dntps  (Qiagen)


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    Structured Review

    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 826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntps - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants"

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

    Journal: Plant Methods

    doi: 10.1186/1746-4811-7-24

    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.
    Figure Legend 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.

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

    Related Articles

    Amplification:

    Article Title: Polymorphic repeat in AIB1 does not alter breast cancer risk
    Article Snippet: PCR amplification of the polymorphic fragment was generated using the following primers: 5'-TTCCGACAACAGAGGGTGG-3' (forward) and 5'-AGTCA CATTAGGTGGGC-3' (reverse). .. Forty nanograms of genomic DNA was used per 22 μ l reaction with 1.7 μ l of each 10 μ mol/l primer, 4 μ l of 10 μ mol/l dNTPs, 2.2 μ l 10 × PCR buffer, 9.0 μ l water and 1.50 U Taq polymerase (Qiagen Incorporated).

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
    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. .. The PCR conditions used for screening the genomic DNA superpools were: pre-incubation at 95°C for 5 min followed by 35 cycles of sequential denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 2 min. PCR products were separated by gel electrophoresis in 1% agarose gels.

    Article Title: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG
    Article Snippet: .. For those viral supernatants that could not be sequenced using this method because of inadequate viral content, the viral RNA was amplified and sequenced using primers KK1 and DR8 (400 nM each) in a one-step RT-PCR reaction containing 10 µl viral RNA, 1.5 µl of each primer, 10 µl 5× buffer, 40 µM dNTPs, 0.1 µl RNAse inhibitor and 2 µl Qiagen Taq (Qiagen), as follows: RT: 30 mins at 50°C, denaturation: 15 mins 95°C, 40 cycles of amplification (15 sec 95°C, 30 sec 55.5°C, 1 min 72°C) and a final 5 min extension step at 72°C, followed by an inner PCR using the same conditions as above. .. V3-loop sequences are available under EMBL Nucleotide Sequence Database with accession numbers: HE972342-HE972511 and JN407569, JN407585, JN407591, JN407601, JN407602, JN407608, JN407609, JN407611, JN407624, JN407629, JN407632, JN407661, JN407676, JN407696, JN407704, JN407706, JN407709, JN407713, JN407726, JN407738, JN407740, JN407745, JN407747, JN407805, JN407808, JN407810, JN407813, JN407814, JN407816, JN407817, JN407836, JN407872, JN407949, JN407971, JN407987, JN407991, JN408004, JN408005, JN408022, JN408023, JN408027, JN408043 and JN408058.

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: Oligonucleotides for PCR amplification and pyrosequencing (Additional File ) were synthesized by Biotez (Buch, Germany). .. Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume.

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. .. The acquired fragments were purified by agarose gel electrophoresis and enriched by PCR amplification.

    Article Title: Association of Human Papilloma Virus Infection and Oral Squamous Cell Carcinoma in Bangladesh
    Article Snippet: .. PCR was carried out in 15 μL reaction volume which contained : 5 μL DNA solution 1.5 mM MgCl2 200 μM dNTPs 500 μM of each primer 1 unit of HotStart Taq DNA polymerase (Qiagen, USA) The PCR conditions were as follows: initial incubation at 94 °C for 15 min followed by 35 cycles of reaction with step of denaturation at 95 °C for 45 seconds, annealing at 57 °C for 45 seconds and elongation at 72 °C for 45 seconds, and the 35th cycle was followed by a step of final elongation at 72 °C for 10 min. PCR product was checked for amplification in a 3% agarose gel stained with ethidium bromide. ..

    Article Title: Dendrochilumhampelii ( Coelogyninae, Epidendroideae, Orchidaceae) traded as ‘Big Pink’ is a new species, not a hybrid: evidence from nrITS, matK and ycf1 sequence data
    Article Snippet: Paragraph title: Amplification and Sanger sequencing ... Each PCR reaction consisted of 25 µl, containing the template DNA, CoralLoad PCR buffer (Qiagen), dNTPs, Taq DNA Polymerase (Qiagen), and both primers.

    Article Title: The alien slipper limpet Crepipatella dilatata (Lamarck, 1819) in northern Spain: A multidisciplinary approach to its taxonomic identification and invasive biology
    Article Snippet: .. The COI gene was amplified using the universal primers HCO2198 and LCO1490 [ ] in 30 μL reactions containing 3 μL of PCR buffer (10X), 3.6 μL MgCl2 (25 mM), 1.5 μL dNTPs (2.5 mM), 0.3 μL BSA (100X), 0.3 μL of each primer (10 μM), 0.25 μL Taq polymerase TopTaq (Quiagen, 5 U μL-1 ) and 3 μL of genomic DNA diluted 1/25. .. PCR amplification occurred in a PTC200 MJ thermocycler with the following parameters: one cycle at 94°C for 60 s, 35 cycles at 95°C for 30 s, 49°C for 55 s and 72°C for 90 s, and a final extension at 72°C f or 10 min. PCR products were sequenced with the forward primer by applying the ABI 3730xl BigDye Terminator Cycle Sequencing 3.1 (Applied Biosystems) standard protocol employed by Macrogen Inc.

