 U of uracil DNA glycosylase and the corresponding 5× buffer (Gibco Life Technologies) were added and incubated for 20 min at 37°C.

Generated:

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR. .. A detailed protocol to generate in situ Hi-C libraries including sequence alignment and Aggregate Peak Analysis can be obtained from ( ).

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction. .. βgal copy numbers from experimental samples were extrapolated from a standard curve generated with the same amplification conditions as above, with cDNA replaced by varying copy numbers (102 –108 ) of plasmid fBgal 3.9 (cloned in our laboratory), which contains the putative open reading frame of feline βgal.

Article Title: Comprehensive Cancer Panel Sequencing Defines Genetic Diversity and Changes in the Mutational Characteristics of Pancreatic Cancer Patients Receiving Neoadjuvant Treatment
Article Snippet: To prevent sequencing artifacts, DNA samples were treated with uracil-DNA glycosylase prior to amplification., Targeted panel sequencing was performed with the Ion AmpliSeq Comprehensive Cancer Panel covering 409 genes (Ion Torrent, Life Technologies, Carlsbad, CA, USA). .. Next-generation sequencing data were generated from 22 tumor samples, seven biopsy samples, and 22 blood samples from the 22 patients with PDA.

DNA Labeling:

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA). .. The fragmented cDNA was labeled by terminal deoxynucleotidyltransferase using the DNA labeling reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA), which was covalently linked to biotin.

Reverse Transcription Polymerase Chain Reaction:

Article Title: A Missense Mutation in CASK Causes FG Syndrome in an Italian Family
Article Snippet: Paragraph title: RT-PCR and Real-Time RT-PCR ... In order to prevent PCR carry-over contamination, Uracil N-glycosylase was used (Amperase UNG, Applied Biosystems).

Binding Assay:

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Because primer 3a anneals in exon 3, the qRT-PCR assay measures transcript levels of βgal only, not the elastin–laminin binding protein (a GLB1 splice variant lacking exons 3, 4 and 6). .. Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction.

DNA Extraction:

Article Title: Transmission Probabilities of Mouse Parvovirus 1 to Sentinel Mice Chronically Exposed to Serial Dilutions of Contaminated Bedding
Article Snippet: DNA was extracted from tissues and feces and screened by an MPV-specific qPCR assay as previously described., Briefly, DNA was extracted using a genomic DNA extraction kit (MagneSil KF, Promega, Madison, WI) and a robotic extraction station (KingFisher, Thermo Labsystems, Franklin, MA) according to the manufacturer's recommendations. qPCR reactions were performed by using an automated system (Mx3000P QPCR System, Stratagene, Cedar Creek, TX), and products were analyzed with the accompanying software. .. Each 20-μl reaction consisted of 1× TaqMan buffer (50 mM KCl, 10 μM EDTA, 10 mM Tris-HCl [pH 8.3], and 60 nM passive reference dye); 5.5 mM MgCl2 ; 200 μM (each) dATP, dCTP, and dGTP; 400 μM dUTP; 300 nM primers; 125 nM probe; 0.2 U uracil-N-glycosylase (AmpErase, PE Applied Biosystems, Foster City, CA); 0.5 U Taq polymerase (AmpliTaq Gold, PE Applied Biosystems, Foster City, CA); and 2 μl template DNA (approximately 50 ng DNA).

Article Title: Molecular Study of Microsporidiosis Due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ †
Article Snippet: Paragraph title: DNA extraction and PCR amplification. ... PCR was performed with a 25-μl volume containing 6 mM MgCl2 , 1× PCR Rxn buffer (Invitrogen), a 200 μM concentration of each deoxynucleoside triphosphate, 0.2 μM FEI1, 0.2 μM REI1, 0.4 μM S.INT, 0.5 U of uracil DNA glycosylase (Invitrogen), 1× Exo Mix IPC and 0.5× IPC DNA (Applied Biosystems), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 5 μl DNA extract.

