Structured Review

Millipore dttp
E295K has no polymerase activity. (A) An in vitro primer extension assay under steady-state conditions. Typically, 5 nM pol β and 300 nM DNA substrate were incubated with 10 mM MgCl 2 and 50 μM each of <t>dATP,</t> dCTP, dGTP, and <t>dTTP</t> at 37°C
Dttp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation"

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation

Journal:

doi: 10.1128/MCB.01883-06

E295K has no polymerase activity. (A) An in vitro primer extension assay under steady-state conditions. Typically, 5 nM pol β and 300 nM DNA substrate were incubated with 10 mM MgCl 2 and 50 μM each of dATP, dCTP, dGTP, and dTTP at 37°C
Figure Legend Snippet: E295K has no polymerase activity. (A) An in vitro primer extension assay under steady-state conditions. Typically, 5 nM pol β and 300 nM DNA substrate were incubated with 10 mM MgCl 2 and 50 μM each of dATP, dCTP, dGTP, and dTTP at 37°C

Techniques Used: Activity Assay, In Vitro, Primer Extension Assay, Incubation

2) Product Images from "DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically"

Article Title: DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically

Journal: Oncotarget

doi: 10.18632/oncotarget.11864

Intracellular 5-methyl-dCTP, dCTP and dTTP concentrations in DCTPP1 -knockdown and control BGC-823 cells. The concentrations of A. 5-methyl-dCTP, B. dCTP, and C. dTTP in DCTPP1 -knockdown and control BGC-823 cells were measured by LC-MS/MS assay. All the values shown were represented as means ± SD. (ns: not significant; **: P
Figure Legend Snippet: Intracellular 5-methyl-dCTP, dCTP and dTTP concentrations in DCTPP1 -knockdown and control BGC-823 cells. The concentrations of A. 5-methyl-dCTP, B. dCTP, and C. dTTP in DCTPP1 -knockdown and control BGC-823 cells were measured by LC-MS/MS assay. All the values shown were represented as means ± SD. (ns: not significant; **: P

Techniques Used: Liquid Chromatography, Mass Spectrometry

3) Product Images from "Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity"

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp201

The Pol β polymorphism R137Q is defective in polymerase activity. ( A ) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP- 32 P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. ( B ) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. ( C ) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32 P as shown in figure. ( D ) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.
Figure Legend Snippet: The Pol β polymorphism R137Q is defective in polymerase activity. ( A ) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP- 32 P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. ( B ) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. ( C ) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32 P as shown in figure. ( D ) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.

Techniques Used: Activity Assay, Labeling, Incubation, Purification, Nucleic Acid Electrophoresis, Avidin-Biotin Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Adsorption

Related Articles

Clone Assay:

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: To create the pMAD_lic vector we oligomerized the primers pMAD_lic (EcoRI) and pMAD_lic (BamHI) listed in Supplementary Table , and cloned the double DNA strand dimer into pMAD using EcoRI and BamHI restriction enzymes. .. To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C.

Amplification:

Article Title: Soybean Resistance to White Mold: Evaluation of Soybean Germplasm Under Different Conditions and Validation of QTL
Article Snippet: The PCR amplification was performed in MJ TetradTM thermal cycler (MJ Research, Waltham, MA). .. Total reaction volume of 15.0 μL contained 50 ng of genomic DNA, 0.3 μM each of forward and reverse primers, 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma-Aldrich, St. Louis, MO), 3.0 mM MgCl2 , 2.5 units of Taq polymerase, and 1.0 × PCR buffer.

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C. .. To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C.

Synthesized:

Article Title: Dysregulation of Amyloid-β Protein Precursor, β-Secretase, Presenilin 1 and 2 Genes in the Rat Selectively Vulnerable CA1 Subfield of Hippocampus Following Transient Global Brain Ischemia
Article Snippet: The cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit, according to manufacturer’s instructions (Applied Biosystems, USA). .. Each reactive mixture contained the following set of reagents: 1×RT buffer, 20 U RNase inhibitor, 50 U reverse transcriptase (Multiscribe Reverse Transcriptase), 1×RT Random Primers, 4 mM of each deoxynucleotide: dATP, dGTP, dTTP, and dCTP plus examined 1 μg RNA in DNase-, RNase-, and protease-free water (Sigma-Aldrich, USA) to complete the volume required for reaction.