    Article Title: A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma
    Article Snippet: Briefly, PCR was carried out in a final volume of 25 μL using the forward primer sequence 5′- TCATAATGCTTGCTCTGATAGGA-3′ and the reverse primer sequence 5′-GGCCAAAAATTTAATCAGTGGA-3′ to give an amplicon size of 224 base pairs. .. PCR reactions contained 10–50 ng of DNA, 0.5 μM of each primer, 200 μM of dNTPs, 3 μM MgCl2 and 0.4 units of Taq polymerase (Qiagen, Australia).

    Synthesized:

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: Oligonucleotides for PCR amplification and pyrosequencing (Additional File ) were synthesized by Biotez (Buch, Germany). .. Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume.

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. ..

    Article Title: Human collectin-11 (COLEC11) and its synergic genetic interaction with MASP2 are associated with the pathophysiology of Chagas Disease
    Article Snippet: The COLEC11 reference sequence (ENST00000349077.8) was retrieved from the Ensembl database ( www.ensembl.org ), primers targeting exon 7 of COLEC11 gene were those utilized by Antony et al. [ ] and were synthesized commercially (Eurofins Genomics, Ebersberg, Germany). .. PCR amplifications were carried out in a 25 μl volume of reaction mixture containing 10x PCR buffer, 2 mM MgCl2 , 0.125 mM of dNTPs, 0.2 μM of each primer, 1 unit of Taq polymerase (Qiagen, Germany), and 20 ng of genomic DNA on a Mastercycler Nexus Gradient (Eppendorf, Germany).

    Nested PCR:

    Article Title: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG
    Article Snippet: For sequencing of viral supernatants, viral RNA was extracted and amplified as described previously and a nested PCR was performed using 2 µl of the Env cDNA, using primers KK1 and DR8 (400 nM each) in a mix containing 5 µl 10× PCR Gold Buffer II, 20 µM dNTPs, 200 µM MgCl2 and 0.5 µl AmpliTaq Gold DNA polymerase (Applied Biosystems), in the following cycling conditions: denaturing for 10 mins at 95°C, followed by 40 amplification cycles (15 sec 95°C, 30 sec 55,5°C, 1 min 72°C) and a final 10 mins extension step at 72°C. .. For those viral supernatants that could not be sequenced using this method because of inadequate viral content, the viral RNA was amplified and sequenced using primers KK1 and DR8 (400 nM each) in a one-step RT-PCR reaction containing 10 µl viral RNA, 1.5 µl of each primer, 10 µl 5× buffer, 40 µM dNTPs, 0.1 µl RNAse inhibitor and 2 µl Qiagen Taq (Qiagen), as follows: RT: 30 mins at 50°C, denaturation: 15 mins 95°C, 40 cycles of amplification (15 sec 95°C, 30 sec 55.5°C, 1 min 72°C) and a final 5 min extension step at 72°C, followed by an inner PCR using the same conditions as above.

    Incubation:

    Article Title: Association of Human Papilloma Virus Infection and Oral Squamous Cell Carcinoma in Bangladesh
    Article Snippet: .. PCR was carried out in 15 μL reaction volume which contained : 5 μL DNA solution 1.5 mM MgCl2 200 μM dNTPs 500 μM of each primer 1 unit of HotStart Taq DNA polymerase (Qiagen, USA) The PCR conditions were as follows: initial incubation at 94 °C for 15 min followed by 35 cycles of reaction with step of denaturation at 95 °C for 45 seconds, annealing at 57 °C for 45 seconds and elongation at 72 °C for 45 seconds, and the 35th cycle was followed by a step of final elongation at 72 °C for 10 min. PCR product was checked for amplification in a 3% agarose gel stained with ethidium bromide. ..

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: Sixteen microlitres of purified RNA was fragmented by addition of 4 μl 5× fragmentation buffer (200 mM Tris acetate (pH 8.2), 500 mM potassium acetate and 150 mM magnesium acetate) and incubated at 94 °C for exactly 90 s. After ethanol precipitation, fragment ed RNA was mixed with 5 μg random hexamers, followed by incubation at 70 °C for 10 min and chilling on ice. .. From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer.

    DNA Methylation Assay:

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: Quantitative DNA methylation analysis of the bisulphite treated DNA was performed by pyrosequencing or - in case of several sequencing primers - by serial pyrosequencing [ ]. .. Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume.

    Methylation:

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: Paragraph title: Methylation assays ... Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume.

    Infection:

    Article Title: Intraspecific variability in the parasitoid wasp Trichogramma chilonis: can we predict the outcome of hybridization?
    Article Snippet: Concentrations of MgCl2 in buffer, dNTPs, primers, and Qiagen Taq polymerase (QIAGEN S.A.S., Courtaboeuf, France) were 1.5 mm , 0.2 mm , 0.5 μm , and 1 U, respectively. .. Positive controls of infection by Wolbachia and Cardinium were, respectively, provided by individuals of Psyttalia lounsburyi ( ) and Bemisia tabaci (provided by L. Mouton, UMR 5558 ‘Biométrie et Biologie Evolutive’ – Lyon I, France).

