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    Structured Review

    Qiagen dntp
    5FU incorporated into <t>DNA</t> is preferentially paired with adenine, followed by guanine. (A) Flowchart of procedure of in vitro analysis of binding partner of 5FU within DNA. (B-E) Frequencies of paired base with 5FU incorporated into DNA. One microgram of 5FdU:C containing dsDNA template PCR were utilized for both pH 8.2 or pH5.7 as a model of intracellular pH (pH i ) of tumor cells or normal cells in acidic tumor microenvironment with <t>dNTP</t> (B), only dCTP and dGTP (C), only dCTP, dGTP, and dATP (D), or only dCTP, dGTP, and dTTP (E). After TA cloning and total colony PCR, paired bases were analyzed by direct sequencing. The frequency of paired bases was calculated as the number of each base where 5FdU was inserted by total number of colonies ( N ≥ 24) with an informative sequence at the 5FdU site. 5FU within DNA is predominantly paired with adenine when both dGTP and dATP are available (B, D), whereas guanine is preferentially paired with 5FU when dATP is absent (C, E).
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    1) Product Images from "Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity"

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity

    Journal: Mutation research

    doi: 10.1016/j.mrfmmm.2013.04.006

    5FU incorporated into DNA is preferentially paired with adenine, followed by guanine. (A) Flowchart of procedure of in vitro analysis of binding partner of 5FU within DNA. (B-E) Frequencies of paired base with 5FU incorporated into DNA. One microgram of 5FdU:C containing dsDNA template PCR were utilized for both pH 8.2 or pH5.7 as a model of intracellular pH (pH i ) of tumor cells or normal cells in acidic tumor microenvironment with dNTP (B), only dCTP and dGTP (C), only dCTP, dGTP, and dATP (D), or only dCTP, dGTP, and dTTP (E). After TA cloning and total colony PCR, paired bases were analyzed by direct sequencing. The frequency of paired bases was calculated as the number of each base where 5FdU was inserted by total number of colonies ( N ≥ 24) with an informative sequence at the 5FdU site. 5FU within DNA is predominantly paired with adenine when both dGTP and dATP are available (B, D), whereas guanine is preferentially paired with 5FU when dATP is absent (C, E).
    Figure Legend Snippet: 5FU incorporated into DNA is preferentially paired with adenine, followed by guanine. (A) Flowchart of procedure of in vitro analysis of binding partner of 5FU within DNA. (B-E) Frequencies of paired base with 5FU incorporated into DNA. One microgram of 5FdU:C containing dsDNA template PCR were utilized for both pH 8.2 or pH5.7 as a model of intracellular pH (pH i ) of tumor cells or normal cells in acidic tumor microenvironment with dNTP (B), only dCTP and dGTP (C), only dCTP, dGTP, and dATP (D), or only dCTP, dGTP, and dTTP (E). After TA cloning and total colony PCR, paired bases were analyzed by direct sequencing. The frequency of paired bases was calculated as the number of each base where 5FdU was inserted by total number of colonies ( N ≥ 24) with an informative sequence at the 5FdU site. 5FU within DNA is predominantly paired with adenine when both dGTP and dATP are available (B, D), whereas guanine is preferentially paired with 5FU when dATP is absent (C, E).

    Techniques Used: In Vitro, Binding Assay, Polymerase Chain Reaction, TA Cloning, Sequencing

    Related Articles

    Clone Assay:

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: Paragraph title: Cloning, sequencing of rat CYP4A1 cDNA. ... The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA).

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: .. Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

    Amplification:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: PCR amplification of the corresponding genomic region surrounding each SNP locus was performed in a Takara PCR thermal cycler (Takara TP600, Dalian, China). .. The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA.

    Article Title: Elbamycellarosea gen. et sp. nov. ( Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea
    Article Snippet: Paragraph title: DNA extraction, PCR amplification, and data assembling ... Reaction mixtures consisted of 60–80 ng DNA template, 10× PCR Buffer (15 mM MgCl2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 μM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 μL final volume.

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The primer sequences for the amplification of the full-length rat CYP4A1 cDNA were 5′-GCT GCA CCA TGA GCG TCT C-3′ (sense) and 5′-GTG CAG GAC ACT GGA CAC-3′ (antisense). .. The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA).

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: .. Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: The primers used for PCR amplification were: 16SF [5'-GTTTGATCATGGCTCAGATTG-3'] and 16SR [5'-CTACCTTGTTACGACTTCACC-3']. .. The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template.

    Article Title: Development and application of a tri-allelic PCR assay for screening Vgsc-L1014F kdr mutations associated with pyrethroid and organochlorine resistance in the mosquito Culex quinquefasciatus
    Article Snippet: .. ETAS-PCR/Vgsc-1014 amplification, endonuclease digestion and genotyping The ETAS-PCR/Vgsc -1014 multiplex reaction was performed in a total volume of 25 µl containing 10 ng of gDNA, 200 µM each dNTP, 1× PCR buffer, 0.2 units HotStartTaq DNA polymerase (Qiagen), 0.6 µM universal reverse primer Vgsc -1014/R, 0.35 µM of the specific forward primers (Vgsc -1014/F-T and Vgsc -1014/F-C) and 0.3 µM of the Vgsc -1014/F-A primer (Table ). .. After initial denaturation at 95 °C for 15 min, amplification was performed for 40 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, followed by a final extension step of 72 °C for 10 min. After amplification, the ETAS-PCR/Vgsc -1014 multiplex products were digested with FastDigest Eco 32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water.

