Structured Review

Thermo Fisher dntps
The presence of RT functional domains from HIV-1 subtype C leads to decreased cDNA accumulation . A - Endogenous reverse transcription (ERT) in permeabilized virions. Purified and p24 CA -normalized virus particles of either the backbone NL4-3 or NL-based chimeric viruses were subjected to ERT with addition of <t>dNTPs</t> and permeabilizing agent melittin. Samples without dNTPs were used as a control. <t>DNA</t> was harvested after the indicated time of incubation. The relative amounts of negative-strand strong-stop DNA were measured using quantitative real-time PCR. Data from the control samples were subtracted. Levels of cDNA are shown as percentages of the peak accumulation detected in virions of NL4-3 at 5 h after initiation of incubation. Error bars show the standard deviation from three independent viral preparations. B - Accumulation of early or strong-stop viral DNA in Sup-T1 cells at 24 h p.i. Untreated or treated with 10 μM nevirapine cells were infected with backbone NL4-3 or the chimeric viruses, containing pol fragments from subtype C 1084i isolate using spinoculation. Relative amounts of reverse transcription products were measured using quantitative real-time PCR analysis of DNA from infected cells after incubation with or without 10 μM nevirapine. Data from nevirapine-treated samples were subtracted. Levels of cDNA are shown as percentages of the maximal accumulation detected for cDNA in cells infected with NL4-3 virus strain. Error bars show the standard deviation from three independent viral preparations. C - Accumulation of early and late reverse transcription products in Sup-T1 cells infected with recombinant viruses carrying protease and RT polymerase domain from 1084i, 2669i, and 1984i isolates of subtype C at 24 h p.i. The cells were infected with the indicated viruses as described in B. Harvested DNA was measured using quantitative real-time PCR analysis. Levels of cDNA are shown as percentages of the maximal accumulation detected for negative strand strong-stop cDNA in cells infected with NL4-3. Error bars indicate the standard deviation from three independent viral preparations.
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1) Product Images from "Subtype-associated differences in HIV-1 reverse transcription affect the viral replication"

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Journal: Retrovirology

doi: 10.1186/1742-4690-7-85

The presence of RT functional domains from HIV-1 subtype C leads to decreased cDNA accumulation . A - Endogenous reverse transcription (ERT) in permeabilized virions. Purified and p24 CA -normalized virus particles of either the backbone NL4-3 or NL-based chimeric viruses were subjected to ERT with addition of dNTPs and permeabilizing agent melittin. Samples without dNTPs were used as a control. DNA was harvested after the indicated time of incubation. The relative amounts of negative-strand strong-stop DNA were measured using quantitative real-time PCR. Data from the control samples were subtracted. Levels of cDNA are shown as percentages of the peak accumulation detected in virions of NL4-3 at 5 h after initiation of incubation. Error bars show the standard deviation from three independent viral preparations. B - Accumulation of early or strong-stop viral DNA in Sup-T1 cells at 24 h p.i. Untreated or treated with 10 μM nevirapine cells were infected with backbone NL4-3 or the chimeric viruses, containing pol fragments from subtype C 1084i isolate using spinoculation. Relative amounts of reverse transcription products were measured using quantitative real-time PCR analysis of DNA from infected cells after incubation with or without 10 μM nevirapine. Data from nevirapine-treated samples were subtracted. Levels of cDNA are shown as percentages of the maximal accumulation detected for cDNA in cells infected with NL4-3 virus strain. Error bars show the standard deviation from three independent viral preparations. C - Accumulation of early and late reverse transcription products in Sup-T1 cells infected with recombinant viruses carrying protease and RT polymerase domain from 1084i, 2669i, and 1984i isolates of subtype C at 24 h p.i. The cells were infected with the indicated viruses as described in B. Harvested DNA was measured using quantitative real-time PCR analysis. Levels of cDNA are shown as percentages of the maximal accumulation detected for negative strand strong-stop cDNA in cells infected with NL4-3. Error bars indicate the standard deviation from three independent viral preparations.
Figure Legend Snippet: The presence of RT functional domains from HIV-1 subtype C leads to decreased cDNA accumulation . A - Endogenous reverse transcription (ERT) in permeabilized virions. Purified and p24 CA -normalized virus particles of either the backbone NL4-3 or NL-based chimeric viruses were subjected to ERT with addition of dNTPs and permeabilizing agent melittin. Samples without dNTPs were used as a control. DNA was harvested after the indicated time of incubation. The relative amounts of negative-strand strong-stop DNA were measured using quantitative real-time PCR. Data from the control samples were subtracted. Levels of cDNA are shown as percentages of the peak accumulation detected in virions of NL4-3 at 5 h after initiation of incubation. Error bars show the standard deviation from three independent viral preparations. B - Accumulation of early or strong-stop viral DNA in Sup-T1 cells at 24 h p.i. Untreated or treated with 10 μM nevirapine cells were infected with backbone NL4-3 or the chimeric viruses, containing pol fragments from subtype C 1084i isolate using spinoculation. Relative amounts of reverse transcription products were measured using quantitative real-time PCR analysis of DNA from infected cells after incubation with or without 10 μM nevirapine. Data from nevirapine-treated samples were subtracted. Levels of cDNA are shown as percentages of the maximal accumulation detected for cDNA in cells infected with NL4-3 virus strain. Error bars show the standard deviation from three independent viral preparations. C - Accumulation of early and late reverse transcription products in Sup-T1 cells infected with recombinant viruses carrying protease and RT polymerase domain from 1084i, 2669i, and 1984i isolates of subtype C at 24 h p.i. The cells were infected with the indicated viruses as described in B. Harvested DNA was measured using quantitative real-time PCR analysis. Levels of cDNA are shown as percentages of the maximal accumulation detected for negative strand strong-stop cDNA in cells infected with NL4-3. Error bars indicate the standard deviation from three independent viral preparations.