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: The RNA mixtures were further added with 4 μL of 5× first-strand buffer, 2 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 4 μg of actinomycin D (USB), 200 U SuperScript III, and 20 U SUPERase-In (Ambion, USA), incubated at room temperature for 10 min followed by 1 h at 55°C to synthesize the first-strand cDNA. .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. First, five times less adapter mixture was ligated to the cDNAs.

Article Title: Dysregulation of Autophagy, Mitophagy, and Apoptotic Genes in the Medial Temporal Lobe Cortex in an Ischemic Model of Alzheimer’s Disease
Article Snippet: The cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit, according to manufacturer’s instructions (Applied Biosystems, USA). .. Each reactive mixture contained the following set of reagents: 1×RT buffer, 20 U RNase inhibitor, 50 U reverse transcriptase (Multiscribe Reverse Transcriptase), 1×RT Random Primers, 4 mM of each deoxynucleotide: dATP, dGTP, dTTP and dCTP plus examined 1μg RNA in DNase-, RNase- and protease-free water (Sigma-Aldrich, USA) to complete the volume required for reaction [ ].

Mouse Assay:

Article Title: No Evidence of Persisting Unrepaired Nuclear DNA Single Strand Breaks in Distinct Types of Cells in the Brain, Kidney, and Liver of Adult Mice after Continuous Eight-Week 50 Hz Magnetic Field Exposure with Flux Density of 0.1 mT or 1.0 mT
Article Snippet: For studying unrepaired nDNA SSB by ISNT (carried out on the same mice used for the analysis of UDS and mtDNA synthesis), 3 µm-thick paraffin sections were mounted on slides coated with 3-aminopropyltriethoxysilane (APES, Sigma-Aldrich, St. Louis, MO, USA) for improved adhesion . .. Subsequently, ISNT was carried out as follows , : sections were incubated for 60 min at 37°C with a reaction solution containing 50 mM Tris-HCl-buffer (pH 7.5), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 200 U/ml of Escherichia coli DNA polymerase-I (endonuclease-free; Roche), 10 µmol/ml each of dATP, dGTP, dCTP, and dTTP (Sigma-Aldrich), and 15 µl 3 H-dTTP/ml (1.03 MBq/ml; American Radiolabeled Chemicals).

Autoradiography:

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β. .. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer.

Construct:

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: A LIC-modified pMAD vector was constructed in order to enable efficient directional cloning without restriction enzyme digestion or ligation reactions ( ). .. To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C.

Electrophoresis:

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation
Article Snippet: Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma). .. Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma).

Article Title: Soybean Resistance to White Mold: Evaluation of Soybean Germplasm Under Different Conditions and Validation of QTL
Article Snippet: Total reaction volume of 15.0 μL contained 50 ng of genomic DNA, 0.3 μM each of forward and reverse primers, 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma-Aldrich, St. Louis, MO), 3.0 mM MgCl2 , 2.5 units of Taq polymerase, and 1.0 × PCR buffer. .. Total reaction volume of 15.0 μL contained 50 ng of genomic DNA, 0.3 μM each of forward and reverse primers, 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma-Aldrich, St. Louis, MO), 3.0 mM MgCl2 , 2.5 units of Taq polymerase, and 1.0 × PCR buffer.

Article Title: Effects of Ravuconazole Treatment on Parasite Load and Immune Response in Dogs Experimentally Infected with Trypanosoma cruzi
Article Snippet: Briefly, 2 μl of blood DNA template was added to 10 mM Tris-HCl (pH 9.0); 75 mM KCl; 3.5 mM MgCl2 ; 0.1% Triton X-100; 0.2 mM each dATP, dCTP, dGTP, and dTTP (Sigma Chemical Co.); 1.0 U of Taq DNA polymerase (Promega); and water in a 20-μl reaction volume. .. Briefly, 2 μl of blood DNA template was added to 10 mM Tris-HCl (pH 9.0); 75 mM KCl; 3.5 mM MgCl2 ; 0.1% Triton X-100; 0.2 mM each dATP, dCTP, dGTP, and dTTP (Sigma Chemical Co.); 1.0 U of Taq DNA polymerase (Promega); and water in a 20-μl reaction volume.