    Article Title: Genome-scale design of PCR primers and long oligomers for DNA microarrays
    Article Snippet: Paragraph title: PCR amplification ... The first PCR round was performed in 50 µl reactions containing 100 ng of genomic DNA, 1× Qiagen PCR buffer (1.5 mM MgCl2 ), 40 µM of each dNTP, 1 U Qiagen Taq polymerase and 20 pmol of each primer.

    Article Title: ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma
    Article Snippet: Unless otherwise stated, all PCR reactions were performed using 1.5 mM MgCl2 , 0.2 mM of each dNTP, 0.3 μM of each primer and 0.5 units HotStarTaq DNA polymerase (Qiagen). .. The upstream promoter region was amplified from sodium bisulfite treated DNA using primers -353: 5'-AGTGGGGGAGTTAAGGAGATATTTT-3' and -136: 5'-CTAAACTCCAAAACCAAAAATACTTAA-3' at a Ta of 63°C.

    Article Title: Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure
    Article Snippet: The DNA from the competitive growth assays was PCR amplified (Platinum PCR Supermix [Invitrogen, USA]) in triplicate using biotinylated primer pairs targeting the region containing the SNP distinguishing the two organisms in the assay, that is mutations in gyrA , parC and aroC ( ). .. PCR amplifications were performed in 60 µl reactions containing 1 × NH4 buffer, 1.5 mM of MgCl2 , 200 µM of dNTP, 10 pM of each primer, 1.25 Units of Hotstart DNA polymerase (Qiagen, USA) and 5 µl of template DNA.

    Article Title: A Novel Locus for Autosomal Recessive Peripheral Neuropathy in the EGR2 Region on 10q23
    Article Snippet: The coding regions of EGR2 were amplified from genomic DNA, with the use of overlapping primers as described elsewhere (Timmerman et al. ; ). .. PCR reactions were performed in a 50-μl volume containing 50 ng DNA, 50 ng each primer, 500 μM each dNTP, 1.5 mM MgCl2 , 1 × Qiagen PCR buffer (Qiagen), and 0.125 U Taq DNA polymerase (Qiagen).

    Polymerase Chain Reaction:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: PCR amplification of the corresponding genomic region surrounding each SNP locus was performed in a Takara PCR thermal cycler (Takara TP600, Dalian, China). .. The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA.

    Article Title: Elbamycellarosea gen. et sp. nov. ( Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea
    Article Snippet: .. Reaction mixtures consisted of 60–80 ng DNA template, 10× PCR Buffer (15 mM MgCl2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 μM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 μL final volume. .. Following visualization of the amplicons on a 1.5% agarose gel stained with 5 mL 100 mL−1 ethidium bromide, PCR products were purified and sequenced at Macrogen Europe Laboratory (Madrid, Spain).

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA. ..

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: .. The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The reaction mixtures were initially denatured at 94o C for 5 min and then cycled 35 times between 94o C for 30 s, 58o C for 30 s, and 72o C for 2 min followed by extension for 10 min at 72o C. The PCR products were separated on 1% agarose gel in a Tris-borate-EDTA (TBE) buffer with 100 mg/ml of ethidium bromide (Sigma, St. Louis, MO), they were visualized, and their density was analyzed using ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA).

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: .. The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template. .. The optimized amplification program began with a 95°C for 15 min enzyme activation step.

    Article Title: Development and application of a tri-allelic PCR assay for screening Vgsc-L1014F kdr mutations associated with pyrethroid and organochlorine resistance in the mosquito Culex quinquefasciatus
    Article Snippet: .. ETAS-PCR/Vgsc-1014 amplification, endonuclease digestion and genotyping The ETAS-PCR/Vgsc -1014 multiplex reaction was performed in a total volume of 25 µl containing 10 ng of gDNA, 200 µM each dNTP, 1× PCR buffer, 0.2 units HotStartTaq DNA polymerase (Qiagen), 0.6 µM universal reverse primer Vgsc -1014/R, 0.35 µM of the specific forward primers (Vgsc -1014/F-T and Vgsc -1014/F-C) and 0.3 µM of the Vgsc -1014/F-A primer (Table ). .. After initial denaturation at 95 °C for 15 min, amplification was performed for 40 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, followed by a final extension step of 72 °C for 10 min. After amplification, the ETAS-PCR/Vgsc -1014 multiplex products were digested with FastDigest Eco 32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water.