Techniques Used: Functional Assay, Purification, Incubation, Real-time Polymerase Chain Reaction, Standard Deviation, Infection, Recombinant

2) Product Images from "Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †"

Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †

Journal: Journal of Virology

doi:

NERT assay for HIV-1 wild-type and tat mutant viruses. Virus stocks for wild-type virus (lanes 1), Δ tat virus trans -complemented with wild-type tat (lanes 2), Δ tat virus (lanes 3), or Δ tat virus produced in the presence of tat mutants [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 4 to 10, respectively) were analyzed for endogenous reverse transcription. Culture supernatant (200 μl) containing approximately 0.75 mU of RT activity was treated with 100 U of DNase I. Half of each reaction mixture was added to 150 μl of stop solution, incubated at 37°C for 10 min, and then boiled for 10 min (B). The remaining half of each reaction mixture was supplemented with 50 μM dNTPs and incubated at 37°C for 90 minutes before the reaction was terminated as described above. (A) PCR to detect HIV-1 negative-strand strong-stop DNA was performed on NERT reaction mixtures as described in Materials and Methods. All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10 2 , 10 3 , and 10 4 ).
Figure Legend Snippet: NERT assay for HIV-1 wild-type and tat mutant viruses. Virus stocks for wild-type virus (lanes 1), Δ tat virus trans -complemented with wild-type tat (lanes 2), Δ tat virus (lanes 3), or Δ tat virus produced in the presence of tat mutants [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 4 to 10, respectively) were analyzed for endogenous reverse transcription. Culture supernatant (200 μl) containing approximately 0.75 mU of RT activity was treated with 100 U of DNase I. Half of each reaction mixture was added to 150 μl of stop solution, incubated at 37°C for 10 min, and then boiled for 10 min (B). The remaining half of each reaction mixture was supplemented with 50 μM dNTPs and incubated at 37°C for 90 minutes before the reaction was terminated as described above. (A) PCR to detect HIV-1 negative-strand strong-stop DNA was performed on NERT reaction mixtures as described in Materials and Methods. All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10 2 , 10 3 , and 10 4 ).

Techniques Used: Mutagenesis, Produced, Activity Assay, Incubation, Polymerase Chain Reaction

Related Articles

Amplification:

Article Title: Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type
Article Snippet: : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No. .. The reverse transcription reaction mix was stored at -20°C until PCR amplification.