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: RNA quantity and quality were assessed using a Nanodrop BD-1000 spectrophotometer (Nanodrop Technologies) and Experion Automated Electrophoresis System (Bio-Rad Laboratories), respectively. .. The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C.

Primer Extension Assay:

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation
Article Snippet: Paragraph title: In vitro primer extension assay. ... Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma).

Incubation:

Article Title: High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics
Article Snippet: Subsequently, this solution containing hairpin-tethered beads is flushed into the flow cell, where it is incubated for 30 min. .. For the experiments in which we observe the effect of dTTP (Sigma Aldrich, Zwijndrecht, The Netherlands) on the hopping transition of the hairpin (see and ), we exchange buffer A from the flow cell with buffer B, which contains 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 7 mM MgCl2 , 3 mM EDTA, 0.01% Tween-20, and varying concentrations of dTTP.

Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
Article Snippet: For the nicking reaction, 900 ng of lambda phage DNA (New England Biolabs) was digested with 30 units of Nt.BspQI nicking enzyme (New England Biolabs) for 2 h at 50 °C in the presence of 3 μL of 10× buffer 3.1 (New England Biolabs) and ultrapure water to a total volume of 30 μL. .. Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL. .. Following labeling, DNA was repaired for 30 min at 45 °C with 12 units of Taq DNA ligase (New England Biolabs) in the presence of 1.5 μL of 10× thermopol buffer, 1 mM NAD+ (New England Biolabs), and ultrapure water to a total reaction volume of 60 μL.

Article Title: Global probabilistic annotation of metabolic networks enables enzyme discovery
Article Snippet: Thus, this term quantifies tendencies of closely related activities to be filled together. .. Different amounts of purified SpsI or SpsJ were incubated at 37 °C in 1 mL of 10 mM potassium phosphate buffer pH 7.4, 2.5 mM MgCl2 , 1 mM glucose-1-phosphate (Sigma-Aldrich, > = 97% purity), 1 mM dTTP (Sigma-Aldrich, > = 96% purity) and 1 U pyrophosphatase . .. The enzyme reaction samples were assayed after 4 hours by flow-injection into a time of flight mass spectrometer (6520 Series QTOF, Agilent Technologies) operated in the negative ionization mode.

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C. .. The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C.

Article Title: No Evidence of Persisting Unrepaired Nuclear DNA Single Strand Breaks in Distinct Types of Cells in the Brain, Kidney, and Liver of Adult Mice after Continuous Eight-Week 50 Hz Magnetic Field Exposure with Flux Density of 0.1 mT or 1.0 mT
Article Snippet: After removing paraffin with xylol, the sections were treated with a solution (Tris-HCl-buffer, pH 8, and 5 mM ethylenediaminetetraacetic acid (EDTA)) containing 25 µg proteinase K (Roche Diagnostics, Mannheim, Germany) per ml. .. Subsequently, ISNT was carried out as follows , : sections were incubated for 60 min at 37°C with a reaction solution containing 50 mM Tris-HCl-buffer (pH 7.5), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 200 U/ml of Escherichia coli DNA polymerase-I (endonuclease-free; Roche), 10 µmol/ml each of dATP, dGTP, dCTP, and dTTP (Sigma-Aldrich), and 15 µl 3 H-dTTP/ml (1.03 MBq/ml; American Radiolabeled Chemicals). .. Incubation was carried out using special in situ chambers consisting of small, custom-made frames of silicon (height = 1 mm; comparable to customary adapters for in situ polymerase chain reaction) and coverslips placed onto the slides.

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: The RNA mixtures were further added with 4 μL of 5× first-strand buffer, 2 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 4 μg of actinomycin D (USB), 200 U SuperScript III, and 20 U SUPERase-In (Ambion, USA), incubated at room temperature for 10 min followed by 1 h at 55°C to synthesize the first-strand cDNA. .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications.