    Article Title: Genome-scale design of PCR primers and long oligomers for DNA microarrays
    Article Snippet: .. The first PCR round was performed in 50 µl reactions containing 100 ng of genomic DNA, 1× Qiagen PCR buffer (1.5 mM MgCl2 ), 40 µM of each dNTP, 1 U Qiagen Taq polymerase and 20 pmol of each primer. .. In the last 20 cycles the elongation time was prolonged by 5 s for each cycle to compensate for decreasing enzymatic activity.

    Article Title: ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma
    Article Snippet: .. Unless otherwise stated, all PCR reactions were performed using 1.5 mM MgCl2 , 0.2 mM of each dNTP, 0.3 μM of each primer and 0.5 units HotStarTaq DNA polymerase (Qiagen). ..

    Article Title: Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure
    Article Snippet: .. PCR amplifications were performed in 60 µl reactions containing 1 × NH4 buffer, 1.5 mM of MgCl2 , 200 µM of dNTP, 10 pM of each primer, 1.25 Units of Hotstart DNA polymerase (Qiagen, USA) and 5 µl of template DNA. .. Reactions were cycled once at 95°C for 15 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, with a final elongation of 72°C for 5 min. All PCR amplifications were visualized on 1% agarose gels prior to pyrosequencing.

    Article Title: A Novel Locus for Autosomal Recessive Peripheral Neuropathy in the EGR2 Region on 10q23
    Article Snippet: .. PCR reactions were performed in a 50-μl volume containing 50 ng DNA, 50 ng each primer, 500 μM each dNTP, 1.5 mM MgCl2 , 1 × Qiagen PCR buffer (Qiagen), and 0.125 U Taq DNA polymerase (Qiagen). .. A PE 9600 thermal cycler (PE Biosystems) was used with the following cycling conditions: initial denaturation at 94°C for 5 min; 35 cycles at 94°C for 30 s, 66°C for 1 min, and 72°C for 1 min; and final extension at 72°C for 7 min. PCR products were purified through QIAquick Spin Columns (Qiagen) and were sequenced using the PCR primers and Big Dye Terminator Cycle Sequencing Ready Reaction Kits (PE Biosystems).

    TA Cloning:

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

    Incubation:

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The full-length CYP4A1 cDNA, purified using a PureLink PCR purification kit, was ligated into pCR4-TOPO sequencing vector (Life Technologies) and incubated at room temperature for 30 min. DH 5α-T1 Escherichia coli- competent cells (Life Technologies) were transformed by adding 2 μl of the ligation reaction and incubated at 42o C for 30 s followed by placing the reactions in an ice bath for 2 min.

    Article Title: Genome-scale design of PCR primers and long oligomers for DNA microarrays
    Article Snippet: The first PCR round was performed in 50 µl reactions containing 100 ng of genomic DNA, 1× Qiagen PCR buffer (1.5 mM MgCl2 ), 40 µM of each dNTP, 1 U Qiagen Taq polymerase and 20 pmol of each primer. .. The plates were incubated for 5 min at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing for 30 s at 60°C and elongation at 72°C for 90 s. In the last 20 cycles the elongation time was prolonged by 5 s for each cycle to compensate for decreasing enzymatic activity.

    Article Title: ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma
    Article Snippet: Unless otherwise stated, all PCR reactions were performed using 1.5 mM MgCl2 , 0.2 mM of each dNTP, 0.3 μM of each primer and 0.5 units HotStarTaq DNA polymerase (Qiagen). .. The resulting 217 bp product was digested into a 56 bp and a 161 bp product by incubation with BstU I.

    Activity Assay:

    Article Title: Genome-scale design of PCR primers and long oligomers for DNA microarrays
    Article Snippet: The first PCR round was performed in 50 µl reactions containing 100 ng of genomic DNA, 1× Qiagen PCR buffer (1.5 mM MgCl2 ), 40 µM of each dNTP, 1 U Qiagen Taq polymerase and 20 pmol of each primer. .. In the last 20 cycles the elongation time was prolonged by 5 s for each cycle to compensate for decreasing enzymatic activity.

    Touchdown PCR:

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template. .. To minimize PCR products derived from mispriming events, the actual amplification was initiated with a 'touchdown' PCR step consisting of 10 cycles at 95°C for 30 second (sec), 72°C-63°C (decreasing 1°C/cycle) for 20 sec and 72°C for 1.00 min followed by 35 cycles of 95°C for 30 sec, 63°C for 20 sec and 72°C for 1.00 min.

    Transformation Assay:

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The full-length CYP4A1 cDNA, purified using a PureLink PCR purification kit, was ligated into pCR4-TOPO sequencing vector (Life Technologies) and incubated at room temperature for 30 min. DH 5α-T1 Escherichia coli- competent cells (Life Technologies) were transformed by adding 2 μl of the ligation reaction and incubated at 42o C for 30 s followed by placing the reactions in an ice bath for 2 min.

    Derivative Assay:

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: CYP4A1 template was generated by adding an aliquot of the RNA (1 μg) to a 15-μl reverse transcription reaction with 0.5 μg of a gene-specific primer (5′-GTG CAG GAC ACT GGA CAC-3′) derived from the rat CYP4A1 ( ) sequence ( ) using a method that we previously described ( ). .. The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA).