Fluorescence:

Article Title: Transmission Probabilities of Mouse Parvovirus 1 to Sentinel Mice Chronically Exposed to Serial Dilutions of Contaminated Bedding
Article Snippet: Each 20-μl reaction consisted of 1× TaqMan buffer (50 mM KCl, 10 μM EDTA, 10 mM Tris-HCl [pH 8.3], and 60 nM passive reference dye); 5.5 mM MgCl2 ; 200 μM (each) dATP, dCTP, and dGTP; 400 μM dUTP; 300 nM primers; 125 nM probe; 0.2 U uracil-N-glycosylase (AmpErase, PE Applied Biosystems, Foster City, CA); 0.5 U Taq polymerase (AmpliTaq Gold, PE Applied Biosystems, Foster City, CA); and 2 μl template DNA (approximately 50 ng DNA). .. Samples were considered positive if they exhibited a mean fluorescence of greater than 0.1 and a cycle threshold of less than 35.

Labeling:

Article Title: Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia ▿
Article Snippet: .. Briefly, 4 μl of DNA (in 10 mM Tris HCl, 0.5 mM EDTA, pH 9.0) was amplified in duplicate in 25-μl volumes with 1 U HotStarTaq Plus DNA polymerase (QIAGEN) using final concentrations of 416 nM (each) primer, 256 nM probe labeled with 6-carboxyfluorescein and a black hole quencher (Biosearch Techonologies), 6 mM MgCl2 , 0.2 mM deoxynucleoside triphosphates (including dUTP at a dUTP/dTTP ratio of 1:9), and 0.25 U uracil DNA glycosylase (Invitrogen). .. Nonacetylated bovine serum albumin at a final concentration of 400 ng/μl was added to bind PCR inhibitors such as humic acids ( ).

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA). .. The fragmented cDNA was labeled by terminal deoxynucleotidyltransferase using the DNA labeling reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA), which was covalently linked to biotin.

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: .. The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix). .. Hybridization was performed using 5 µg of biotinylated target, which was incubated with the GeneChip Human gene 2.0 array (Affymetrix) at 45°C for 16–20 h. After hybridization, non–specifically bound material was removed by washing and specifically bound target was detected using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix).

Purification:

Article Title: Evolution of the Mycobacterial SigK Regulon ▿
Article Snippet: After incubation for 16 h at 15°C, 0.5 U of uracil DNA glycosylase and the corresponding 5× buffer (Gibco Life Technologies) were added and incubated for 20 min at 37°C. .. The PCR products were purified by gel extraction kit (Qiagen) and sequenced.

Polymerase Chain Reaction:

Article Title: A Missense Mutation in CASK Causes FG Syndrome in an Italian Family
Article Snippet: .. In order to prevent PCR carry-over contamination, Uracil N-glycosylase was used (Amperase UNG, Applied Biosystems). .. Each assay was performed in triplicate and results were normalized and analyzed with SDS2.1 software (Applied Biosystems).

Article Title: Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia ▿
Article Snippet: Briefly, 4 μl of DNA (in 10 mM Tris HCl, 0.5 mM EDTA, pH 9.0) was amplified in duplicate in 25-μl volumes with 1 U HotStarTaq Plus DNA polymerase (QIAGEN) using final concentrations of 416 nM (each) primer, 256 nM probe labeled with 6-carboxyfluorescein and a black hole quencher (Biosearch Techonologies), 6 mM MgCl2 , 0.2 mM deoxynucleoside triphosphates (including dUTP at a dUTP/dTTP ratio of 1:9), and 0.25 U uracil DNA glycosylase (Invitrogen). .. Nonacetylated bovine serum albumin at a final concentration of 400 ng/μl was added to bind PCR inhibitors such as humic acids ( ).