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C. .. RNA was then hydrolyzed during 15 min at 37°C by 2 units of RNase H. The cDNAs were purified with Microcon-G30 (Millipore), dried and resuspended in 10 µL water.

Activity Assay:

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: WT and R137Q genes were inserted into the pET28b vector and expressed in Escherichia coli strain BL21. .. The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β. .. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer.

Article Title: Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation
Article Snippet: Restriction enzymes, T4 DNA polymerase and coupling enzymes for determination of UMPK activity were purchased from Roche Applied Science. .. ATP, GTP, UTP, dUTP, dGTP and dTTP were purchased from Sigma.

Derivative Assay:

Article Title: Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains
Article Snippet: DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight. .. To couple the amino-allyl cDNA with fluorescent labels, Cy3 or Cy5 mono-reactive dye (Amersham) was added for 1 h. Uncoupled label was removed using the Qiagen QIAquick PCR purification kit (Valencia, CA).

Hybridization:

Article Title: Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains
Article Snippet: Paragraph title: Generation of probes for microarray experiments, microarray hybridization, normalization and data analysis ... DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight.

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C. .. The fluorescently labeled cDNAs were purified using a Nucleospin Extract II kit (Macherey-Nagel) and dye incorporation was measured using Nanodrop BD-1000 spectrophotometer (Nanodrop Technologies).

Flow Cytometry:

Article Title: High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics
Article Snippet: Rinsing the flow cell subsequently with buffer A removes the remaining nonspecifically attached beads. .. For the experiments in which we observe the effect of dTTP (Sigma Aldrich, Zwijndrecht, The Netherlands) on the hopping transition of the hairpin (see and ), we exchange buffer A from the flow cell with buffer B, which contains 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 7 mM MgCl2 , 3 mM EDTA, 0.01% Tween-20, and varying concentrations of dTTP. .. Thermal noise arises from the fact that the DNA-tethered bead experiences Brownian fluctuations in a (approximately) harmonic potential ( , , ).

Gas Chromatography:

Article Title: DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically
Article Snippet: The GC cell line BGC-823 was purchased from the Shanghai Institute for Biological Sciences Chinese Academy of Sciences (Shanghai, China) and routinely maintained in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) in a 5% CO2 humidified atmosphere at 37°C. .. Rabbit anti-Phospho-BRCA1 Ab (Cat# 9009), rabbit anti-Phospho-H2A.X Ab (Cat# 2197), rabbit anti-caspase-3 (8G10) mAb (Cat# 9665), rabbit anti-cleaved caspase-3 (Asp 175) mAb (Cat# 9664), rabbit anti-Bax pAb (Cat#2772), rabbit anti-Bcl-2 (50E3) mAb (Cat# 2870), HRP-linked anti-rabbit IgG Ab (Cat# 7074) and HRP-linked anti-mouse IgG Ab (Cat# 7076) were purchased from Cell Signaling Technology (Beverly, MA, USA). dCTP, dTTP and 5-FU were purchased from Sigma and 5-methyl-dCTP was purchased from New England BioLabs (Ipswich, MA, USA).

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. Second, 1 U USER (New England Biolabs, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

Article Title: Genetic and Biochemical Characterizations of Enzymes Involved in Streptococcus pneumoniae Serotype 2 Capsule Synthesis Demonstrate that Cps2T (WchF) Catalyzes the Committed Step by Addition of ?1-4 Rhamnose, the Second Sugar Residue in the Repeat Unit
Article Snippet: UDP-[14 C]Glc (293 mCi/mmol) was obtained from Amersham Biosciences. .. Inorganic pyrophosphatase, NAD+ , NADH, dTTP, Glc-1-P, and dTDP-Glc were obtained from Sigma-Aldrich. .. Nonidet P-40 (NP-40) was obtained from Pierce Chemical Company.

Ligation:

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: A LIC-modified pMAD vector was constructed in order to enable efficient directional cloning without restriction enzyme digestion or ligation reactions ( ). .. To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C.