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: Derived primer sequences were evaluated for predicted efficiency using the NetPrimer computer software (Premier Biosoft International, Palo Alto, CA, USA). .. The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template.

    Transfection:

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: Ten days after transfection, genomic DNA was isolated using a Wizerd® Genomic DNA Purification Kit (Promega). .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA.

    Ligation:

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The full-length CYP4A1 cDNA, purified using a PureLink PCR purification kit, was ligated into pCR4-TOPO sequencing vector (Life Technologies) and incubated at room temperature for 30 min. DH 5α-T1 Escherichia coli- competent cells (Life Technologies) were transformed by adding 2 μl of the ligation reaction and incubated at 42o C for 30 s followed by placing the reactions in an ice bath for 2 min.

    Generated:

    Article Title: Elbamycellarosea gen. et sp. nov. ( Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea
    Article Snippet: Reaction mixtures consisted of 60–80 ng DNA template, 10× PCR Buffer (15 mM MgCl2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 μM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 μL final volume. .. Newly generated sequences were deposited in GenBank (Table ).

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: CYP4A1 template was generated by adding an aliquot of the RNA (1 μg) to a 15-μl reverse transcription reaction with 0.5 μg of a gene-specific primer (5′-GTG CAG GAC ACT GGA CAC-3′) derived from the rat CYP4A1 ( ) sequence ( ) using a method that we previously described ( ). .. The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA).

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: Cloning for Analytical Sensitivity Analysis For cloning, a segment of viral genome covering a part of the N gene (positions 1–933 according to the Pasteur virus genome accession number X03673) of CVS-11 was generated using primers designed for this purpose (TWclon-F and TWclon-R, ). .. Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C.

    Imaging:

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The reaction mixtures were initially denatured at 94o C for 5 min and then cycled 35 times between 94o C for 30 s, 58o C for 30 s, and 72o C for 2 min followed by extension for 10 min at 72o C. The PCR products were separated on 1% agarose gel in a Tris-borate-EDTA (TBE) buffer with 100 mg/ml of ethidium bromide (Sigma, St. Louis, MO), they were visualized, and their density was analyzed using ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA).

    Sequencing:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA. .. Cycling conditions were as follows: One cycle at 95°C for 2 min; 11 cycles at 94°C for 20 sec; 65-0.5°C for 40 sec, 72°C for 1.5 min; and 24 cycles at 94°C for 20 sec, 59°C for 30 sec and 72°C for 1.5 min; and a final extension at 72°C for 2 min. PCR products were purified by the PCR purification kit containing 1X shrimp alkaline phsophatase (SAP) and 1X Exonuclease I (Qiagen, Hilden, Germany) and were used as DNA templates for cycle sequencing.

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA. ..

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: Paragraph title: Cloning, sequencing of rat CYP4A1 cDNA. ... The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA).

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: Paragraph title: 16 S rRNA gene sequencing ... The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template.

    Article Title: A Novel Locus for Autosomal Recessive Peripheral Neuropathy in the EGR2 Region on 10q23
    Article Snippet: Paragraph title: Sequence Analysis of the EGR2 Gene ... PCR reactions were performed in a 50-μl volume containing 50 ng DNA, 50 ng each primer, 500 μM each dNTP, 1.5 mM MgCl2 , 1 × Qiagen PCR buffer (Qiagen), and 0.125 U Taq DNA polymerase (Qiagen).

    Binding Assay:

    Article Title: Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure
    Article Snippet: PCR amplifications were performed in 60 µl reactions containing 1 × NH4 buffer, 1.5 mM of MgCl2 , 200 µM of dNTP, 10 pM of each primer, 1.25 Units of Hotstart DNA polymerase (Qiagen, USA) and 5 µl of template DNA. .. PCR amplicons were combined with 56 µl of binding buffer and 4 µl of streptavidin sepharose beads.

    DNA Sequencing:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA. .. Direct DNA sequencing was performed using 5 μl SNaPshot Multiplex kit (ABI) in 10-μl volumes containing 2 μl primer and 2 μl DNA template.

    DNA Extraction:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: Polymorphism genotyping Genomic DNA was isolated from peripheral blood leukocytes using a DNA extraction kit (Tiangen, Beijing, China). .. The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA.

    Article Title: Elbamycellarosea gen. et sp. nov. ( Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea
    Article Snippet: Paragraph title: DNA extraction, PCR amplification, and data assembling ... Reaction mixtures consisted of 60–80 ng DNA template, 10× PCR Buffer (15 mM MgCl2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 μM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 μL final volume.

    Article Title: Acute myeloid leukemia patient with FLT3-ITD and NPM1 double mutation should undergo allogeneic hematopoietic stem cell transplantation in CR1 for better prognosis
    Article Snippet: Gene mutation analyses Bone marrow mononuclear cells were isolated and the DNA extracted using a DNA Extraction kit (Invitrogen, Shanghai, People's Republic of China). .. The total volume of the PCR reaction system was 20 µL, including 200 ng DNA, 20 pmol PCR primers, 25 mmol/L MgCl2 , 2.5 mmol/L dNTP, 2 µL 10× PCR buffer, 0.2 µL HotTaq DNA polymerase (Qiagen, Shanghai, People's Republic of China).