    Generated:

    Article Title: Polymorphic repeat in AIB1 does not alter breast cancer risk
    Article Snippet: PCR amplification of the polymorphic fragment was generated using the following primers: 5'-TTCCGACAACAGAGGGTGG-3' (forward) and 5'-AGTCA CATTAGGTGGGC-3' (reverse). .. Forty nanograms of genomic DNA was used per 22 μ l reaction with 1.7 μ l of each 10 μ mol/l primer, 4 μ l of 10 μ mol/l dNTPs, 2.2 μ l 10 × PCR buffer, 9.0 μ l water and 1.50 U Taq polymerase (Qiagen Incorporated).

    Polymerase Chain Reaction:

    Article Title: Polymorphic repeat in AIB1 does not alter breast cancer risk
    Article Snippet: .. Forty nanograms of genomic DNA was used per 22 μ l reaction with 1.7 μ l of each 10 μ mol/l primer, 4 μ l of 10 μ mol/l dNTPs, 2.2 μ l 10 × PCR buffer, 9.0 μ l water and 1.50 U Taq polymerase (Qiagen Incorporated). .. Amplification conditions were 2 min of initial denaturation at 94°C followed by 35 cycles of 30 s at 94°C, 90 s at 60°C and 30 s at 72°C, followed by a final extension at 72°C for 8 min. Two fluorescent 5' -labeled primers were utilized, allowing two samples per lane.

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
    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. .. The previously designed RB1 (5'-ATGGGGCGGTATCGGAGGAAAAG-3') and RB2 (5'-TACCGGCTGTTGGACGAGTTCTTCTG-3') primers [ ] specifically anneal to the AphVIII gene and were used as specific marker gene primers (Figure ).

    Article Title: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG
    Article Snippet: .. For those viral supernatants that could not be sequenced using this method because of inadequate viral content, the viral RNA was amplified and sequenced using primers KK1 and DR8 (400 nM each) in a one-step RT-PCR reaction containing 10 µl viral RNA, 1.5 µl of each primer, 10 µl 5× buffer, 40 µM dNTPs, 0.1 µl RNAse inhibitor and 2 µl Qiagen Taq (Qiagen), as follows: RT: 30 mins at 50°C, denaturation: 15 mins 95°C, 40 cycles of amplification (15 sec 95°C, 30 sec 55.5°C, 1 min 72°C) and a final 5 min extension step at 72°C, followed by an inner PCR using the same conditions as above. .. V3-loop sequences are available under EMBL Nucleotide Sequence Database with accession numbers: HE972342-HE972511 and JN407569, JN407585, JN407591, JN407601, JN407602, JN407608, JN407609, JN407611, JN407624, JN407629, JN407632, JN407661, JN407676, JN407696, JN407704, JN407706, JN407709, JN407713, JN407726, JN407738, JN407740, JN407745, JN407747, JN407805, JN407808, JN407810, JN407813, JN407814, JN407816, JN407817, JN407836, JN407872, JN407949, JN407971, JN407987, JN407991, JN408004, JN408005, JN408022, JN408023, JN408027, JN408043 and JN408058.

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: Oligonucleotides for PCR amplification and pyrosequencing (Additional File ) were synthesized by Biotez (Buch, Germany). .. Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume.

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. ..

    Article Title: Intraspecific variability in the parasitoid wasp Trichogramma chilonis: can we predict the outcome of hybridization?
    Article Snippet: PCR were performed in a final volume of 10 μL using 1 μL of DNA template. .. Concentrations of MgCl2 in buffer, dNTPs, primers, and Qiagen Taq polymerase (QIAGEN S.A.S., Courtaboeuf, France) were 1.5 mm , 0.2 mm , 0.5 μm , and 1 U, respectively.

    Article Title: Association of Human Papilloma Virus Infection and Oral Squamous Cell Carcinoma in Bangladesh
    Article Snippet: .. PCR was carried out in 15 μL reaction volume which contained : 5 μL DNA solution 1.5 mM MgCl2 200 μM dNTPs 500 μM of each primer 1 unit of HotStart Taq DNA polymerase (Qiagen, USA) The PCR conditions were as follows: initial incubation at 94 °C for 15 min followed by 35 cycles of reaction with step of denaturation at 95 °C for 45 seconds, annealing at 57 °C for 45 seconds and elongation at 72 °C for 45 seconds, and the 35th cycle was followed by a step of final elongation at 72 °C for 10 min. PCR product was checked for amplification in a 3% agarose gel stained with ethidium bromide. ..

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer. .. Before the final PCR, a band corresponding to ~300 bp (DNA + adaptor) was collected and incubated with 1 U USER enzyme (NEB) at 37 °C for 15 min, followed by 5 min at 95 °C.