Generated:

Article Title: Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains
Article Snippet: Total RNA was extracted from homogenized and lysed tissues as described by the manufacturer (Ambion). .. DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight. .. The RNA template was then hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1 M, respectively, followed by incubatation at 70°C for 15 min. Unincorporated aa-dUTP was removed with a QIAquick column (Qiagen).

other:

Article Title: FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein
Article Snippet: The deoxynucleotide (dNTP) mixture was prepared by mixing each of UltraPure dATP, dCTP, dGTP and dTTP, purchased from Sigma Aldrich.

Article Title: Molecular Mechanisms of Action of Herbal Antifungal Alkaloid Berberine, in Candida albicans
Article Snippet: AMB, CAS, CR, CFW, SDS, TRB were purchased from Sigma chemicals Co. (St. Louis, MO) Superscript-II Reverse transcriptase enzyme, oligo dt(18) primer and random primer from Invitrogen, USA. dATP, dGTP, dTTP and dCTP were bought from Sigma.

Polymerase Chain Reaction:

Article Title: Soybean Resistance to White Mold: Evaluation of Soybean Germplasm Under Different Conditions and Validation of QTL
Article Snippet: The PCR amplification was performed in MJ TetradTM thermal cycler (MJ Research, Waltham, MA). .. Total reaction volume of 15.0 μL contained 50 ng of genomic DNA, 0.3 μM each of forward and reverse primers, 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma-Aldrich, St. Louis, MO), 3.0 mM MgCl2 , 2.5 units of Taq polymerase, and 1.0 × PCR buffer. .. The PCR was performed using a regular program as follows; an initial denaturation at 95°C for 2 min, followed by 38 cycles of denaturation at 94°C for 25 s, 25 s of annealing at primer specific annealing temperature, 45 s of extension at 70°C, a final extension at 72°C for 10 min, followed by a final hold at 4°C.

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: The PCR products AB and CD were used to obtain an overlapping PCR product named AD, which was cloned into the shuttle vector pMAD ( ) or pMAD_lic. .. To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C.

Article Title: Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains
Article Snippet: DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight. .. DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight.

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications.

Article Title: Effects of Ravuconazole Treatment on Parasite Load and Immune Response in Dogs Experimentally Infected with Trypanosoma cruzi
Article Snippet: Paragraph title: (iii) PCR assay. ... Briefly, 2 μl of blood DNA template was added to 10 mM Tris-HCl (pH 9.0); 75 mM KCl; 3.5 mM MgCl2 ; 0.1% Triton X-100; 0.2 mM each dATP, dCTP, dGTP, and dTTP (Sigma Chemical Co.); 1.0 U of Taq DNA polymerase (Promega); and water in a 20-μl reaction volume.

Labeling:

Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
Article Snippet: Paragraph title: Measuring the Labeling Efficiency of 5-hmC ... Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL.

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C. .. The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C.

Purification:

Article Title: Global probabilistic annotation of metabolic networks enables enzyme discovery
Article Snippet: Thus, this term quantifies tendencies of closely related activities to be filled together. .. Different amounts of purified SpsI or SpsJ were incubated at 37 °C in 1 mL of 10 mM potassium phosphate buffer pH 7.4, 2.5 mM MgCl2 , 1 mM glucose-1-phosphate (Sigma-Aldrich, > = 97% purity), 1 mM dTTP (Sigma-Aldrich, > = 96% purity) and 1 U pyrophosphatase . .. The enzyme reaction samples were assayed after 4 hours by flow-injection into a time of flight mass spectrometer (6520 Series QTOF, Agilent Technologies) operated in the negative ionization mode.

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C. .. PCR products with complementary overhangs were created using Phusion enzyme (Thermo Scientific) by building appropriate 5′ extensions into the primers.

Article Title: Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains
Article Snippet: DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight. .. DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight.

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs, USA) and 2 M betaine (Sigma, USA).

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C. .. The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C.