    Nucleic Acid Electrophoresis:

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

    Combined Bisulfite Restriction Analysis Assay:

    Article Title: ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma
    Article Snippet: Methylation of an upstream (-353/-136 relative to transcription start site) and a downstream (-69/+187 relative to transcription start site) fragment from this CpG island was determined by COBRA (combined bisulfite and restriction analysis). .. Unless otherwise stated, all PCR reactions were performed using 1.5 mM MgCl2 , 0.2 mM of each dNTP, 0.3 μM of each primer and 0.5 units HotStarTaq DNA polymerase (Qiagen).

    Methylation:

    Article Title: ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma
    Article Snippet: Paragraph title: Methylation analysis of the ECRG4 promoter region ... Unless otherwise stated, all PCR reactions were performed using 1.5 mM MgCl2 , 0.2 mM of each dNTP, 0.3 μM of each primer and 0.5 units HotStarTaq DNA polymerase (Qiagen).

    Mutagenesis:

    Article Title: Acute myeloid leukemia patient with FLT3-ITD and NPM1 double mutation should undergo allogeneic hematopoietic stem cell transplantation in CR1 for better prognosis
    Article Snippet: Paragraph title: Gene mutation analyses ... The total volume of the PCR reaction system was 20 µL, including 200 ng DNA, 20 pmol PCR primers, 25 mmol/L MgCl2 , 2.5 mmol/L dNTP, 2 µL 10× PCR buffer, 0.2 µL HotTaq DNA polymerase (Qiagen, Shanghai, People's Republic of China).

    Isolation:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: Polymorphism genotyping Genomic DNA was isolated from peripheral blood leukocytes using a DNA extraction kit (Tiangen, Beijing, China). .. The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA.

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: Ten days after transfection, genomic DNA was isolated using a Wizerd® Genomic DNA Purification Kit (Promega). .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA.

    Article Title: Acute myeloid leukemia patient with FLT3-ITD and NPM1 double mutation should undergo allogeneic hematopoietic stem cell transplantation in CR1 for better prognosis
    Article Snippet: Gene mutation analyses Bone marrow mononuclear cells were isolated and the DNA extracted using a DNA Extraction kit (Invitrogen, Shanghai, People's Republic of China). .. The total volume of the PCR reaction system was 20 µL, including 200 ng DNA, 20 pmol PCR primers, 25 mmol/L MgCl2 , 2.5 mmol/L dNTP, 2 µL 10× PCR buffer, 0.2 µL HotTaq DNA polymerase (Qiagen, Shanghai, People's Republic of China).

    Size-exclusion Chromatography:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA. .. Cycling conditions were as follows: One cycle at 95°C for 2 min; 11 cycles at 94°C for 20 sec; 65-0.5°C for 40 sec, 72°C for 1.5 min; and 24 cycles at 94°C for 20 sec, 59°C for 30 sec and 72°C for 1.5 min; and a final extension at 72°C for 2 min. PCR products were purified by the PCR purification kit containing 1X shrimp alkaline phsophatase (SAP) and 1X Exonuclease I (Qiagen, Hilden, Germany) and were used as DNA templates for cycle sequencing.

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template. .. To minimize PCR products derived from mispriming events, the actual amplification was initiated with a 'touchdown' PCR step consisting of 10 cycles at 95°C for 30 second (sec), 72°C-63°C (decreasing 1°C/cycle) for 20 sec and 72°C for 1.00 min followed by 35 cycles of 95°C for 30 sec, 63°C for 20 sec and 72°C for 1.00 min.

    Purification:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA. .. Cycling conditions were as follows: One cycle at 95°C for 2 min; 11 cycles at 94°C for 20 sec; 65-0.5°C for 40 sec, 72°C for 1.5 min; and 24 cycles at 94°C for 20 sec, 59°C for 30 sec and 72°C for 1.5 min; and a final extension at 72°C for 2 min. PCR products were purified by the PCR purification kit containing 1X shrimp alkaline phsophatase (SAP) and 1X Exonuclease I (Qiagen, Hilden, Germany) and were used as DNA templates for cycle sequencing.

    Article Title: Elbamycellarosea gen. et sp. nov. ( Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea
    Article Snippet: Reaction mixtures consisted of 60–80 ng DNA template, 10× PCR Buffer (15 mM MgCl2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 μM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 μL final volume. .. Following visualization of the amplicons on a 1.5% agarose gel stained with 5 mL 100 mL−1 ethidium bromide, PCR products were purified and sequenced at Macrogen Europe Laboratory (Madrid, Spain).

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA. ..

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The full-length CYP4A1 cDNA, purified using a PureLink PCR purification kit, was ligated into pCR4-TOPO sequencing vector (Life Technologies) and incubated at room temperature for 30 min. DH 5α-T1 Escherichia coli- competent cells (Life Technologies) were transformed by adding 2 μl of the ligation reaction and incubated at 42o C for 30 s followed by placing the reactions in an ice bath for 2 min.