    Article Title: Dendrochilumhampelii ( Coelogyninae, Epidendroideae, Orchidaceae) traded as ‘Big Pink’ is a new species, not a hybrid: evidence from nrITS, matK and ycf1 sequence data
    Article Snippet: .. Each PCR reaction consisted of 25 µl, containing the template DNA, CoralLoad PCR buffer (Qiagen), dNTPs, Taq DNA Polymerase (Qiagen), and both primers. .. The PCR reactions were carried out using a MyCycler Thermal Cycler (Bio-Rad) or a C1000 Touch Thermal Cycler (Bio-Rad).

    Article Title: The alien slipper limpet Crepipatella dilatata (Lamarck, 1819) in northern Spain: A multidisciplinary approach to its taxonomic identification and invasive biology
    Article Snippet: .. The COI gene was amplified using the universal primers HCO2198 and LCO1490 [ ] in 30 μL reactions containing 3 μL of PCR buffer (10X), 3.6 μL MgCl2 (25 mM), 1.5 μL dNTPs (2.5 mM), 0.3 μL BSA (100X), 0.3 μL of each primer (10 μM), 0.25 μL Taq polymerase TopTaq (Quiagen, 5 U μL-1 ) and 3 μL of genomic DNA diluted 1/25. .. PCR amplification occurred in a PTC200 MJ thermocycler with the following parameters: one cycle at 94°C for 60 s, 35 cycles at 95°C for 30 s, 49°C for 55 s and 72°C for 90 s, and a final extension at 72°C f or 10 min. PCR products were sequenced with the forward primer by applying the ABI 3730xl BigDye Terminator Cycle Sequencing 3.1 (Applied Biosystems) standard protocol employed by Macrogen Inc.

    Article Title: Human collectin-11 (COLEC11) and its synergic genetic interaction with MASP2 are associated with the pathophysiology of Chagas Disease
    Article Snippet: .. PCR amplifications were carried out in a 25 μl volume of reaction mixture containing 10x PCR buffer, 2 mM MgCl2 , 0.125 mM of dNTPs, 0.2 μM of each primer, 1 unit of Taq polymerase (Qiagen, Germany), and 20 ng of genomic DNA on a Mastercycler Nexus Gradient (Eppendorf, Germany). ..

    Article Title: A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma
    Article Snippet: .. PCR reactions contained 10–50 ng of DNA, 0.5 μM of each primer, 200 μM of dNTPs, 3 μM MgCl2 and 0.4 units of Taq polymerase (Qiagen, Australia). .. The PCR conditions were an initial denaturation period of 5 minutes at 94°C, followed by 40 cycles of denaturation at 94°C for 40 sec, annealing at 60°C for 40 sec and extension at 72°C for 40 sec.

    Sequencing:

    Article Title: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG
    Article Snippet: Paragraph title: Sequencing ... For those viral supernatants that could not be sequenced using this method because of inadequate viral content, the viral RNA was amplified and sequenced using primers KK1 and DR8 (400 nM each) in a one-step RT-PCR reaction containing 10 µl viral RNA, 1.5 µl of each primer, 10 µl 5× buffer, 40 µM dNTPs, 0.1 µl RNAse inhibitor and 2 µl Qiagen Taq (Qiagen), as follows: RT: 30 mins at 50°C, denaturation: 15 mins 95°C, 40 cycles of amplification (15 sec 95°C, 30 sec 55.5°C, 1 min 72°C) and a final 5 min extension step at 72°C, followed by an inner PCR using the same conditions as above.

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: Quantitative DNA methylation analysis of the bisulphite treated DNA was performed by pyrosequencing or - in case of several sequencing primers - by serial pyrosequencing [ ]. .. Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume.

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: Paragraph title: 4.2. RNA Isolation, Illumina Sequencing and Raw Data Processing ... The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition.

    Article Title: Association of Human Papilloma Virus Infection and Oral Squamous Cell Carcinoma in Bangladesh
    Article Snippet: Polymerase chain reaction (PCR) for HPV All samples were subjected to PCR, using primers specific for consensus sequence spanning the E6 open reading frame of high-risk HPV type 16, 18, 31, and 33. .. PCR was carried out in 15 μL reaction volume which contained : 5 μL DNA solution 1.5 mM MgCl2 200 μM dNTPs 500 μM of each primer 1 unit of HotStart Taq DNA polymerase (Qiagen, USA) The PCR conditions were as follows: initial incubation at 94 °C for 15 min followed by 35 cycles of reaction with step of denaturation at 95 °C for 45 seconds, annealing at 57 °C for 45 seconds and elongation at 72 °C for 45 seconds, and the 35th cycle was followed by a step of final elongation at 72 °C for 10 min. PCR product was checked for amplification in a 3% agarose gel stained with ethidium bromide.

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: Paragraph title: mRNA sequencing ... From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer.