Article Title: Sensitivity of PCR Targeting the IS2404 Insertion Sequence of Mycobacterium ulcerans in an Assay Using Punch Biopsy Specimens for Diagnosis of Buruli Ulcer
Article Snippet: .. The composition of the PCR mixture was similar to that described by Kox et al. , with final reaction concentrations of 10 mM Tris, 50 mM KCl, 0.01% gelatin, 1.5 mM MgCl2 , 0.1% Triton X-100, 0.2 mM (each) deoxynucleoside diphosphate (dATP, dCTP, dGTP, and dUTP) (Roche Diagnostics GmbH, Germany), 0.2 μM each of forward primer PU4F (5′-GCGCAGATCAACTTCGCGGT 3′) and reverse primer PU7Rbio (5′-GCCCGATTGGTGCTCGGTCA-3′) (gene positions 548 to 567 bp and 702 to 683 bp, respectively), 1 unit of Taq DNA polymerase (HT Biotechnology, United Kingdom), and 0.1 U of uracil DNA glycosylase (UDG; Invitrogen, United Kingdom) per 50-μl reaction volume. ..

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR. .. A detailed protocol to generate in situ Hi-C libraries including sequence alignment and Aggregate Peak Analysis can be obtained from ( ).

Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
Article Snippet: .. Each individual reaction contained 5 μl 2X TaqMan universal PCR master mix with uracil-N-glycosylase (Applied Biosystems), 1 μl 200 nmol/L fluorogenic probe, 1 μl primer ratio, 0.65 ng human blood DNA, and up to 3 μl HPV DNA or sterile HPLC-grade water in non-template controls. .. The amplification profile was initiated by a 2-minute incubation at 50°C, followed by a 10-minute incubation at 95°C, and a two-step amplification of 15 seconds at 95°C and 60 seconds at 59.3°C or 54.1°C for 45 cycles.

Article Title: Molecular Study of Microsporidiosis Due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ †
Article Snippet: .. PCR was performed with a 25-μl volume containing 6 mM MgCl2 , 1× PCR Rxn buffer (Invitrogen), a 200 μM concentration of each deoxynucleoside triphosphate, 0.2 μM FEI1, 0.2 μM REI1, 0.4 μM S.INT, 0.5 U of uracil DNA glycosylase (Invitrogen), 1× Exo Mix IPC and 0.5× IPC DNA (Applied Biosystems), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 5 μl DNA extract. .. PCR conditions were as follows: after 2 min at 50°C and initial denaturation at 95°C for 10 min, amplification consisted of 45 cycles of 15 s of denaturation followed by 60 s of annealing and extension at 63°C.

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: .. Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction. .. After initial incubations of 2 min at 50 °C and 10 min at 95 °C to activate uracil- N -glycosylase and Taq Gold, respectively, 40 cycles of amplification were performed as follows: 30 s at 95 °C, 30 s at 60 °C and 45 s at 72 °C.

Article Title: Evolution of the Mycobacterial SigK Regulon ▿
Article Snippet: To amplify the flanking region of putative mpk70 and/or mpk83 , we did mixed-linker PCR by modifying a previously described method ( , ). .. After incubation for 16 h at 15°C, 0.5 U of uracil DNA glycosylase and the corresponding 5× buffer (Gibco Life Technologies) were added and incubated for 20 min at 37°C.

Gel Extraction:

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: PCR reactions were cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear 200–500bp was cut and gel purify using QIAquick Gel Extraction Kit (Qiagen). .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR.

Article Title: Evolution of the Mycobacterial SigK Regulon ▿
Article Snippet: After incubation for 16 h at 15°C, 0.5 U of uracil DNA glycosylase and the corresponding 5× buffer (Gibco Life Technologies) were added and incubated for 20 min at 37°C. .. The PCR products were purified by gel extraction kit (Qiagen) and sequenced.

Concentration Assay:

Article Title: Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia ▿
Article Snippet: Briefly, 4 μl of DNA (in 10 mM Tris HCl, 0.5 mM EDTA, pH 9.0) was amplified in duplicate in 25-μl volumes with 1 U HotStarTaq Plus DNA polymerase (QIAGEN) using final concentrations of 416 nM (each) primer, 256 nM probe labeled with 6-carboxyfluorescein and a black hole quencher (Biosearch Techonologies), 6 mM MgCl2 , 0.2 mM deoxynucleoside triphosphates (including dUTP at a dUTP/dTTP ratio of 1:9), and 0.25 U uracil DNA glycosylase (Invitrogen). .. Nonacetylated bovine serum albumin at a final concentration of 400 ng/μl was added to bind PCR inhibitors such as humic acids ( ).