    Article Title: Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure
    Article Snippet: Calculation of allele frequencies by pyrosequencing Bacterial cells from each time point were thawed and DNA was extracted using heat treatment and phenol-chloroform purification. .. PCR amplifications were performed in 60 µl reactions containing 1 × NH4 buffer, 1.5 mM of MgCl2 , 200 µM of dNTP, 10 pM of each primer, 1.25 Units of Hotstart DNA polymerase (Qiagen, USA) and 5 µl of template DNA.

    Article Title: A Novel Locus for Autosomal Recessive Peripheral Neuropathy in the EGR2 Region on 10q23
    Article Snippet: PCR reactions were performed in a 50-μl volume containing 50 ng DNA, 50 ng each primer, 500 μM each dNTP, 1.5 mM MgCl2 , 1 × Qiagen PCR buffer (Qiagen), and 0.125 U Taq DNA polymerase (Qiagen). .. A PE 9600 thermal cycler (PE Biosystems) was used with the following cycling conditions: initial denaturation at 94°C for 5 min; 35 cycles at 94°C for 30 s, 66°C for 1 min, and 72°C for 1 min; and final extension at 72°C for 7 min. PCR products were purified through QIAquick Spin Columns (Qiagen) and were sequenced using the PCR primers and Big Dye Terminator Cycle Sequencing Ready Reaction Kits (PE Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: .. Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

    Staining:

    Article Title: Elbamycellarosea gen. et sp. nov. ( Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea
    Article Snippet: Reaction mixtures consisted of 60–80 ng DNA template, 10× PCR Buffer (15 mM MgCl2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 μM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 μL final volume. .. Following visualization of the amplicons on a 1.5% agarose gel stained with 5 mL 100 mL−1 ethidium bromide, PCR products were purified and sequenced at Macrogen Europe Laboratory (Madrid, Spain).

    Concentration Assay:

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: .. The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template. .. The optimized amplification program began with a 95°C for 15 min enzyme activation step.

    Activated Clotting Time Assay:

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The primer sequences for the amplification of the full-length rat CYP4A1 cDNA were 5′-GCT GCA CCA TGA GCG TCT C-3′ (sense) and 5′-GTG CAG GAC ACT GGA CAC-3′ (antisense). .. The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA).

    Plasmid Preparation:

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: Cells were co-transfected with 5 μg of 5FdU:G containing dsDNA, T:G mismatched dsDNA, or C:G matched dsDNA, and 1 μg of puromycin selection plasmid by using Nucleofector Kit V (for SW480 and HCT116) (Amaxa, Cologne, Germany), following the manufacturer's instructions. .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA.

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The full-length CYP4A1 cDNA, purified using a PureLink PCR purification kit, was ligated into pCR4-TOPO sequencing vector (Life Technologies) and incubated at room temperature for 30 min. DH 5α-T1 Escherichia coli- competent cells (Life Technologies) were transformed by adding 2 μl of the ligation reaction and incubated at 42o C for 30 s followed by placing the reactions in an ice bath for 2 min.

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

    Software:

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: Derived primer sequences were evaluated for predicted efficiency using the NetPrimer computer software (Premier Biosoft International, Palo Alto, CA, USA). .. The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template.

    Multiplex Assay:

    Article Title: ADAM33 polymorphisms are associated with asthma and a distinctive palm dermatoglyphic pattern
    Article Snippet: The reaction was performed in a final volume of 10 μl, which contained 3.0 mM Mg2+ , 0.3 mM dNTP, 1X HotStarTaq polymerase (Qiagen Inc., Hilden, Germany), 1 μl each primer and 1 μl (10 ng) genomic DNA. .. Direct DNA sequencing was performed using 5 μl SNaPshot Multiplex kit (ABI) in 10-μl volumes containing 2 μl primer and 2 μl DNA template.

    Article Title: Development and application of a tri-allelic PCR assay for screening Vgsc-L1014F kdr mutations associated with pyrethroid and organochlorine resistance in the mosquito Culex quinquefasciatus
    Article Snippet: .. ETAS-PCR/Vgsc-1014 amplification, endonuclease digestion and genotyping The ETAS-PCR/Vgsc -1014 multiplex reaction was performed in a total volume of 25 µl containing 10 ng of gDNA, 200 µM each dNTP, 1× PCR buffer, 0.2 units HotStartTaq DNA polymerase (Qiagen), 0.6 µM universal reverse primer Vgsc -1014/R, 0.35 µM of the specific forward primers (Vgsc -1014/F-T and Vgsc -1014/F-C) and 0.3 µM of the Vgsc -1014/F-A primer (Table ). .. After initial denaturation at 95 °C for 15 min, amplification was performed for 40 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, followed by a final extension step of 72 °C for 10 min. After amplification, the ETAS-PCR/Vgsc -1014 multiplex products were digested with FastDigest Eco 32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water.