    Article Title: Dendrochilumhampelii ( Coelogyninae, Epidendroideae, Orchidaceae) traded as ‘Big Pink’ is a new species, not a hybrid: evidence from nrITS, matK and ycf1 sequence data
    Article Snippet: Paragraph title: Amplification and Sanger sequencing ... Each PCR reaction consisted of 25 µl, containing the template DNA, CoralLoad PCR buffer (Qiagen), dNTPs, Taq DNA Polymerase (Qiagen), and both primers.

    Article Title: The alien slipper limpet Crepipatella dilatata (Lamarck, 1819) in northern Spain: A multidisciplinary approach to its taxonomic identification and invasive biology
    Article Snippet: The COI gene was amplified using the universal primers HCO2198 and LCO1490 [ ] in 30 μL reactions containing 3 μL of PCR buffer (10X), 3.6 μL MgCl2 (25 mM), 1.5 μL dNTPs (2.5 mM), 0.3 μL BSA (100X), 0.3 μL of each primer (10 μM), 0.25 μL Taq polymerase TopTaq (Quiagen, 5 U μL-1 ) and 3 μL of genomic DNA diluted 1/25. .. PCR amplification occurred in a PTC200 MJ thermocycler with the following parameters: one cycle at 94°C for 60 s, 35 cycles at 95°C for 30 s, 49°C for 55 s and 72°C for 90 s, and a final extension at 72°C f or 10 min. PCR products were sequenced with the forward primer by applying the ABI 3730xl BigDye Terminator Cycle Sequencing 3.1 (Applied Biosystems) standard protocol employed by Macrogen Inc.

    Article Title: Human collectin-11 (COLEC11) and its synergic genetic interaction with MASP2 are associated with the pathophysiology of Chagas Disease
    Article Snippet: The COLEC11 reference sequence (ENST00000349077.8) was retrieved from the Ensembl database ( www.ensembl.org ), primers targeting exon 7 of COLEC11 gene were those utilized by Antony et al. [ ] and were synthesized commercially (Eurofins Genomics, Ebersberg, Germany). .. PCR amplifications were carried out in a 25 μl volume of reaction mixture containing 10x PCR buffer, 2 mM MgCl2 , 0.125 mM of dNTPs, 0.2 μM of each primer, 1 unit of Taq polymerase (Qiagen, Germany), and 20 ng of genomic DNA on a Mastercycler Nexus Gradient (Eppendorf, Germany).

    Article Title: A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma
    Article Snippet: Paragraph title: Sanger sequencing (Perth laboratory) ... PCR reactions contained 10–50 ng of DNA, 0.5 μM of each primer, 200 μM of dNTPs, 3 μM MgCl2 and 0.4 units of Taq polymerase (Qiagen, Australia).

    DNA Extraction:

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
    Article Snippet: Paragraph title: DNA isolation and PCR procedures ... 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.

    Nucleic Acid Electrophoresis:

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
    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. .. The PCR conditions used for screening the genomic DNA superpools were: pre-incubation at 95°C for 5 min followed by 35 cycles of sequential denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 2 min. PCR products were separated by gel electrophoresis in 1% agarose gels.

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: The concentration and quality of the RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and 2% gel electrophoresis was used to determine the quality and integrity of the total RNA. .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition.

    Magnetic Beads:

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: Briefly, mRNA was enriched from total RNA using oligo (dT) magnetic beads. .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition.

    Isolation:

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
    Article Snippet: Isolation of genomic DNA was performed using a standard phenol-chloroform extraction method as described previously [ ]. .. 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.

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: Paragraph title: 4.2. RNA Isolation, Illumina Sequencing and Raw Data Processing ... The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition.

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: After washing in Ca2+ - and Mg2+ -containing PBS, RNA was isolated and purified using the RNAeasy kit (Qiagen), followed by DNase treatment (Qiagen). .. From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer.

    Size-exclusion Chromatography:

    Article Title: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG
    Article Snippet: .. For those viral supernatants that could not be sequenced using this method because of inadequate viral content, the viral RNA was amplified and sequenced using primers KK1 and DR8 (400 nM each) in a one-step RT-PCR reaction containing 10 µl viral RNA, 1.5 µl of each primer, 10 µl 5× buffer, 40 µM dNTPs, 0.1 µl RNAse inhibitor and 2 µl Qiagen Taq (Qiagen), as follows: RT: 30 mins at 50°C, denaturation: 15 mins 95°C, 40 cycles of amplification (15 sec 95°C, 30 sec 55.5°C, 1 min 72°C) and a final 5 min extension step at 72°C, followed by an inner PCR using the same conditions as above. .. V3-loop sequences are available under EMBL Nucleotide Sequence Database with accession numbers: HE972342-HE972511 and JN407569, JN407585, JN407591, JN407601, JN407602, JN407608, JN407609, JN407611, JN407624, JN407629, JN407632, JN407661, JN407676, JN407696, JN407704, JN407706, JN407709, JN407713, JN407726, JN407738, JN407740, JN407745, JN407747, JN407805, JN407808, JN407810, JN407813, JN407814, JN407816, JN407817, JN407836, JN407872, JN407949, JN407971, JN407987, JN407991, JN408004, JN408005, JN408022, JN408023, JN408027, JN408043 and JN408058.

