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Roche dntp mix
Dntp Mix, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dntp mix - by Bioz Stars, 2020-02
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Clone Assay:

Article Title: Development of a Multiplexed Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Identify Common Members of the Subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala
Article Snippet: .. An approximately 1-kb region of rDNA gene array including both internal transcribed spacer regions was amplified from voucher specimens for cloning and sequencing., A 100 μL reaction was prepared with 1X of Gene Amp PCR Buffer I (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.01% wt:vol gelatin) (Applied Biosystems, Foster City, CA), 1 mM of dNTP mix (Roche Diagnostics, Indianapolis, IN), 120 nM of each primer, 2.5 U of Amplitaq DNA polymerase (Applied Biosystems), and 60 ng of template DNA. .. Amplification was conducted under the following thermal cycler conditions: one cycle at 97°C for 4 minutes, followed by 30 cycles of 96°C for 30 seconds, 48°C for 30 seconds and 72°C for 2 minutes, and ending with a 5 minute extension step at 72°C.

Centrifugation:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: Reactions were incubated at 16°C for 2hr and the resulting cDNAs were purified. cDNA binding buffer (250μL) was added to each reaction which was then mixed and passed through a cDNA filter cartridge by centrifugation at 10,000 × g for 1 min. Filters were washed with wash Buffer (500μL) and dried by centrifugation for an additional minute. cDNA was eluted using 2 × 10μL aliquots of Nuclease-free Water at 55°C. .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme.

Amplification:

Article Title: Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma
Article Snippet: .. Amplification and sequencing of MTAP -Exon 3 For PCR amplification of exon 3 of MTAP , 1 μL cDNA (extracted from tissue or cell lines, or 2 μL genomic DNA (extracted from tissue or cell lines) were added to 0.5 μL of forward and reverse primers (MTAP-for89: 5’-GCCCACTGCAGATTCCTTTC-3’ ; MTAP-rev416: 5’-GGTCTCATAGTGGTCCTGTC-3’ ; each 20 μM), 5 μL PCR reaction buffer (10x), 0.5 μL dNTP mix (each 10 mM), and 0.5 μL (2.5 Units) FastStart Taq DNA Polymerase dNTPack (Roche, Mannheim, Germany) in a total reaction volume of 50 μL. .. The following PCR program was used: 5 min at 95°C (initial denaturation); 60 s at 95°C (denaturation); 30 s at 61°C (annealing), 30 s at 72°C (elongation), repeated 35 times.

Article Title: Development of a Multiplexed Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Identify Common Members of the Subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala
Article Snippet: .. An approximately 1-kb region of rDNA gene array including both internal transcribed spacer regions was amplified from voucher specimens for cloning and sequencing., A 100 μL reaction was prepared with 1X of Gene Amp PCR Buffer I (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.01% wt:vol gelatin) (Applied Biosystems, Foster City, CA), 1 mM of dNTP mix (Roche Diagnostics, Indianapolis, IN), 120 nM of each primer, 2.5 U of Amplitaq DNA polymerase (Applied Biosystems), and 60 ng of template DNA. .. Amplification was conducted under the following thermal cycler conditions: one cycle at 97°C for 4 minutes, followed by 30 cycles of 96°C for 30 seconds, 48°C for 30 seconds and 72°C for 2 minutes, and ending with a 5 minute extension step at 72°C.

Article Title: scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells
Article Snippet: Purified products were resuspended in 50 μl of second strand mastermix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT) then heated to 98 °C for 2 min and cooled on ice. .. Second strand products were purified using a 0.75:1 ratio of AMPure XP beads and resuspended in 50 μl of PCR mastermix (1× KAPA HiFi Readymix, 0.2 μM PE1.0 primer, 0.2 μM iTAG index primer) and amplified with 14 cycles.

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: RNA samples, with A260:A280 ratios between 1.7 and 2.1, and with integrity confirmed by denaturing agarose gel electrophoresis, were labeled using the Illumina® RNA Amplification kit (Ambion, Austin, TX). .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme.

Article Title: Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention
Article Snippet: Retinoic acid receptor alpha cDNA sequences (RARα) were amplified in separate reactions as positive cDNA controls. .. LightCycler‐FastStart DNA master SYBR Green, which contains Taq DNA polymerase, dNTP mix, SYBR Green 1 dye and MgCl2 (Roche), was used as a reaction mix for PCR.

Article Title: Microbial Community of High Arsenic Groundwater in Agricultural Irrigation Area of Hetao Plain, Inner Mongolia
Article Snippet: The 25 μL PCR reaction mix consisted of 2.5 μL of Takara 10× Ex Taq Buffer, 2 μL of dNTP Mix (2.5 mM each), 0.7 μL of Roche bovine serum albumin (20 mg/mL), 0.125 μL of Ex Taq DNA polymerase (Takara, 5 U/μL), 0.5 μL of 10 μM primer 515F, 0.5 μL of 10 μM barcode primer 806R, 1 μL of template DNA, and 17.675 μL of PCR grade water. .. The PCR amplification condition was: initial denaturation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 10 min. All samples were run in triplicate and then pooled together.

Article Title: A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci
Article Snippet: Real-time PCR for high resolution melting analysis DNA amplification was performed on the LightCycler 480 real-time PCR platform (Roche Applied Science) using pan-bacterial 16S rRNA primers (V1-forward: GYGGCGNACGGGTGAGTAA and V1-reverse: TTACCCCACCAACTAGC primers). .. PCR reactions in a final volume of 20 μL contained the following: DNA extract (250 fg-250 ng), 0.3 μM forward and reverse primers, MgCl2 at a final concentration of 3 μM and 10 μL (1X) LightCycler 480 High Resolution Melting Master mix containing FastStart Taq DNA polymerase, reaction buffer and dNTP mix (Roche Diagnostics; Burgess Hill, UK).

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: Paragraph title: DNA extraction, PCR amplification and DNA sequencing ... The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm.

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: .. Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome . .. Thermocycler parameters for RT-PCR was 95 °C for 3 min followed by 40 cycles of 94 °C for 45 s, 42 °C for 45 s, and 72 °C for 1 min.