    Selection:

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: Selection with 0.75 mg/mL puromycin-containing culture medium adjusted to pH 7.4 or pH 6.5 commenced 12 h after transfection. .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA.

    Agarose Gel Electrophoresis:

    Article Title: Elbamycellarosea gen. et sp. nov. ( Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea
    Article Snippet: Reaction mixtures consisted of 60–80 ng DNA template, 10× PCR Buffer (15 mM MgCl2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 μM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 μL final volume. .. Following visualization of the amplicons on a 1.5% agarose gel stained with 5 mL 100 mL−1 ethidium bromide, PCR products were purified and sequenced at Macrogen Europe Laboratory (Madrid, Spain).

    Article Title: Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat
    Article Snippet: The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2 , 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). .. The reaction mixtures were initially denatured at 94o C for 5 min and then cycled 35 times between 94o C for 30 s, 58o C for 30 s, and 72o C for 2 min followed by extension for 10 min at 72o C. The PCR products were separated on 1% agarose gel in a Tris-borate-EDTA (TBE) buffer with 100 mg/ml of ethidium bromide (Sigma, St. Louis, MO), they were visualized, and their density was analyzed using ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA).

    Next-Generation Sequencing:

    Article Title: Acute myeloid leukemia patient with FLT3-ITD and NPM1 double mutation should undergo allogeneic hematopoietic stem cell transplantation in CR1 for better prognosis
    Article Snippet: The total volume of the PCR reaction system was 20 µL, including 200 ng DNA, 20 pmol PCR primers, 25 mmol/L MgCl2 , 2.5 mmol/L dNTP, 2 µL 10× PCR buffer, 0.2 µL HotTaq DNA polymerase (Qiagen, Shanghai, People's Republic of China). .. CEBPA, KIT, IDH1 /IDH2 and TET2 mutations were analyzed by next-generation sequencing technology (San Valley Diagnostics).

    Activation Assay:

    Article Title: PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species
    Article Snippet: The PCR was performed in a 50 μl volume with HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) containing 400 μM dNTP (each of dATP, dCTP, dGTP and dTTP), 5 U of HotStart Taq Polymerase (Qiagen), 1x Taq polymerase buffer (Qiagen), 2.5 mM MgCl2 and a 300 nM concentration of each primer with ~100 ng of DNA template. .. The optimized amplification program began with a 95°C for 15 min enzyme activation step.

    DNA Purification:

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity
    Article Snippet: Ten days after transfection, genomic DNA was isolated using a Wizerd® Genomic DNA Purification Kit (Promega). .. Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA.

    Gel Extraction:

    Article Title: Real-Time RT-PCR for the Detection of Lyssavirus Species
    Article Snippet: Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μ L of RNase-free water, 10 μ L 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μ M of the cloning primers, 10 U RNasin (40 U/μ L), and 2 μ L of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μ L of extracted RNA to a total volume of 50 μ L. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. .. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.

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  • 99
    Qiagen dntps
    Determination of marker gene copy number by Southern blot hybridization . Southern-blot analysis of digested ( PstI ) genomic <t>DNA</t> from 9 randomly isolated paromomycin resistant mutants. A fragment of the AphVIII gene labeled with 32 <t>P-dNTPs</t> was used as probe. As shown, most transformants have a single copy of the integrated marker gene. Only the transformant represented in lane 4 may have two insertions (indicated by the two hybridizing bands). p.s., parental strain used to obtain the transformants.
    Dntps, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 867 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntps/product/Qiagen
    Average 99 stars, based on 867 article reviews
    Price from $9.99 to $1999.99
    dntps - by Bioz Stars, 2020-04
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    Qiagen residual dntps
    Confirmation of cleanup and enzymatic hydrolysis. ( A ) Removal of <t>dNTPs.</t> Samples were subjected to CE before and after a conventional <t>PCR</t> purification process. No peak was detected even in the 2-fold concentrated sample after purification. ( B ) Removal of primers. dNMP peaks resulting from residual primers were not detected in the 4-fold concentrated sample after purification. ( C ) CE profile of hydrolysis of a PCR product during a time course of phosphodiesterase treatment. dIMP was spiked as an internal standard for comparison. ( D ) Normalized dNMP peak intensities during a time course of phosphodiesterase treatment.
    Residual Dntps, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Determination of marker gene copy number by Southern blot hybridization . Southern-blot analysis of digested ( PstI ) genomic DNA from 9 randomly isolated paromomycin resistant mutants. A fragment of the AphVIII gene labeled with 32 P-dNTPs was used as probe. As shown, most transformants have a single copy of the integrated marker gene. Only the transformant represented in lane 4 may have two insertions (indicated by the two hybridizing bands). p.s., parental strain used to obtain the transformants.

    Journal: Plant Methods

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

    doi: 10.1186/1746-4811-7-24

    Figure Lengend Snippet: Determination of marker gene copy number by Southern blot hybridization . Southern-blot analysis of digested ( PstI ) genomic DNA from 9 randomly isolated paromomycin resistant mutants. A fragment of the AphVIII gene labeled with 32 P-dNTPs was used as probe. As shown, most transformants have a single copy of the integrated marker gene. Only the transformant represented in lane 4 may have two insertions (indicated by the two hybridizing bands). p.s., parental strain used to obtain the transformants.