    Article Title: Dendrochilumhampelii ( Coelogyninae, Epidendroideae, Orchidaceae) traded as ‘Big Pink’ is a new species, not a hybrid: evidence from nrITS, matK and ycf1 sequence data
    Article Snippet: Each PCR reaction consisted of 25 µl, containing the template DNA, CoralLoad PCR buffer (Qiagen), dNTPs, Taq DNA Polymerase (Qiagen), and both primers. .. The thermal cycling protocol began with 5 min initial denaturation at 95 °C followed by 35 amplification cycles, each with 30 sec denaturation at 95 °C, 30 sec annealing at 50 °C, and 1 min extension at 72 °C, which were concluded by a 7 min final extension at 72 °C.

    Article Title: A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma
    Article Snippet: PCR reactions contained 10–50 ng of DNA, 0.5 μM of each primer, 200 μM of dNTPs, 3 μM MgCl2 and 0.4 units of Taq polymerase (Qiagen, Australia). .. The PCR conditions were an initial denaturation period of 5 minutes at 94°C, followed by 40 cycles of denaturation at 94°C for 40 sec, annealing at 60°C for 40 sec and extension at 72°C for 40 sec.

    Labeling:

    Article Title: Polymorphic repeat in AIB1 does not alter breast cancer risk
    Article Snippet: Forty nanograms of genomic DNA was used per 22 μ l reaction with 1.7 μ l of each 10 μ mol/l primer, 4 μ l of 10 μ mol/l dNTPs, 2.2 μ l 10 × PCR buffer, 9.0 μ l water and 1.50 U Taq polymerase (Qiagen Incorporated). .. Amplification conditions were 2 min of initial denaturation at 94°C followed by 35 cycles of 30 s at 94°C, 90 s at 60°C and 30 s at 72°C, followed by a final extension at 72°C for 8 min. Two fluorescent 5' -labeled primers were utilized, allowing two samples per lane.

    Purification:

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. ..

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: Sixteen microlitres of purified RNA was fragmented by addition of 4 μl 5× fragmentation buffer (200 mM Tris acetate (pH 8.2), 500 mM potassium acetate and 150 mM magnesium acetate) and incubated at 94 °C for exactly 90 s. After ethanol precipitation, fragment ed RNA was mixed with 5 μg random hexamers, followed by incubation at 70 °C for 10 min and chilling on ice. .. From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer.

    Article Title: Human collectin-11 (COLEC11) and its synergic genetic interaction with MASP2 are associated with the pathophysiology of Chagas Disease
    Article Snippet: PCR amplifications were carried out in a 25 μl volume of reaction mixture containing 10x PCR buffer, 2 mM MgCl2 , 0.125 mM of dNTPs, 0.2 μM of each primer, 1 unit of Taq polymerase (Qiagen, Germany), and 20 ng of genomic DNA on a Mastercycler Nexus Gradient (Eppendorf, Germany). .. PCR products were purified using Exo-SAP-IT (USB-Affymetrix, Santa Clara, USA) and the purified products were directly used as templates for sequencing using the BigDye terminator cycle sequencing kit (v.3.1; Applied Biosystems, Texas, USA) on an ABI 3130XL DNA Analyzer.

    Article Title: A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma
    Article Snippet: PCR reactions contained 10–50 ng of DNA, 0.5 μM of each primer, 200 μM of dNTPs, 3 μM MgCl2 and 0.4 units of Taq polymerase (Qiagen, Australia). .. PCR products were purified using the QIAquick PCR purification Kit (Qiagen, Australia).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG
    Article Snippet: .. For those viral supernatants that could not be sequenced using this method because of inadequate viral content, the viral RNA was amplified and sequenced using primers KK1 and DR8 (400 nM each) in a one-step RT-PCR reaction containing 10 µl viral RNA, 1.5 µl of each primer, 10 µl 5× buffer, 40 µM dNTPs, 0.1 µl RNAse inhibitor and 2 µl Qiagen Taq (Qiagen), as follows: RT: 30 mins at 50°C, denaturation: 15 mins 95°C, 40 cycles of amplification (15 sec 95°C, 30 sec 55.5°C, 1 min 72°C) and a final 5 min extension step at 72°C, followed by an inner PCR using the same conditions as above. .. V3-loop sequences are available under EMBL Nucleotide Sequence Database with accession numbers: HE972342-HE972511 and JN407569, JN407585, JN407591, JN407601, JN407602, JN407608, JN407609, JN407611, JN407624, JN407629, JN407632, JN407661, JN407676, JN407696, JN407704, JN407706, JN407709, JN407713, JN407726, JN407738, JN407740, JN407745, JN407747, JN407805, JN407808, JN407810, JN407813, JN407814, JN407816, JN407817, JN407836, JN407872, JN407949, JN407971, JN407987, JN407991, JN408004, JN408005, JN408022, JN408023, JN408027, JN408043 and JN408058.