Synthesized:

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: .. Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome . .. Thermocycler parameters for RT-PCR was 95 °C for 3 min followed by 40 cycles of 94 °C for 45 s, 42 °C for 45 s, and 72 °C for 1 min.

Quantitative RT-PCR:

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: Paragraph title: RNA Extraction, Enterovirus Detection by qRT-PCR and VP1 R T-PCR Amplification ... Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome .

Article Title: Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling
Article Snippet: Paragraph title: Real time quantitative RT‐PCR analysis ... Total RNA was reverse transcribed in 20 μl of a reaction mixture that contained reverse transcriptase, 5× reaction buffer, RNase inhibitor, dNTP mix and random hexamer primers, using the Transcriptor First Strand cDNA Synthesis Kit (Roche), run at 25°C for 10 min., 50°C for 1 hr, 85°C for 5 min. and then stored at 4°C.

SYBR Green Assay:

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: Quantitative real-time PCR (RT-PCR) was performed using SYBR green. .. RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis.

Article Title: Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention
Article Snippet: .. LightCycler‐FastStart DNA master SYBR Green, which contains Taq DNA polymerase, dNTP mix, SYBR Green 1 dye and MgCl2 (Roche), was used as a reaction mix for PCR. .. LightCycler Primer set (Roche, Indianapolis, IN) was used for the primers for porphobilinogen deaminase (PBGD) as a housekeeping gene.

Article Title: Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling
Article Snippet: Total RNA was reverse transcribed in 20 μl of a reaction mixture that contained reverse transcriptase, 5× reaction buffer, RNase inhibitor, dNTP mix and random hexamer primers, using the Transcriptor First Strand cDNA Synthesis Kit (Roche), run at 25°C for 10 min., 50°C for 1 hr, 85°C for 5 min. and then stored at 4°C. .. Quantification of gene expression was performed with real‐time quantitative RT‐PCR (Real‐Time PCR System; Bio‐Rad, Hercules, CA, USA) using the Fast Start Universal SYBR Green Master Mix Kit (Roche) and specific primers (Table ).

Microarray:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: Paragraph title: Illumina Microarray analysis ... The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme.

Incubation:

Article Title: scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells
Article Snippet: Reactions were diluted to 100 μl and 20U of exonuclease I (NEB) added and incubated at 37 °C before purification using a 0.75:1 ratio of AMPure XP beads. .. Purified products were resuspended in 50 μl of second strand mastermix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT) then heated to 98 °C for 2 min and cooled on ice.

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: Reactions were incubated at 16°C for 2hr and the resulting cDNAs were purified. cDNA binding buffer (250μL) was added to each reaction which was then mixed and passed through a cDNA filter cartridge by centrifugation at 10,000 × g for 1 min. Filters were washed with wash Buffer (500μL) and dried by centrifugation for an additional minute. cDNA was eluted using 2 × 10μL aliquots of Nuclease-free Water at 55°C. .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme.

Expressing:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme. .. Hybridization, washing and detection were performed using the Illumina Gene Expression System Buffer Kit for HumanRef-8 BeadChips (Illumina, San Diego, CA) according to the manufacturer's protocol.

Article Title: Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling
Article Snippet: Total RNA was reverse transcribed in 20 μl of a reaction mixture that contained reverse transcriptase, 5× reaction buffer, RNase inhibitor, dNTP mix and random hexamer primers, using the Transcriptor First Strand cDNA Synthesis Kit (Roche), run at 25°C for 10 min., 50°C for 1 hr, 85°C for 5 min. and then stored at 4°C. .. Quantification of gene expression was performed with real‐time quantitative RT‐PCR (Real‐Time PCR System; Bio‐Rad, Hercules, CA, USA) using the Fast Start Universal SYBR Green Master Mix Kit (Roche) and specific primers (Table ).

Transformation Assay:

Article Title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
Article Snippet: Paragraph title: Site-directed mutagenesis and bacteria transformation ... PCR was carried out in 25 μl of reaction mixture containing the following components: 14.25 μl ddH2 O, 5 μl plasmid template (25 ng of total plasmid templates per reaction; when using combinations of templates, equal amounts of each plasmid were mixed to give 25 ng), 2.5 μl buffer 10X Pwo DNA polymerase (Roche), 1.25 μl dNTP mix (4 X 2.5 mM), 0.8 μM of mutagenic primers mix, and 0.1 μl Pwo DNA polymerase (5 U/μl; Roche).

Derivative Assay:

Article Title: Accessory Gene Regulator Group Polymorphisms in Methicillin-Resistant Staphylococcus aureus: An Association with Clinical Significance
Article Snippet: PCR was done in 50-µL volumes containing 3µL DNA extract, 0.2mM dNTP mix, 1.5mM magnesium chloride, 2.5U AmpliTaq DNA polymerase (Roche Diagnostics), and 20 pmol each of the forward and reverse primers. .. The nucleotide coordinates of the forward (agr 1801-1818) and reverse (agr 3668-3685) primers were derived from the agr locus sequence of S. aureus RN639019 (GenBank accession no. X52543) and consist of the sequences 5'-ACCAGT TTGCCACGTATC-3' and 5'-TAAACCACGACCT TCACC-3', respectively.

Hybridization:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme. .. Hybridization, washing and detection were performed using the Illumina Gene Expression System Buffer Kit for HumanRef-8 BeadChips (Illumina, San Diego, CA) according to the manufacturer's protocol.

High Performance Liquid Chromatography:

Article Title: A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci
Article Snippet: PCR reactions in a final volume of 20 μL contained the following: DNA extract (250 fg-250 ng), 0.3 μM forward and reverse primers, MgCl2 at a final concentration of 3 μM and 10 μL (1X) LightCycler 480 High Resolution Melting Master mix containing FastStart Taq DNA polymerase, reaction buffer and dNTP mix (Roche Diagnostics; Burgess Hill, UK). .. PCR reactions in a final volume of 20 μL contained the following: DNA extract (250 fg-250 ng), 0.3 μM forward and reverse primers, MgCl2 at a final concentration of 3 μM and 10 μL (1X) LightCycler 480 High Resolution Melting Master mix containing FastStart Taq DNA polymerase, reaction buffer and dNTP mix (Roche Diagnostics; Burgess Hill, UK).