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR.

Article Title: Molecular Study of Microsporidiosis Due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ †
Article Snippet: .. PCR was performed with a 25-μl volume containing 6 mM MgCl2 , 1× PCR Rxn buffer (Invitrogen), a 200 μM concentration of each deoxynucleoside triphosphate, 0.2 μM FEI1, 0.2 μM REI1, 0.4 μM S.INT, 0.5 U of uracil DNA glycosylase (Invitrogen), 1× Exo Mix IPC and 0.5× IPC DNA (Applied Biosystems), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 5 μl DNA extract. .. PCR conditions were as follows: after 2 min at 50°C and initial denaturation at 95°C for 10 min, amplification consisted of 45 cycles of 15 s of denaturation followed by 60 s of annealing and extension at 63°C.

Article Title: Evolution of the Mycobacterial SigK Regulon ▿
Article Snippet: The ligation step was performed in 20 μl, by adding 1 ng of digested genomic DNA, annealing primers (final concentration, 0.67 pmol/μl), 5× T4 DNA ligase buffer, and 1 U of T4 DNA ligase (Invitrogen). .. After incubation for 16 h at 15°C, 0.5 U of uracil DNA glycosylase and the corresponding 5× buffer (Gibco Life Technologies) were added and incubated for 20 min at 37°C.

Chromatin Immunoprecipitation:

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: Paragraph title: 4.3. RNA Extraction, cDNA Preparation and Gene Chip ... A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA).

Plasmid Preparation:

Article Title: Molecular Study of Microsporidiosis Due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam ▿ †
Article Snippet: PCR was performed with a 25-μl volume containing 6 mM MgCl2 , 1× PCR Rxn buffer (Invitrogen), a 200 μM concentration of each deoxynucleoside triphosphate, 0.2 μM FEI1, 0.2 μM REI1, 0.4 μM S.INT, 0.5 U of uracil DNA glycosylase (Invitrogen), 1× Exo Mix IPC and 0.5× IPC DNA (Applied Biosystems), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 5 μl DNA extract. .. Both methods showed high sensitivity (data not shown), as determined by serial 10-fold dilutions of a plasmid DNA carrying the SSU rRNA fragment, which allowed the detection of less than 20 copies/μl of E. bieneusi and E. intestinalis .

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction. .. βgal copy numbers from experimental samples were extrapolated from a standard curve generated with the same amplification conditions as above, with cDNA replaced by varying copy numbers (102 –108 ) of plasmid fBgal 3.9 (cloned in our laboratory), which contains the putative open reading frame of feline βgal.

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: For SV40-immortalized fibroblasts, 48 h after seeding of single guide RNA against BLM (sgBLM) and empty vector (EV) cells, total RNA was extracted with a Nucleospin kit and eluted in 40 µl of RNase/DNase free water. .. The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix).

Software:

Article Title: A Missense Mutation in CASK Causes FG Syndrome in an Italian Family
Article Snippet: In order to prevent PCR carry-over contamination, Uracil N-glycosylase was used (Amperase UNG, Applied Biosystems). .. Each assay was performed in triplicate and results were normalized and analyzed with SDS2.1 software (Applied Biosystems).

Article Title: Transmission Probabilities of Mouse Parvovirus 1 to Sentinel Mice Chronically Exposed to Serial Dilutions of Contaminated Bedding
Article Snippet: DNA was extracted from tissues and feces and screened by an MPV-specific qPCR assay as previously described., Briefly, DNA was extracted using a genomic DNA extraction kit (MagneSil KF, Promega, Madison, WI) and a robotic extraction station (KingFisher, Thermo Labsystems, Franklin, MA) according to the manufacturer's recommendations. qPCR reactions were performed by using an automated system (Mx3000P QPCR System, Stratagene, Cedar Creek, TX), and products were analyzed with the accompanying software. .. Each 20-μl reaction consisted of 1× TaqMan buffer (50 mM KCl, 10 μM EDTA, 10 mM Tris-HCl [pH 8.3], and 60 nM passive reference dye); 5.5 mM MgCl2 ; 200 μM (each) dATP, dCTP, and dGTP; 400 μM dUTP; 300 nM primers; 125 nM probe; 0.2 U uracil-N-glycosylase (AmpErase, PE Applied Biosystems, Foster City, CA); 0.5 U Taq polymerase (AmpliTaq Gold, PE Applied Biosystems, Foster City, CA); and 2 μl template DNA (approximately 50 ng DNA).