    Article Snippet: PCR reactions using DNA from "superpools" as template were performed in a final volume of 25 μL and contained 0.4 pmoles of each primer, 0.2 mM of each of the dNTPs, 0.2 U of Taq DNA polymerase (Qiagen, Valencia, CA), 2.5 μL of 10 × Qiagen Taq DNA polymerase buffer, 5 μL Q solution (Qiagen, Valencia, CA), 100 ng of DNA template, and distilled water to make up the remainder of the 25 μL volume.

    Techniques: Marker, Southern Blot, Hybridization, Isolation, Labeling

    5FU incorporated into DNA is preferentially paired with adenine, followed by guanine. (A) Flowchart of procedure of in vitro analysis of binding partner of 5FU within DNA. (B-E) Frequencies of paired base with 5FU incorporated into DNA. One microgram of 5FdU:C containing dsDNA template PCR were utilized for both pH 8.2 or pH5.7 as a model of intracellular pH (pH i ) of tumor cells or normal cells in acidic tumor microenvironment with dNTP (B), only dCTP and dGTP (C), only dCTP, dGTP, and dATP (D), or only dCTP, dGTP, and dTTP (E). After TA cloning and total colony PCR, paired bases were analyzed by direct sequencing. The frequency of paired bases was calculated as the number of each base where 5FdU was inserted by total number of colonies ( N ≥ 24) with an informative sequence at the 5FdU site. 5FU within DNA is predominantly paired with adenine when both dGTP and dATP are available (B, D), whereas guanine is preferentially paired with 5FU when dATP is absent (C, E).

    Journal: Mutation research

    Article Title: Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity

    doi: 10.1016/j.mrfmmm.2013.04.006

    Figure Lengend Snippet: 5FU incorporated into DNA is preferentially paired with adenine, followed by guanine. (A) Flowchart of procedure of in vitro analysis of binding partner of 5FU within DNA. (B-E) Frequencies of paired base with 5FU incorporated into DNA. One microgram of 5FdU:C containing dsDNA template PCR were utilized for both pH 8.2 or pH5.7 as a model of intracellular pH (pH i ) of tumor cells or normal cells in acidic tumor microenvironment with dNTP (B), only dCTP and dGTP (C), only dCTP, dGTP, and dATP (D), or only dCTP, dGTP, and dTTP (E). After TA cloning and total colony PCR, paired bases were analyzed by direct sequencing. The frequency of paired bases was calculated as the number of each base where 5FdU was inserted by total number of colonies ( N ≥ 24) with an informative sequence at the 5FdU site. 5FU within DNA is predominantly paired with adenine when both dGTP and dATP are available (B, D), whereas guanine is preferentially paired with 5FU when dATP is absent (C, E).

    Article Snippet: Purified genomic DNA was used for a PCR reaction mixture with 10 mM Tris–HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2 , 200 μM dNTP, 5U of HotStarTaq DNA polymerase (QIAGEN), 0.25 μM forward primer (5′-AGAACCCACTGCTTACTGGC-3′) and reverse primer (5′-ATGGCTGGCAACTAGAAGGC-3/ ) whose sequence is identical with each end of 5FdU:G, T:G, or C:G matched dsDNA.

    Techniques: In Vitro, Binding Assay, Polymerase Chain Reaction, TA Cloning, Sequencing

    Confirmation of cleanup and enzymatic hydrolysis. ( A ) Removal of dNTPs. Samples were subjected to CE before and after a conventional PCR purification process. No peak was detected even in the 2-fold concentrated sample after purification. ( B ) Removal of primers. dNMP peaks resulting from residual primers were not detected in the 4-fold concentrated sample after purification. ( C ) CE profile of hydrolysis of a PCR product during a time course of phosphodiesterase treatment. dIMP was spiked as an internal standard for comparison. ( D ) Normalized dNMP peak intensities during a time course of phosphodiesterase treatment.

    Journal: Nucleic Acids Research

    Article Title: Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

    doi: 10.1093/nar/gkl257

    Figure Lengend Snippet: Confirmation of cleanup and enzymatic hydrolysis. ( A ) Removal of dNTPs. Samples were subjected to CE before and after a conventional PCR purification process. No peak was detected even in the 2-fold concentrated sample after purification. ( B ) Removal of primers. dNMP peaks resulting from residual primers were not detected in the 4-fold concentrated sample after purification. ( C ) CE profile of hydrolysis of a PCR product during a time course of phosphodiesterase treatment. dIMP was spiked as an internal standard for comparison. ( D ) Normalized dNMP peak intensities during a time course of phosphodiesterase treatment.

    Article Snippet: PCR products were cleaned up to remove residual dNTPs and primers using a conventional PCR purification kit (Qiagen, Hilden, Germany).

    Techniques: Polymerase Chain Reaction, Purification