Sequencing:

Article Title: Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma
Article Snippet: .. Amplification and sequencing of MTAP -Exon 3 For PCR amplification of exon 3 of MTAP , 1 μL cDNA (extracted from tissue or cell lines, or 2 μL genomic DNA (extracted from tissue or cell lines) were added to 0.5 μL of forward and reverse primers (MTAP-for89: 5’-GCCCACTGCAGATTCCTTTC-3’ ; MTAP-rev416: 5’-GGTCTCATAGTGGTCCTGTC-3’ ; each 20 μM), 5 μL PCR reaction buffer (10x), 0.5 μL dNTP mix (each 10 mM), and 0.5 μL (2.5 Units) FastStart Taq DNA Polymerase dNTPack (Roche, Mannheim, Germany) in a total reaction volume of 50 μL. .. The following PCR program was used: 5 min at 95°C (initial denaturation); 60 s at 95°C (denaturation); 30 s at 61°C (annealing), 30 s at 72°C (elongation), repeated 35 times.

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis. .. Sequence-specific primers were designed to span exon-exon boundaries using Primer Express software version 1.5 (Applied Biosystems) and were obtained from Sigma-Aldrich.

Article Title: Development of a Multiplexed Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Identify Common Members of the Subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala
Article Snippet: .. An approximately 1-kb region of rDNA gene array including both internal transcribed spacer regions was amplified from voucher specimens for cloning and sequencing., A 100 μL reaction was prepared with 1X of Gene Amp PCR Buffer I (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.01% wt:vol gelatin) (Applied Biosystems, Foster City, CA), 1 mM of dNTP mix (Roche Diagnostics, Indianapolis, IN), 120 nM of each primer, 2.5 U of Amplitaq DNA polymerase (Applied Biosystems), and 60 ng of template DNA. .. Amplification was conducted under the following thermal cycler conditions: one cycle at 97°C for 4 minutes, followed by 30 cycles of 96°C for 30 seconds, 48°C for 30 seconds and 72°C for 2 minutes, and ending with a 5 minute extension step at 72°C.

Article Title: scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells
Article Snippet: Purified products were resuspended in 50 μl of second strand mastermix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT) then heated to 98 °C for 2 min and cooled on ice. .. Finally, scBS-seq libraries were purified using a 0.7:1 volumetric ratio of AMPure XP beads before pooling and sequencing.

Article Title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
Article Snippet: Q97H introduces an XbaI site and D107A eliminates a BglII site in the human PTEN cDNA sequence (NM_000314). .. PCR was carried out in 25 μl of reaction mixture containing the following components: 14.25 μl ddH2 O, 5 μl plasmid template (25 ng of total plasmid templates per reaction; when using combinations of templates, equal amounts of each plasmid were mixed to give 25 ng), 2.5 μl buffer 10X Pwo DNA polymerase (Roche), 1.25 μl dNTP mix (4 X 2.5 mM), 0.8 μM of mutagenic primers mix, and 0.1 μl Pwo DNA polymerase (5 U/μl; Roche).

Article Title: Competition Between Conjugation and M13 Phage Infection in Escherichia coli in the Absence of Selection Pressure: A Kinetic Study
Article Snippet: Quantitative PCR assay All qPCR assays used a master mix consisting of final concentration: 2 mM MgCl2 , 200 µM dNTP mix, 1U (per 25 μL volume) Roche FastStart Enzyme blend (Roche Diagnostics, Mannheim, Germany), 1× Roche FastStartBuffer (Roche Diagnostics), 0.4 μM forward and reverse primers, 2 μM SYTO 9 (Life Technologies, Carlsbad, CA), and 1X ROX reference dye (Life Technologies). .. We use SYTO9 dye for double stranded DNA quantification, as it has been shown to have fewer sequence and concentration artifacts ( ).

Article Title: Microbial Community of High Arsenic Groundwater in Agricultural Irrigation Area of Hetao Plain, Inner Mongolia
Article Snippet: Paragraph title: DNA Extraction, PCR, and Illumina Sequencing ... The 25 μL PCR reaction mix consisted of 2.5 μL of Takara 10× Ex Taq Buffer, 2 μL of dNTP Mix (2.5 mM each), 0.7 μL of Roche bovine serum albumin (20 mg/mL), 0.125 μL of Ex Taq DNA polymerase (Takara, 5 U/μL), 0.5 μL of 10 μM primer 515F, 0.5 μL of 10 μM barcode primer 806R, 1 μL of template DNA, and 17.675 μL of PCR grade water.

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm. .. DNA sequencing was conducted at First BASE Laboratories Sdn Bhd (Selangor, Malaysia) using the BigDye terminator v3.1 cycle-sequencing kit (ABI), and both strands were directly sequenced using the PCR primers as sequencing primers (Model 310 or 3100, Applied Biosystems).

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: All screened NPEVs were further characterized by sequencing of VP1 gene through RT-PCR. .. Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome .

Article Title: Accessory Gene Regulator Group Polymorphisms in Methicillin-Resistant Staphylococcus aureus: An Association with Clinical Significance
Article Snippet: PCR was done in 50-µL volumes containing 3µL DNA extract, 0.2mM dNTP mix, 1.5mM magnesium chloride, 2.5U AmpliTaq DNA polymerase (Roche Diagnostics), and 20 pmol each of the forward and reverse primers. .. The nucleotide coordinates of the forward (agr 1801-1818) and reverse (agr 3668-3685) primers were derived from the agr locus sequence of S. aureus RN639019 (GenBank accession no. X52543) and consist of the sequences 5'-ACCAGT TTGCCACGTATC-3' and 5'-TAAACCACGACCT TCACC-3', respectively.

Concentration Assay:

Article Title: Competition Between Conjugation and M13 Phage Infection in Escherichia coli in the Absence of Selection Pressure: A Kinetic Study
Article Snippet: .. Quantitative PCR assay All qPCR assays used a master mix consisting of final concentration: 2 mM MgCl2 , 200 µM dNTP mix, 1U (per 25 μL volume) Roche FastStart Enzyme blend (Roche Diagnostics, Mannheim, Germany), 1× Roche FastStartBuffer (Roche Diagnostics), 0.4 μM forward and reverse primers, 2 μM SYTO 9 (Life Technologies, Carlsbad, CA), and 1X ROX reference dye (Life Technologies). .. We use SYTO9 dye for double stranded DNA quantification, as it has been shown to have fewer sequence and concentration artifacts ( ).