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix). .. The arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix), and raw data were extracted from the scanned images and analy zed with the Affymetrix Power Tools software package.

SYBR Green Assay:

Article Title: A Missense Mutation in CASK Causes FG Syndrome in an Italian Family
Article Snippet: Real-time RT-PCR experiments were performed on ABI Prism 7900HT System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems) according to manufacturer's instructions. .. In order to prevent PCR carry-over contamination, Uracil N-glycosylase was used (Amperase UNG, Applied Biosystems).

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: .. Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction. .. After initial incubations of 2 min at 50 °C and 10 min at 95 °C to activate uracil- N -glycosylase and Taq Gold, respectively, 40 cycles of amplification were performed as follows: 30 s at 95 °C, 30 s at 60 °C and 45 s at 72 °C.

RNA Extraction:

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: Paragraph title: 4.3. RNA Extraction, cDNA Preparation and Gene Chip ... A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA).

Agarose Gel Electrophoresis:

Article Title: Genome Organization Drives Chromosome Fragility
Article Snippet: PCR reactions were cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear 200–500bp was cut and gel purify using QIAquick Gel Extraction Kit (Qiagen). .. Alternatively, for the triptolide experiment strand specific nascent-RNA libraries were generated by using in the second-strand synthesis reaction 1.2 mM of dUTP instead of 0.6 mM of dTTP, and the elution of the ligation reaction was treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 minutes at 37°C before the PCR.

Next-Generation Sequencing:

Article Title: Comprehensive Cancer Panel Sequencing Defines Genetic Diversity and Changes in the Mutational Characteristics of Pancreatic Cancer Patients Receiving Neoadjuvant Treatment
Article Snippet: To prevent sequencing artifacts, DNA samples were treated with uracil-DNA glycosylase prior to amplification., Targeted panel sequencing was performed with the Ion AmpliSeq Comprehensive Cancer Panel covering 409 genes (Ion Torrent, Life Technologies, Carlsbad, CA, USA). .. Next-generation sequencing data were generated from 22 tumor samples, seven biopsy samples, and 22 blood samples from the 22 patients with PDA.

Evaporation:

Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
Article Snippet: A total of 5.5 μg of the amplified product was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Thermo Fisher Scientific, USA). .. The mixed hybridization buffer was loaded into a microarray, and then the both septa were covered by round labels to prevent leaks and evaporation.

Activation Assay:

Article Title: Transmission Probabilities of Mouse Parvovirus 1 to Sentinel Mice Chronically Exposed to Serial Dilutions of Contaminated Bedding
Article Snippet: Each 20-μl reaction consisted of 1× TaqMan buffer (50 mM KCl, 10 μM EDTA, 10 mM Tris-HCl [pH 8.3], and 60 nM passive reference dye); 5.5 mM MgCl2 ; 200 μM (each) dATP, dCTP, and dGTP; 400 μM dUTP; 300 nM primers; 125 nM probe; 0.2 U uracil-N-glycosylase (AmpErase, PE Applied Biosystems, Foster City, CA); 0.5 U Taq polymerase (AmpliTaq Gold, PE Applied Biosystems, Foster City, CA); and 2 μl template DNA (approximately 50 ng DNA). .. Thermal cycling conditions consisted of 50 °C for 2 min for incubation of uracil-N-glycosylase, polymerase activation at 95 °C for 10 min, and then 45 cycles of 95 °C for 15 s followed by 60 °C for 1 min.

Standard Deviation:

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Data are expressed as means ± standard deviation (SD). .. Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction.