Sequencing:

Article Title: Soybean Resistance to White Mold: Evaluation of Soybean Germplasm Under Different Conditions and Validation of QTL
Article Snippet: A total of 132 simple sequence repeat (SSR) primer pairs were selected from the integrated soybean linkage map (Song et al., ; Choi et al., ). .. Total reaction volume of 15.0 μL contained 50 ng of genomic DNA, 0.3 μM each of forward and reverse primers, 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma-Aldrich, St. Louis, MO), 3.0 mM MgCl2 , 2.5 units of Taq polymerase, and 1.0 × PCR buffer.

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: The RNA mixtures were further added with 4 μL of 5× first-strand buffer, 2 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 4 μg of actinomycin D (USB), 200 U SuperScript III, and 20 U SUPERase-In (Ambion, USA), incubated at room temperature for 10 min followed by 1 h at 55°C to synthesize the first-strand cDNA. .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. First, five times less adapter mixture was ligated to the cDNAs.

Polyacrylamide Gel Electrophoresis:

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β. .. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer.

Chloramphenicol Acetyltransferase Assay:

Article Title: DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically
Article Snippet: Mouse anti-α-tublin Ab (Cat# T6074) was purchased from Sigma (Louis, MO, USA). .. Rabbit anti-Phospho-BRCA1 Ab (Cat# 9009), rabbit anti-Phospho-H2A.X Ab (Cat# 2197), rabbit anti-caspase-3 (8G10) mAb (Cat# 9665), rabbit anti-cleaved caspase-3 (Asp 175) mAb (Cat# 9664), rabbit anti-Bax pAb (Cat2772), rabbit anti-Bcl-2 (50E3) mAb (Cat# 2870), HRP-linked anti-rabbit IgG Ab (Cat# 7074) and HRP-linked anti-mouse IgG Ab (Cat# 7076) were purchased from Cell Signaling Technology (Beverly, MA, USA). dCTP, dTTP and 5-FU were purchased from Sigma and 5-methyl-dCTP was purchased from New England BioLabs (Ipswich, MA, USA). .. Stable DCTPP1 -knockdown BGC-823 cells were constructed by transfecting RNAi-Ready pSIREN-RetroQ retroviral vector (Clontech, CA, USA) containing shRNA oligonucleotides targeting DCTPP1 as described in our previous report [ ].

Silver Staining:

Article Title: Effects of Ravuconazole Treatment on Parasite Load and Immune Response in Dogs Experimentally Infected with Trypanosoma cruzi
Article Snippet: Briefly, 2 μl of blood DNA template was added to 10 mM Tris-HCl (pH 9.0); 75 mM KCl; 3.5 mM MgCl2 ; 0.1% Triton X-100; 0.2 mM each dATP, dCTP, dGTP, and dTTP (Sigma Chemical Co.); 1.0 U of Taq DNA polymerase (Promega); and water in a 20-μl reaction volume. .. Briefly, 2 μl of blood DNA template was added to 10 mM Tris-HCl (pH 9.0); 75 mM KCl; 3.5 mM MgCl2 ; 0.1% Triton X-100; 0.2 mM each dATP, dCTP, dGTP, and dTTP (Sigma Chemical Co.); 1.0 U of Taq DNA polymerase (Promega); and water in a 20-μl reaction volume.

Plasmid Preparation:

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: To create the pMAD_lic vector we oligomerized the primers pMAD_lic (EcoRI) and pMAD_lic (BamHI) listed in Supplementary Table , and cloned the double DNA strand dimer into pMAD using EcoRI and BamHI restriction enzymes. .. To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C. .. PCR products with complementary overhangs were created using Phusion enzyme (Thermo Scientific) by building appropriate 5′ extensions into the primers.

Article Title: Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains
Article Snippet: DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight. .. To couple the amino-allyl cDNA with fluorescent labels, Cy3 or Cy5 mono-reactive dye (Amersham) was added for 1 h. Uncoupled label was removed using the Qiagen QIAquick PCR purification kit (Valencia, CA).

RNA Extraction:

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: Paragraph title: RNA Extraction and cDNA Synthesis ... The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C.