Article Title: A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci
Article Snippet: .. PCR reactions in a final volume of 20 μL contained the following: DNA extract (250 fg-250 ng), 0.3 μM forward and reverse primers, MgCl2 at a final concentration of 3 μM and 10 μL (1X) LightCycler 480 High Resolution Melting Master mix containing FastStart Taq DNA polymerase, reaction buffer and dNTP mix (Roche Diagnostics; Burgess Hill, UK). .. Negative controls containing PCR grade water instead of bacterial DNA extract were included.

Infection:

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: RNA Extraction, Enterovirus Detection by qRT-PCR and VP1 R T-PCR Amplification RNA was extracted from the supernatant of infected cells using the QIAamp Viral RNA extraction Kit (Qiagen, Valencia, CA) as per the manufacturer’s instruction. .. Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome .

Generated:

Article Title: Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity
Article Snippet: Single B cell RT-PCR with random primers Sort plates were thawed on ice and briefly centrifuged before adding 11.7 µl per well of ice-cold RT-PCR master mix containing 5.8 µl nuclease free water, 4.8 µl 5× FS buffer (Life Technologies), 1 µl 25 mM dNTP mix (Roche Applied Science, Indianapolis, Indiana), and 0.1 µl random primers (Life Technologies, catalog no. 48190-011) per well. .. Then 8.3 µl of ice-cold RT-PCR master mix (2) containing 4.8 µl nuclease-free water, 0.8 µl 5× FS buffer, 0.2 µl RNasin, 2 µl 0.1M dTT, and 0.5 µl SuperScript III RT (Life Technologies) were added per well, and cDNA was generated with the following PCR program: 1 cycle for 5 minutes at 25°C, 1 cycle for 60 minutes at 50°C, 1 cycle for 15 minutes at 70°C, and 4°C forever. cDNA was stored at 4°C (short-term) or −20°C (long-term).

DNA Sequencing:

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: Paragraph title: DNA extraction, PCR amplification and DNA sequencing ... The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm.

Polymerase Chain Reaction:

Article Title: Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma
Article Snippet: .. Amplification and sequencing of MTAP -Exon 3 For PCR amplification of exon 3 of MTAP , 1 μL cDNA (extracted from tissue or cell lines, or 2 μL genomic DNA (extracted from tissue or cell lines) were added to 0.5 μL of forward and reverse primers (MTAP-for89: 5’-GCCCACTGCAGATTCCTTTC-3’ ; MTAP-rev416: 5’-GGTCTCATAGTGGTCCTGTC-3’ ; each 20 μM), 5 μL PCR reaction buffer (10x), 0.5 μL dNTP mix (each 10 mM), and 0.5 μL (2.5 Units) FastStart Taq DNA Polymerase dNTPack (Roche, Mannheim, Germany) in a total reaction volume of 50 μL. .. The following PCR program was used: 5 min at 95°C (initial denaturation); 60 s at 95°C (denaturation); 30 s at 61°C (annealing), 30 s at 72°C (elongation), repeated 35 times.

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis. .. RT-PCR was performed on an ABI Prism 7900HT Fast PCR System (Applied Biosystems, Foster City, CA).

Article Title: Development of a Multiplexed Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Identify Common Members of the Subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala
Article Snippet: .. An approximately 1-kb region of rDNA gene array including both internal transcribed spacer regions was amplified from voucher specimens for cloning and sequencing., A 100 μL reaction was prepared with 1X of Gene Amp PCR Buffer I (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.01% wt:vol gelatin) (Applied Biosystems, Foster City, CA), 1 mM of dNTP mix (Roche Diagnostics, Indianapolis, IN), 120 nM of each primer, 2.5 U of Amplitaq DNA polymerase (Applied Biosystems), and 60 ng of template DNA. .. Amplification was conducted under the following thermal cycler conditions: one cycle at 97°C for 4 minutes, followed by 30 cycles of 96°C for 30 seconds, 48°C for 30 seconds and 72°C for 2 minutes, and ending with a 5 minute extension step at 72°C.

Article Title: scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells
Article Snippet: Purified products were resuspended in 50 μl of second strand mastermix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT) then heated to 98 °C for 2 min and cooled on ice. .. Second strand products were purified using a 0.75:1 ratio of AMPure XP beads and resuspended in 50 μl of PCR mastermix (1× KAPA HiFi Readymix, 0.2 μM PE1.0 primer, 0.2 μM iTAG index primer) and amplified with 14 cycles.

Article Title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
Article Snippet: .. PCR was carried out in 25 μl of reaction mixture containing the following components: 14.25 μl ddH2 O, 5 μl plasmid template (25 ng of total plasmid templates per reaction; when using combinations of templates, equal amounts of each plasmid were mixed to give 25 ng), 2.5 μl buffer 10X Pwo DNA polymerase (Roche), 1.25 μl dNTP mix (4 X 2.5 mM), 0.8 μM of mutagenic primers mix, and 0.1 μl Pwo DNA polymerase (5 U/μl; Roche). .. For individual mutagenesis, 1 μl of each mutagenic primer (at 10 μM in ddH2 O; 2 complementary mutagenic primers per mutation) (Invitrogen) was used.

Article Title: Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention
Article Snippet: .. LightCycler‐FastStart DNA master SYBR Green, which contains Taq DNA polymerase, dNTP mix, SYBR Green 1 dye and MgCl2 (Roche), was used as a reaction mix for PCR. .. LightCycler Primer set (Roche, Indianapolis, IN) was used for the primers for porphobilinogen deaminase (PBGD) as a housekeeping gene.