Staining:

Article Title: Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS
Article Snippet: The sense cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease-1 and biotin labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix). .. Hybridization was performed using 5 µg of biotinylated target, which was incubated with the GeneChip Human gene 2.0 array (Affymetrix) at 45°C for 16–20 h. After hybridization, non–specifically bound material was removed by washing and specifically bound target was detected using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix).

Variant Assay:

Article Title: Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis
Article Snippet: Because primer 3a anneals in exon 3, the qRT-PCR assay measures transcript levels of βgal only, not the elastin–laminin binding protein (a GLB1 splice variant lacking exons 3, 4 and 6). .. Quadruplicate 25 μl PCR reactions were performed with 1 × Sybr Green buffer, 3 mM MgCl2 , 0.2 mM dNTP’s, 0.625 U Taq Gold, 0.25 U uracil- N -glycosylase (all from Applied Biosystems), 0.4 μM each primer and 0.5 μl cDNA template per reaction.

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    Thermo Fisher gene exp smug1 mm00452897 m1
    Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , <t>Smug1</t> , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.
    Gene Exp Smug1 Mm00452897 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp smug1 mm00452897 m1/product/Thermo Fisher
    Average 83 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gene exp smug1 mm00452897 m1 - by Bioz Stars, 2020-02
    83/100 stars
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    84
    Thermo Fisher uracil dna glycosylase
    Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , <t>Smug1</t> , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.
    Uracil Dna Glycosylase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/uracil dna glycosylase/product/Thermo Fisher
    Average 84 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2020-02
    84/100 stars
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    Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , Smug1 , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.

    Journal: The Journal of Experimental Medicine

    Article Title: Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

    doi: 10.1084/jem.20161576

    Figure Lengend Snippet: Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , Smug1 , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.

    Article Snippet: Relative transcript abundance was assessed by real-time PCR on a PCR machine (7500 Fast System Real-Time; Applied Biosystems; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 10 s at 95°C and 1 min at 60°C) in universal PCR master mix (No AmpErase UNG; Applied Biosystems), with the following validated TaqMan assays: Mm00437762_m1 (β2-microglobulin), Mm01225357_g1 (Tdg), Mm01184338_m1 (Mbd4), and Mm00452897_m1 (Smug1).

    Techniques: Mutagenesis, Isolation, Mouse Assay, Injection, Two Tailed Test, MANN-WHITNEY, shRNA, Transduction, Inhibition

    Analysis of uracil glycosylase expression levels by qRT-PCR. (A) Relative expression levels of Mbd4 , Tdg , and Smug1 in naive B cells (B220 + GL7 − CD95 − ) from spleen of immunized WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice, compared with β-2 microglobulin (b2m). (B) Relative expression of Mbd4 , Tdg , and Smug1 in GC B cells (B220 + GL7 + CD95 + ) compared with naive B cells from spleens of WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice. The dotted line represents a ratio of 1 between GC and naive B cells. Quantification was performed in triplicates for RNA samples from three to four different mice. Error bars represent SD.

    Journal: The Journal of Experimental Medicine

    Article Title: Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

    doi: 10.1084/jem.20161576

    Figure Lengend Snippet: Analysis of uracil glycosylase expression levels by qRT-PCR. (A) Relative expression levels of Mbd4 , Tdg , and Smug1 in naive B cells (B220 + GL7 − CD95 − ) from spleen of immunized WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice, compared with β-2 microglobulin (b2m). (B) Relative expression of Mbd4 , Tdg , and Smug1 in GC B cells (B220 + GL7 + CD95 + ) compared with naive B cells from spleens of WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice. The dotted line represents a ratio of 1 between GC and naive B cells. Quantification was performed in triplicates for RNA samples from three to four different mice. Error bars represent SD.

    Article Snippet: Relative transcript abundance was assessed by real-time PCR on a PCR machine (7500 Fast System Real-Time; Applied Biosystems; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 10 s at 95°C and 1 min at 60°C) in universal PCR master mix (No AmpErase UNG; Applied Biosystems), with the following validated TaqMan assays: Mm00437762_m1 (β2-microglobulin), Mm01225357_g1 (Tdg), Mm01184338_m1 (Mbd4), and Mm00452897_m1 (Smug1).