Agarose Gel Electrophoresis:

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: The quality of the total RNA was verified by agarose gel electrophoresis, and the concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, USA). .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications.

In Vitro:

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: Paragraph title: In vitro polymerase activity assay ... The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β.

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation
Article Snippet: Paragraph title: In vitro primer extension assay. ... Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma).

Microarray:

Article Title: Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains
Article Snippet: Total RNA was extracted from homogenized and lysed tissues as described by the manufacturer (Ambion). .. DNA probes for microarray experiments were generated by adding 2 μg of total RNA to a mixture containing 6 μg of random hexamers (Invitrogen), 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (Invitrogen) at 42°C overnight. .. The RNA template was then hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1 M, respectively, followed by incubatation at 70°C for 15 min. Unincorporated aa-dUTP was removed with a QIAquick column (Qiagen).

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: For microarray experiments, total RNAs were extracted with TriReagent™ solution (Sigma) and contaminating DNA was digested using DNA-free ™ DNase (Ambion) following the manufacturer's instructions. .. The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C.

Ethanol Precipitation:

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: The RNA mixtures were further added with 4 μL of 5× first-strand buffer, 2 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 4 μg of actinomycin D (USB), 200 U SuperScript III, and 20 U SUPERase-In (Ambion, USA), incubated at room temperature for 10 min followed by 1 h at 55°C to synthesize the first-strand cDNA. .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. First, five times less adapter mixture was ligated to the cDNAs.

Spectrophotometry:

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: The quality of the total RNA was verified by agarose gel electrophoresis, and the concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, USA). .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications.

Article Title: Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium
Article Snippet: RNA quantity and quality were assessed using a Nanodrop BD-1000 spectrophotometer (Nanodrop Technologies) and Experion Automated Electrophoresis System (Bio-Rad Laboratories), respectively. .. The reverse transcription reactions were performed with 400 units of Superscript II reverse transcriptase (Invitrogen), RNA (20 µg), random hexamers (6 µg), 1.5 mM dATP, dCTP, and dGTP, 1.2 mM dTTP, and 0.3 mM amino allyl-dUTP (Sigma) for 1.5 hrs at 42°C.

Concentration Assay:

Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
Article Snippet: Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL. .. Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL.

Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
Article Snippet: The quality of the total RNA was verified by agarose gel electrophoresis, and the concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, USA). .. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications.

Staining:

Article Title: Soybean Resistance to White Mold: Evaluation of Soybean Germplasm Under Different Conditions and Validation of QTL
Article Snippet: Total reaction volume of 15.0 μL contained 50 ng of genomic DNA, 0.3 μM each of forward and reverse primers, 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma-Aldrich, St. Louis, MO), 3.0 mM MgCl2 , 2.5 units of Taq polymerase, and 1.0 × PCR buffer. .. Total reaction volume of 15.0 μL contained 50 ng of genomic DNA, 0.3 μM each of forward and reverse primers, 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma-Aldrich, St. Louis, MO), 3.0 mM MgCl2 , 2.5 units of Taq polymerase, and 1.0 × PCR buffer.

Article Title: No Evidence of Persisting Unrepaired Nuclear DNA Single Strand Breaks in Distinct Types of Cells in the Brain, Kidney, and Liver of Adult Mice after Continuous Eight-Week 50 Hz Magnetic Field Exposure with Flux Density of 0.1 mT or 1.0 mT
Article Snippet: For studying UDS and mtDNA synthesis, sections were mounted on low potassium slides (Kindler, Freiburg, Germany), deparaffinized with xylol, and Feulgen stained (1N HCl at 60°C for 6 min). .. Subsequently, ISNT was carried out as follows , : sections were incubated for 60 min at 37°C with a reaction solution containing 50 mM Tris-HCl-buffer (pH 7.5), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 200 U/ml of Escherichia coli DNA polymerase-I (endonuclease-free; Roche), 10 µmol/ml each of dATP, dGTP, dCTP, and dTTP (Sigma-Aldrich), and 15 µl 3 H-dTTP/ml (1.03 MBq/ml; American Radiolabeled Chemicals).

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    White Dttp, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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