Article Title: Microbial Community of High Arsenic Groundwater in Agricultural Irrigation Area of Hetao Plain, Inner Mongolia
Article Snippet: .. The 25 μL PCR reaction mix consisted of 2.5 μL of Takara 10× Ex Taq Buffer, 2 μL of dNTP Mix (2.5 mM each), 0.7 μL of Roche bovine serum albumin (20 mg/mL), 0.125 μL of Ex Taq DNA polymerase (Takara, 5 U/μL), 0.5 μL of 10 μM primer 515F, 0.5 μL of 10 μM barcode primer 806R, 1 μL of template DNA, and 17.675 μL of PCR grade water. .. The PCR amplification condition was: initial denaturation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 10 min. All samples were run in triplicate and then pooled together.

Article Title: A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci
Article Snippet: .. PCR reactions in a final volume of 20 μL contained the following: DNA extract (250 fg-250 ng), 0.3 μM forward and reverse primers, MgCl2 at a final concentration of 3 μM and 10 μL (1X) LightCycler 480 High Resolution Melting Master mix containing FastStart Taq DNA polymerase, reaction buffer and dNTP mix (Roche Diagnostics; Burgess Hill, UK). .. Negative controls containing PCR grade water instead of bacterial DNA extract were included.

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: .. The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm. .. For the SSU rRNA and the COI amplifications, the PCR conditions were initial denaturation at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 1 min, annealing at 40°C for 1 min and extension at 68°C for 2 min, then 30 cycles of denaturation at 94°C for 1 min, annealing at 45°C for 2 min, extension at 68°C for 2 min and a final extension at 68°C for 7 min. For the ITS2 amplification, the PCR conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec and final extension at 72°C for 7 min. Amplified products were separated by electrophoresis on a 1.0% (w/v) agarose gel, stained with ethidium bromide, and visualized under ultraviolet light.

Article Title: Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity
Article Snippet: Single B cell RT-PCR with random primers Sort plates were thawed on ice and briefly centrifuged before adding 11.7 µl per well of ice-cold RT-PCR master mix containing 5.8 µl nuclease free water, 4.8 µl 5× FS buffer (Life Technologies), 1 µl 25 mM dNTP mix (Roche Applied Science, Indianapolis, Indiana), and 0.1 µl random primers (Life Technologies, catalog no. 48190-011) per well. .. Then 8.3 µl of ice-cold RT-PCR master mix (2) containing 4.8 µl nuclease-free water, 0.8 µl 5× FS buffer, 0.2 µl RNasin, 2 µl 0.1M dTT, and 0.5 µl SuperScript III RT (Life Technologies) were added per well, and cDNA was generated with the following PCR program: 1 cycle for 5 minutes at 25°C, 1 cycle for 60 minutes at 50°C, 1 cycle for 15 minutes at 70°C, and 4°C forever. cDNA was stored at 4°C (short-term) or −20°C (long-term).

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome . .. Specific RT-PCR products were purified using the Qiaquick PCR purification kit (Qiagen) and stored at −20 °C till further procedure.

Article Title: Accessory Gene Regulator Group Polymorphisms in Methicillin-Resistant Staphylococcus aureus: An Association with Clinical Significance
Article Snippet: .. PCR was done in 50-µL volumes containing 3µL DNA extract, 0.2mM dNTP mix, 1.5mM magnesium chloride, 2.5U AmpliTaq DNA polymerase (Roche Diagnostics), and 20 pmol each of the forward and reverse primers. .. The nucleotide coordinates of the forward (agr 1801-1818) and reverse (agr 3668-3685) primers were derived from the agr locus sequence of S. aureus RN639019 (GenBank accession no. X52543) and consist of the sequences 5'-ACCAGT TTGCCACGTATC-3' and 5'-TAAACCACGACCT TCACC-3', respectively.

Article Title: Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling
Article Snippet: Total RNA was reverse transcribed in 20 μl of a reaction mixture that contained reverse transcriptase, 5× reaction buffer, RNase inhibitor, dNTP mix and random hexamer primers, using the Transcriptor First Strand cDNA Synthesis Kit (Roche), run at 25°C for 10 min., 50°C for 1 hr, 85°C for 5 min. and then stored at 4°C. .. The 25 μl total PCR reaction volumes consisted of specific primers (0.2 μM final concentrations), 50 ng of cNDA template, 2× SYBR Green master mix and PCR‐grade water.

Binding Assay:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: Reactions were incubated at 16°C for 2hr and the resulting cDNAs were purified. cDNA binding buffer (250μL) was added to each reaction which was then mixed and passed through a cDNA filter cartridge by centrifugation at 10,000 × g for 1 min. Filters were washed with wash Buffer (500μL) and dried by centrifugation for an additional minute. cDNA was eluted using 2 × 10μL aliquots of Nuclease-free Water at 55°C. .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme.

DNA Extraction:

Article Title: Microbial Community of High Arsenic Groundwater in Agricultural Irrigation Area of Hetao Plain, Inner Mongolia
Article Snippet: Paragraph title: DNA Extraction, PCR, and Illumina Sequencing ... The 25 μL PCR reaction mix consisted of 2.5 μL of Takara 10× Ex Taq Buffer, 2 μL of dNTP Mix (2.5 mM each), 0.7 μL of Roche bovine serum albumin (20 mg/mL), 0.125 μL of Ex Taq DNA polymerase (Takara, 5 U/μL), 0.5 μL of 10 μM primer 515F, 0.5 μL of 10 μM barcode primer 806R, 1 μL of template DNA, and 17.675 μL of PCR grade water.

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: Paragraph title: DNA extraction, PCR amplification and DNA sequencing ... The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm.

Article Title: Accessory Gene Regulator Group Polymorphisms in Methicillin-Resistant Staphylococcus aureus: An Association with Clinical Significance
Article Snippet: Detection of the agr locus restriction fragment length polymorphisms (RFLPs) DNA extraction was performed using the Qiagen tissue kit (Qiagen, Hilden, Germany). .. PCR was done in 50-µL volumes containing 3µL DNA extract, 0.2mM dNTP mix, 1.5mM magnesium chloride, 2.5U AmpliTaq DNA polymerase (Roche Diagnostics), and 20 pmol each of the forward and reverse primers.