    Techniques: Expressing, Quantitative RT-PCR, Mouse Assay

    A model whereby both uracil glycosylases and the Pms2/Mlh1 complex enable A-T base pair mutagenesis with a distinct strand bias. (A) Overlapping enzymatic activities generate entry sites for ExoI in MMR-mediated A-T mutagenesis. (Right) Recognition of AID deamination products by Msh2–Msh6 stimulates the endonuclease activity of the Pms2–Mlh1 complex, which acts at distance on either DNA strands. (Left) In a situation of processive deamination, nicks introduced by uracil glycosylase–mediated base excision (UNG, SMUG1, and TDG) and APEX2 strand incision can participate in MMR-mediated error-prone repair. Glycosylases may also act in the context of two independent deamination events on both DNA strands or, possibly, in the context of a single uracil (see Discussion). (B) Pms2 versus uracil glycosylase contribution estimated from A/T mutation ratios. The 1.8- to twofold A over T ratio observed in hypermutation was estimated to correspond to a 3:1 strand bias in error-prone DNA synthesis, taking into account the intrinsic T over A mutation preference of Pol η (a 4:1 higher mutation frequency opposite T than opposite A; Zivojnovic et al., 2014 ). This strand bias is represented by arrows of unequal length, representing the relative A:T mutagenic patch on each strand, without aiming at indicating whether the patch is shorter or less frequent on the transcribed strand or differently located on the locus. If one considers, from mutations observed in Pms2 −/− mice, that uracil glycosylases operate mainly on the coding, nontranscribed strand and that PMS2 endonuclease operates without strand bias and is responsible for most mutations introduced in the transcribed strand, both pathways appear to contribute equally to the generation of Msh2/6-driven A-T mutations.

    Journal: The Journal of Experimental Medicine

    Article Title: Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

    doi: 10.1084/jem.20161576

    Figure Lengend Snippet: A model whereby both uracil glycosylases and the Pms2/Mlh1 complex enable A-T base pair mutagenesis with a distinct strand bias. (A) Overlapping enzymatic activities generate entry sites for ExoI in MMR-mediated A-T mutagenesis. (Right) Recognition of AID deamination products by Msh2–Msh6 stimulates the endonuclease activity of the Pms2–Mlh1 complex, which acts at distance on either DNA strands. (Left) In a situation of processive deamination, nicks introduced by uracil glycosylase–mediated base excision (UNG, SMUG1, and TDG) and APEX2 strand incision can participate in MMR-mediated error-prone repair. Glycosylases may also act in the context of two independent deamination events on both DNA strands or, possibly, in the context of a single uracil (see Discussion). (B) Pms2 versus uracil glycosylase contribution estimated from A/T mutation ratios. The 1.8- to twofold A over T ratio observed in hypermutation was estimated to correspond to a 3:1 strand bias in error-prone DNA synthesis, taking into account the intrinsic T over A mutation preference of Pol η (a 4:1 higher mutation frequency opposite T than opposite A; Zivojnovic et al., 2014 ). This strand bias is represented by arrows of unequal length, representing the relative A:T mutagenic patch on each strand, without aiming at indicating whether the patch is shorter or less frequent on the transcribed strand or differently located on the locus. If one considers, from mutations observed in Pms2 −/− mice, that uracil glycosylases operate mainly on the coding, nontranscribed strand and that PMS2 endonuclease operates without strand bias and is responsible for most mutations introduced in the transcribed strand, both pathways appear to contribute equally to the generation of Msh2/6-driven A-T mutations.

    Article Snippet: Relative transcript abundance was assessed by real-time PCR on a PCR machine (7500 Fast System Real-Time; Applied Biosystems; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 10 s at 95°C and 1 min at 60°C) in universal PCR master mix (No AmpErase UNG; Applied Biosystems), with the following validated TaqMan assays: Mm00437762_m1 (β2-microglobulin), Mm01225357_g1 (Tdg), Mm01184338_m1 (Mbd4), and Mm00452897_m1 (Smug1).

    Techniques: Mutagenesis, Activity Assay, Activated Clotting Time Assay, DNA Synthesis, Mouse Assay