    Salting Out:

    Article Title: The alien slipper limpet Crepipatella dilatata (Lamarck, 1819) in northern Spain: A multidisciplinary approach to its taxonomic identification and invasive biology
    Article Snippet: The COI sequences of the non-native specimens were obtained by extracting total genomic DNA from samples of their foot muscle tissue following the salting-out protocol [ ]. .. The COI gene was amplified using the universal primers HCO2198 and LCO1490 [ ] in 30 μL reactions containing 3 μL of PCR buffer (10X), 3.6 μL MgCl2 (25 mM), 1.5 μL dNTPs (2.5 mM), 0.3 μL BSA (100X), 0.3 μL of each primer (10 μM), 0.25 μL Taq polymerase TopTaq (Quiagen, 5 U μL-1 ) and 3 μL of genomic DNA diluted 1/25.

    cDNA Library Assay:

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: The construction of the cDNA library and sequencing were performed as previously reported with minor modifications [ , ]. .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition.

    Agarose Gel Electrophoresis:

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. .. The acquired fragments were purified by agarose gel electrophoresis and enriched by PCR amplification.

    Article Title: Association of Human Papilloma Virus Infection and Oral Squamous Cell Carcinoma in Bangladesh
    Article Snippet: .. PCR was carried out in 15 μL reaction volume which contained : 5 μL DNA solution 1.5 mM MgCl2 200 μM dNTPs 500 μM of each primer 1 unit of HotStart Taq DNA polymerase (Qiagen, USA) The PCR conditions were as follows: initial incubation at 94 °C for 15 min followed by 35 cycles of reaction with step of denaturation at 95 °C for 45 seconds, annealing at 57 °C for 45 seconds and elongation at 72 °C for 45 seconds, and the 35th cycle was followed by a step of final elongation at 72 °C for 10 min. PCR product was checked for amplification in a 3% agarose gel stained with ethidium bromide. ..

    Article Title: Human collectin-11 (COLEC11) and its synergic genetic interaction with MASP2 are associated with the pathophysiology of Chagas Disease
    Article Snippet: PCR amplifications were carried out in a 25 μl volume of reaction mixture containing 10x PCR buffer, 2 mM MgCl2 , 0.125 mM of dNTPs, 0.2 μM of each primer, 1 unit of Taq polymerase (Qiagen, Germany), and 20 ng of genomic DNA on a Mastercycler Nexus Gradient (Eppendorf, Germany). .. PCR fragments were stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, USA) and visualized in a 1.5% agarose gel.

    Software:

    Article Title: Polymorphic repeat in AIB1 does not alter breast cancer risk
    Article Snippet: Forty nanograms of genomic DNA was used per 22 μ l reaction with 1.7 μ l of each 10 μ mol/l primer, 4 μ l of 10 μ mol/l dNTPs, 2.2 μ l 10 × PCR buffer, 9.0 μ l water and 1.50 U Taq polymerase (Qiagen Incorporated). .. The size of the amplified products was determined relative to an internal size standard using Genescan and Genotyper Analysis software (Perkin-Elmer).

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
    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. .. Target gene specific primers were designed largely with PrimerSelect software (DNAstar 7.1.0, Lasergene) using the default setting for hairpin formation and dimer duplexing; primer lengths were between 18 and 26 nucleotides.

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume. .. Quantitative DNA methylation analysis was carried out on a PSQ 96MD system with the PyroGold SQA Reagent Kit (Pyrosequencing) and results were analyzed using the Q-CpG software (V.1.0.9, Pyrosequencing AB).

    Article Title: Intraspecific variability in the parasitoid wasp Trichogramma chilonis: can we predict the outcome of hybridization?
    Article Snippet: Pairwise genetic distances were calculated using the p-distance with the software MEGA 4 ( ). .. Concentrations of MgCl2 in buffer, dNTPs, primers, and Qiagen Taq polymerase (QIAGEN S.A.S., Courtaboeuf, France) were 1.5 mm , 0.2 mm , 0.5 μm , and 1 U, respectively.

    Sample Prep:

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer. .. Second-strand cDNA was synthesised by adding 91.8 μl H2 O, 5 μg random hexamers, 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 4 μl 10 mM dNTPs with dTTP replaced by dUTP, 30 μl 5× second-strand buffer, 40 U E. coli DNA polymerase, 10 U E . coli DNA ligase and 2 U E . coli RNase H, and incubated at 16 °C for 2 h, followed by incubation with 10 U T4 polymerase at 16 °C for 10 min. Double-stranded cDNA was purified using a Qiagen MinElute column and used for Illumina sample preparation and sequencing according to the Illumina protocol.

    Ethanol Precipitation:

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: Sixteen microlitres of purified RNA was fragmented by addition of 4 μl 5× fragmentation buffer (200 mM Tris acetate (pH 8.2), 500 mM potassium acetate and 150 mM magnesium acetate) and incubated at 94 °C for exactly 90 s. After ethanol precipitation, fragment ed RNA was mixed with 5 μg random hexamers, followed by incubation at 70 °C for 10 min and chilling on ice. .. From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer.