Fluorescence:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme. .. Reactions were incubated at 37°C for 14 hr after which volumes were adjusted to 100μL by addition of Nuclease-free water. cRNA Binding Buffer (350μL) and 100% ethanol (250μL) were added and mixed by pipetting before passing through a cRNA filter cartridge under centrifugation at 10,000 × g for 1 min. Filters were washed with wash Buffer (650μL) and dried by centrifugation for an additional minute. cDNA was eluted using 100μL of Nuclease-free Water at 55°C. cRNA was quantified using nce-based assay the RiboGreen® fluorescence-based assay (Invitrogen, Carlsbad, CA).

Methylation:

Article Title: scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells
Article Snippet: Bisulfite conversion was carried out using EZ Methylation Direct MagBead kit (Zymo) according the manufacturers’ instructions but with half volumes. .. Purified products were resuspended in 50 μl of second strand mastermix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT) then heated to 98 °C for 2 min and cooled on ice.

Mutagenesis:

Article Title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
Article Snippet: Paragraph title: Site-directed mutagenesis and bacteria transformation ... PCR was carried out in 25 μl of reaction mixture containing the following components: 14.25 μl ddH2 O, 5 μl plasmid template (25 ng of total plasmid templates per reaction; when using combinations of templates, equal amounts of each plasmid were mixed to give 25 ng), 2.5 μl buffer 10X Pwo DNA polymerase (Roche), 1.25 μl dNTP mix (4 X 2.5 mM), 0.8 μM of mutagenic primers mix, and 0.1 μl Pwo DNA polymerase (5 U/μl; Roche).

Isolation:

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: In brief, frozen rat hindlimb tissue, stored at −80°C, was homogenized (Polytron, Kinematica GmbH, Littau, Switzerland), and total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY). .. RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis.

Article Title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
Article Snippet: Plasmid templates (pRK5-PTEN, 6 kbp; pYES2-PTEN, 7.1 kbp; pENTR-PTPRZ-B, 7.3 kbp; pCDNA3-PTPRZ-B, 10 kbp) were isolated from the dam+ DH5α E . coli strain and dissolved in double-distilled water (ddH2 O) at 5 ng/μl. .. PCR was carried out in 25 μl of reaction mixture containing the following components: 14.25 μl ddH2 O, 5 μl plasmid template (25 ng of total plasmid templates per reaction; when using combinations of templates, equal amounts of each plasmid were mixed to give 25 ng), 2.5 μl buffer 10X Pwo DNA polymerase (Roche), 1.25 μl dNTP mix (4 X 2.5 mM), 0.8 μM of mutagenic primers mix, and 0.1 μl Pwo DNA polymerase (5 U/μl; Roche).

Article Title: Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling
Article Snippet: Real time quantitative RT‐PCR analysis After the 60 min. reperfusion period, total RNA was isolated from the LV myocardial tissue using Trizol reagent according to the standard protocol. .. Total RNA was reverse transcribed in 20 μl of a reaction mixture that contained reverse transcriptase, 5× reaction buffer, RNase inhibitor, dNTP mix and random hexamer primers, using the Transcriptor First Strand cDNA Synthesis Kit (Roche), run at 25°C for 10 min., 50°C for 1 hr, 85°C for 5 min. and then stored at 4°C.

Size-exclusion Chromatography:

Article Title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
Article Snippet: PCR was carried out in 25 μl of reaction mixture containing the following components: 14.25 μl ddH2 O, 5 μl plasmid template (25 ng of total plasmid templates per reaction; when using combinations of templates, equal amounts of each plasmid were mixed to give 25 ng), 2.5 μl buffer 10X Pwo DNA polymerase (Roche), 1.25 μl dNTP mix (4 X 2.5 mM), 0.8 μM of mutagenic primers mix, and 0.1 μl Pwo DNA polymerase (5 U/μl; Roche). .. PCR conditions were: 1 min at 95°C to denature the template, followed by 18 cycles of 50 sec at 95°C, 50 sec at 60°C, and 5 min at 68°C (7 min at 68°C for PTPRZ-B plasmids), and finally a 7 min extension at 68°C.

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm. .. For the SSU rRNA and the COI amplifications, the PCR conditions were initial denaturation at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 1 min, annealing at 40°C for 1 min and extension at 68°C for 2 min, then 30 cycles of denaturation at 94°C for 1 min, annealing at 45°C for 2 min, extension at 68°C for 2 min and a final extension at 68°C for 7 min. For the ITS2 amplification, the PCR conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec and final extension at 72°C for 7 min. Amplified products were separated by electrophoresis on a 1.0% (w/v) agarose gel, stained with ethidium bromide, and visualized under ultraviolet light.

Labeling:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme. .. Reactions were incubated at 37°C for 14 hr after which volumes were adjusted to 100μL by addition of Nuclease-free water. cRNA Binding Buffer (350μL) and 100% ethanol (250μL) were added and mixed by pipetting before passing through a cRNA filter cartridge under centrifugation at 10,000 × g for 1 min. Filters were washed with wash Buffer (650μL) and dried by centrifugation for an additional minute. cDNA was eluted using 100μL of Nuclease-free Water at 55°C. cRNA was quantified using nce-based assay the RiboGreen® fluorescence-based assay (Invitrogen, Carlsbad, CA).

Purification:

Article Title: Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma
Article Snippet: Amplification and sequencing of MTAP -Exon 3 For PCR amplification of exon 3 of MTAP , 1 μL cDNA (extracted from tissue or cell lines, or 2 μL genomic DNA (extracted from tissue or cell lines) were added to 0.5 μL of forward and reverse primers (MTAP-for89: 5’-GCCCACTGCAGATTCCTTTC-3’ ; MTAP-rev416: 5’-GGTCTCATAGTGGTCCTGTC-3’ ; each 20 μM), 5 μL PCR reaction buffer (10x), 0.5 μL dNTP mix (each 10 mM), and 0.5 μL (2.5 Units) FastStart Taq DNA Polymerase dNTPack (Roche, Mannheim, Germany) in a total reaction volume of 50 μL. .. Before sequencing, amplicons were purified by precipitation with 30% PEG-8000 (Sigma-Aldrich) to remove unincorporated primers and dNTPs.

Article Title: scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells
Article Snippet: .. Purified products were resuspended in 50 μl of second strand mastermix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT) then heated to 98 °C for 2 min and cooled on ice. .. 50U of klenow exo- (Enzymatics) was added and the mixture incubated on a thermocycler at 37 °C for 90 min after slowly ramping from 4 °C.

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme. .. Reactions were incubated at 37°C for 14 hr after which volumes were adjusted to 100μL by addition of Nuclease-free water. cRNA Binding Buffer (350μL) and 100% ethanol (250μL) were added and mixed by pipetting before passing through a cRNA filter cartridge under centrifugation at 10,000 × g for 1 min. Filters were washed with wash Buffer (650μL) and dried by centrifugation for an additional minute. cDNA was eluted using 100μL of Nuclease-free Water at 55°C. cRNA was quantified using nce-based assay the RiboGreen® fluorescence-based assay (Invitrogen, Carlsbad, CA).

Article Title: Microbial Community of High Arsenic Groundwater in Agricultural Irrigation Area of Hetao Plain, Inner Mongolia
Article Snippet: The 25 μL PCR reaction mix consisted of 2.5 μL of Takara 10× Ex Taq Buffer, 2 μL of dNTP Mix (2.5 mM each), 0.7 μL of Roche bovine serum albumin (20 mg/mL), 0.125 μL of Ex Taq DNA polymerase (Takara, 5 U/μL), 0.5 μL of 10 μM primer 515F, 0.5 μL of 10 μM barcode primer 806R, 1 μL of template DNA, and 17.675 μL of PCR grade water. .. PCR products were purified using the Agencourt AMPure XP PCR purification system (Beckman Coulter, Brea, CA, USA).

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome . .. Specific RT-PCR products were purified using the Qiaquick PCR purification kit (Qiagen) and stored at −20 °C till further procedure.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: Quantitative real-time PCR (RT-PCR) was performed using SYBR green. .. RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis.

Article Title: Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity
Article Snippet: .. Single B cell RT-PCR with random primers Sort plates were thawed on ice and briefly centrifuged before adding 11.7 µl per well of ice-cold RT-PCR master mix containing 5.8 µl nuclease free water, 4.8 µl 5× FS buffer (Life Technologies), 1 µl 25 mM dNTP mix (Roche Applied Science, Indianapolis, Indiana), and 0.1 µl random primers (Life Technologies, catalog no. 48190-011) per well. .. Then 8.3 µl of ice-cold RT-PCR master mix (2) containing 4.8 µl nuclease-free water, 0.8 µl 5× FS buffer, 0.2 µl RNasin, 2 µl 0.1M dTT, and 0.5 µl SuperScript III RT (Life Technologies) were added per well, and cDNA was generated with the following PCR program: 1 cycle for 5 minutes at 25°C, 1 cycle for 60 minutes at 50°C, 1 cycle for 15 minutes at 70°C, and 4°C forever. cDNA was stored at 4°C (short-term) or −20°C (long-term).

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: All screened NPEVs were further characterized by sequencing of VP1 gene through RT-PCR. .. Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome .

Plasmid Preparation:

Article Title: One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections
Article Snippet: .. PCR was carried out in 25 μl of reaction mixture containing the following components: 14.25 μl ddH2 O, 5 μl plasmid template (25 ng of total plasmid templates per reaction; when using combinations of templates, equal amounts of each plasmid were mixed to give 25 ng), 2.5 μl buffer 10X Pwo DNA polymerase (Roche), 1.25 μl dNTP mix (4 X 2.5 mM), 0.8 μM of mutagenic primers mix, and 0.1 μl Pwo DNA polymerase (5 U/μl; Roche). .. For individual mutagenesis, 1 μl of each mutagenic primer (at 10 μM in ddH2 O; 2 complementary mutagenic primers per mutation) (Invitrogen) was used.

Software:

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis. .. Sequence-specific primers were designed to span exon-exon boundaries using Primer Express software version 1.5 (Applied Biosystems) and were obtained from Sigma-Aldrich.

Real-time Polymerase Chain Reaction:

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: Paragraph title: RNA Extraction and Quantitative Real-Time PCR ... RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis.

Article Title: Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention
Article Snippet: Paragraph title: Real‐time PCR of leptin and MMP‐13 mRNA ... LightCycler‐FastStart DNA master SYBR Green, which contains Taq DNA polymerase, dNTP mix, SYBR Green 1 dye and MgCl2 (Roche), was used as a reaction mix for PCR.

Article Title: Competition Between Conjugation and M13 Phage Infection in Escherichia coli in the Absence of Selection Pressure: A Kinetic Study
Article Snippet: .. Quantitative PCR assay All qPCR assays used a master mix consisting of final concentration: 2 mM MgCl2 , 200 µM dNTP mix, 1U (per 25 μL volume) Roche FastStart Enzyme blend (Roche Diagnostics, Mannheim, Germany), 1× Roche FastStartBuffer (Roche Diagnostics), 0.4 μM forward and reverse primers, 2 μM SYTO 9 (Life Technologies, Carlsbad, CA), and 1X ROX reference dye (Life Technologies). .. We use SYTO9 dye for double stranded DNA quantification, as it has been shown to have fewer sequence and concentration artifacts ( ).

Article Title: A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci
Article Snippet: Paragraph title: Real-time PCR for high resolution melting analysis ... PCR reactions in a final volume of 20 μL contained the following: DNA extract (250 fg-250 ng), 0.3 μM forward and reverse primers, MgCl2 at a final concentration of 3 μM and 10 μL (1X) LightCycler 480 High Resolution Melting Master mix containing FastStart Taq DNA polymerase, reaction buffer and dNTP mix (Roche Diagnostics; Burgess Hill, UK).

Article Title: Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling
Article Snippet: Total RNA was reverse transcribed in 20 μl of a reaction mixture that contained reverse transcriptase, 5× reaction buffer, RNase inhibitor, dNTP mix and random hexamer primers, using the Transcriptor First Strand cDNA Synthesis Kit (Roche), run at 25°C for 10 min., 50°C for 1 hr, 85°C for 5 min. and then stored at 4°C. .. Quantification of gene expression was performed with real‐time quantitative RT‐PCR (Real‐Time PCR System; Bio‐Rad, Hercules, CA, USA) using the Fast Start Universal SYBR Green Master Mix Kit (Roche) and specific primers (Table ).