    Spectrophotometry:

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: The concentration and quality of the RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and 2% gel electrophoresis was used to determine the quality and integrity of the total RNA. .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition.

    Sampling:

    Article Title: The alien slipper limpet Crepipatella dilatata (Lamarck, 1819) in northern Spain: A multidisciplinary approach to its taxonomic identification and invasive biology
    Article Snippet: A total of ten COI sequences from non-native individuals collected here, five from each sampling site , were analysed phylogenetically together with all of the COI sequences for C . dilatata and the sister species Crepipatella peruviana (formerly Crepipatella fecunda Gallardo, 1979) available in GenBank. .. The COI gene was amplified using the universal primers HCO2198 and LCO1490 [ ] in 30 μL reactions containing 3 μL of PCR buffer (10X), 3.6 μL MgCl2 (25 mM), 1.5 μL dNTPs (2.5 mM), 0.3 μL BSA (100X), 0.3 μL of each primer (10 μM), 0.25 μL Taq polymerase TopTaq (Quiagen, 5 U μL-1 ) and 3 μL of genomic DNA diluted 1/25.

    Concentration Assay:

    Article Title: DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response
    Article Snippet: DNA concentrations were determined using the Quant-iT™ dsDNA broad range assay kit (Invitrogen, Cergy Pontoise, France) and normalized to a concentration of 50 ng/μl. .. Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mM MgCl2 , 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume.

    Article Title: An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium ‘Zhongshanshan’
    Article Snippet: The concentration and quality of the RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and 2% gel electrophoresis was used to determine the quality and integrity of the total RNA. .. The double-stranded cDNA was synthesized with buffer, dNTPs, RNase H and DNA polymerase I, purified with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition.

    Article Title: E-cadherin loss induces targetable autocrine activation of growth factor signalling in lobular breast cancer
    Article Snippet: After measurement of RNA concentration using a Qubit fluorometer (Invitrogen), 250 ng of total RNA was treated using a Ribo-Zero rRNA removal kit (Epicenter) to remove ribosomal RNAs. .. From this RNA primer mix, first-strand cDNA was synthesised by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng actinomycin D and 200 U SuperScript III in a Qiagen MinElute column to remove dNTPs and eluted in 34 μl elution buffer.

    Marker:

    Article Title: Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants
    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. .. The previously designed RB1 (5'-ATGGGGCGGTATCGGAGGAAAAG-3') and RB2 (5'-TACCGGCTGTTGGACGAGTTCTTCTG-3') primers [ ] specifically anneal to the AphVIII gene and were used as specific marker gene primers (Figure ).

    Article Title: The alien slipper limpet Crepipatella dilatata (Lamarck, 1819) in northern Spain: A multidisciplinary approach to its taxonomic identification and invasive biology
    Article Snippet: As genetic marker, we selected the cytochrome c oxidase subunit I (COI) gene, because of its significant discriminatory power between species in barcoding studies of calyptraeids [ , ]. .. The COI gene was amplified using the universal primers HCO2198 and LCO1490 [ ] in 30 μL reactions containing 3 μL of PCR buffer (10X), 3.6 μL MgCl2 (25 mM), 1.5 μL dNTPs (2.5 mM), 0.3 μL BSA (100X), 0.3 μL of each primer (10 μM), 0.25 μL Taq polymerase TopTaq (Quiagen, 5 U μL-1 ) and 3 μL of genomic DNA diluted 1/25.

    Staining:

    Article Title: Association of Human Papilloma Virus Infection and Oral Squamous Cell Carcinoma in Bangladesh
    Article Snippet: .. PCR was carried out in 15 μL reaction volume which contained : 5 μL DNA solution 1.5 mM MgCl2 200 μM dNTPs 500 μM of each primer 1 unit of HotStart Taq DNA polymerase (Qiagen, USA) The PCR conditions were as follows: initial incubation at 94 °C for 15 min followed by 35 cycles of reaction with step of denaturation at 95 °C for 45 seconds, annealing at 57 °C for 45 seconds and elongation at 72 °C for 45 seconds, and the 35th cycle was followed by a step of final elongation at 72 °C for 10 min. PCR product was checked for amplification in a 3% agarose gel stained with ethidium bromide. ..

    Article Title: Human collectin-11 (COLEC11) and its synergic genetic interaction with MASP2 are associated with the pathophysiology of Chagas Disease
    Article Snippet: PCR amplifications were carried out in a 25 μl volume of reaction mixture containing 10x PCR buffer, 2 mM MgCl2 , 0.125 mM of dNTPs, 0.2 μM of each primer, 1 unit of Taq polymerase (Qiagen, Germany), and 20 ng of genomic DNA on a Mastercycler Nexus Gradient (Eppendorf, Germany). .. PCR fragments were stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, USA) and visualized in a 1.5% agarose gel.

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    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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    86
    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
    https://www.bioz.com/result/residual dntps/product/Qiagen
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    residual dntps - by Bioz Stars, 2020-04
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    Image Search Results


    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