RNA Extraction:

Article Title: Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function
Article Snippet: Paragraph title: RNA Extraction and Quantitative Real-Time PCR ... RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis.

Article Title: Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan
Article Snippet: Paragraph title: RNA Extraction, Enterovirus Detection by qRT-PCR and VP1 R T-PCR Amplification ... Initially, complementary DNA (cDNA) was synthesized in a 20 μL reaction mixture comprising of 11 μL of each viral RNA, 4 μL of 5x transcriptase buffer, 2 μL of 10 mM DTT, 0.5 μL of 25 mM each dNTP mix (Roche), 1 μL of 40U of RNase Inhibitor and 1 μL of 20U AMV reverse transcriptase (Thermo Scientific) at 42 °C for 45 min. For amplification of VP1 region RCR was performed by using 2 μL of cDNA, 5 μL of 10xTaq buffer, 3.5 μL of MgCl2 (50 mM), 1.0 μL of 12.5 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 units/ μL), 37 μL of water and 0.5 μL of each primer (10 μM) 224–222 targeting VP3 (nt 2204–2223) and VP1 (nt 2969–2951) region of virus genome .

Agarose Gel Electrophoresis:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: RNA samples, with A260:A280 ratios between 1.7 and 2.1, and with integrity confirmed by denaturing agarose gel electrophoresis, were labeled using the Illumina® RNA Amplification kit (Ambion, Austin, TX). .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme.

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm. .. For the SSU rRNA and the COI amplifications, the PCR conditions were initial denaturation at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 1 min, annealing at 40°C for 1 min and extension at 68°C for 2 min, then 30 cycles of denaturation at 94°C for 1 min, annealing at 45°C for 2 min, extension at 68°C for 2 min and a final extension at 68°C for 7 min. For the ITS2 amplification, the PCR conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec and final extension at 72°C for 7 min. Amplified products were separated by electrophoresis on a 1.0% (w/v) agarose gel, stained with ethidium bromide, and visualized under ultraviolet light.

In Vitro:

Article Title: Changes in the Peripheral Blood Transcriptome Associated with Occupational Benzene Exposure Identified by Cross-Comparison on Two Microarray Platforms
Article Snippet: .. The purified cDNA was dried to completion in a vacuum centrifuge concentrator set to medium heat and resuspended in 10μL in vitro transcription (IVT) reaction mix comprising 1X reaction buffer, dNTP mix, biotin labeled UTP (10 mM; Roche Applied Science, Indianapolis, IN), and T7 enzyme. .. Reactions were incubated at 37°C for 14 hr after which volumes were adjusted to 100μL by addition of Nuclease-free water. cRNA Binding Buffer (350μL) and 100% ethanol (250μL) were added and mixed by pipetting before passing through a cRNA filter cartridge under centrifugation at 10,000 × g for 1 min. Filters were washed with wash Buffer (650μL) and dried by centrifugation for an additional minute. cDNA was eluted using 100μL of Nuclease-free Water at 55°C. cRNA was quantified using nce-based assay the RiboGreen® fluorescence-based assay (Invitrogen, Carlsbad, CA).

Electrophoresis:

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm. .. For the SSU rRNA and the COI amplifications, the PCR conditions were initial denaturation at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 1 min, annealing at 40°C for 1 min and extension at 68°C for 2 min, then 30 cycles of denaturation at 94°C for 1 min, annealing at 45°C for 2 min, extension at 68°C for 2 min and a final extension at 68°C for 7 min. For the ITS2 amplification, the PCR conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec and final extension at 72°C for 7 min. Amplified products were separated by electrophoresis on a 1.0% (w/v) agarose gel, stained with ethidium bromide, and visualized under ultraviolet light.

Random Hexamer Labeling:

Article Title: Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling
Article Snippet: .. Total RNA was reverse transcribed in 20 μl of a reaction mixture that contained reverse transcriptase, 5× reaction buffer, RNase inhibitor, dNTP mix and random hexamer primers, using the Transcriptor First Strand cDNA Synthesis Kit (Roche), run at 25°C for 10 min., 50°C for 1 hr, 85°C for 5 min. and then stored at 4°C. .. Quantification of gene expression was performed with real‐time quantitative RT‐PCR (Real‐Time PCR System; Bio‐Rad, Hercules, CA, USA) using the Fast Start Universal SYBR Green Master Mix Kit (Roche) and specific primers (Table ).

Activation Assay:

Article Title: A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci
Article Snippet: PCR reactions in a final volume of 20 μL contained the following: DNA extract (250 fg-250 ng), 0.3 μM forward and reverse primers, MgCl2 at a final concentration of 3 μM and 10 μL (1X) LightCycler 480 High Resolution Melting Master mix containing FastStart Taq DNA polymerase, reaction buffer and dNTP mix (Roche Diagnostics; Burgess Hill, UK). .. The same conditions were used for all PCR reactions: activation step at 95°C for 10 minutes followed by 30 cycles of 95°C for 10 seconds, 62°C for 10 seconds and 72°C for 10 seconds.

Staining:

Article Title: Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails
Article Snippet: The reaction mixture was prepared in a total volume of 25 μL containing 2.5 μL of 10x high fidelity PCR buffer, 2.0 μL MgCl2 (25 mM), 0.5μL of dNTP mix (10 mM), 0.5 μL of each primer (10 μM), 0.125 U of Taq high-fidelity PCR system (Roche Applied Science, Mannheim, Germany), adjusted to 25 μL with deionized water and 5 μL of DNA extracted from an individual worm. .. For the SSU rRNA and the COI amplifications, the PCR conditions were initial denaturation at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 1 min, annealing at 40°C for 1 min and extension at 68°C for 2 min, then 30 cycles of denaturation at 94°C for 1 min, annealing at 45°C for 2 min, extension at 68°C for 2 min and a final extension at 68°C for 7 min. For the ITS2 amplification, the PCR conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec and final extension at 72°C for 7 min. Amplified products were separated by electrophoresis on a 1.0% (w/v) agarose gel, stained with ethidium bromide, and visualized under ultraviolet light.

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