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No. .. The reverse transcription reaction mix was stored at −20 °C until PCR amplification.

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: .. Library amplification was performed on one half of the Pippin eluate in 1× Phusion GC buffer with 0.2 mM dNTPs, 0.1 μM forward primer (IDT), 0.1 μM reverse primer, 1 unit of Phusion Hot Start II Polymerase (Thermo Fisher Scientific). .. The amplified library was cleaned using a 1× volume of AMPure XP beads and QC was run with the Agilent Bioanalyzer DNA 1000 kit, followed by concentration determination by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems).

Article Title: Effect of Nicotine on CYP2B1 Expression in a Glioma Animal Model and Analysis of CYP2B6 Expression in Pediatric Gliomas
Article Snippet: Multiplex PCR First strand cDNA was used for multiplex PCR amplification of Cyp2b1/2 , 18s and GAPDH . .. All reactions were performed in a final volume of 50 μL using PCR buffer (1×; BioTecMol, Mexico City, Mexico), magnesium chloride (liver: 1.5 mM; brain: 4 mM; BioTecMol), dNTPs (liver: 200 μM; brain: 400 μM; Thermo Fisher Scientific), oligonucleotides for Cyp2b1/2 (liver: 300 nM; brain: 600 nM), Gapdh (liver: 120 nM; brain: 200 nM) and 18s (200 nM), Taq polymerase (2.5 U; BioTecMol) and 1 μg of cDNA for each sample.

Article Title: Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms
Article Snippet: .. Linear amplification reactions were set up using approximately 4 ng of genomic DNA, and final concentrations of 1× Buffer with 15 mM MgCl2 (Qiagen Inc., Valencia, California), 0.125 mM dNTPs (Fermentas Inc., Geln Burnie, Maryland), 0.04 Units/µl HotStar Taq DNA polymerase (Qiagen Inc., Valencia, California), and GC-rich primers at a final concentration of 0.6 µM, or 1× Buffer (Qiagen Inc., Valencia, California), 0.1 mM dNTPs (Fermentas Inc., Geln Burnie, Maryland), 100 ng/µl BSA (New England Biolabs, Ipswich, Massachusetts), 2 µl of Pfu Ultra II Fusion HS DNA polymerase (Qiagen Inc., Valencia, California), 0.2 µM GC-rich primers (Sigma Genosys, The Woodlands, Texas), and nuclease free water in a final reaction volume of 50 µl. ..

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: Paragraph title: Direct amplification and sequencing of shDNA fragments from human cells ... In the first PCR, we used 10 µM shDNA-specific primer pair with a universal tag sequence, 1 × PCR buffer, 2.0 mM MgCl2 , 2.5 mM dNTPs, and 1 unit of AmpliTaq Gold DNA Polymerase (Thermo Fisher, Cat. No. 8080241).

Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †
Article Snippet: The reverse transcription reactions were amplified by PCR with primer pairs specific for tRNA3 Lys (5′-ATAGCTCAGTCGGTAGAGCAT [sense] and 5′-GCCGAACAGGGACTTGAT [antisense]) and HIV-1 genomic RNA (5′-CAAGTAGTGTGTGCCCGTCTGTT [sense] and 5′-CGAGAGAGCTCCTCTGGTTCTAC [antisense]). .. Total viral RNA was annealed to an oligonucleotide (5′-GACTGCGAATCGTTCTAG-3′, antisense) complementary to sequences in the gag open reading frame at 75°C for 10 min and placed on ice, and cDNA was made by using the supplied buffers, 0.2 mM dNTPs, and M-MLV RT (Life Technologies) at 37°C for 60 min. Each cDNA reaction was assayed by PCR for the internal control (IC) cDNA (reverse transcribed from IC RNA) by using a 32 P-labeled oligonucleotide specific for pGem4z sequences (5′-GGGAGACAAGCTTGCATGCCTG, sense) and an unlabeled HIV-1-specific oligonucleotide (5′-GCAGTGGGTTCCCTAGTTAGC, antisense) for 25 cycles at 93°C for 1 min and 65°C for 2 min.

Real-time Polymerase Chain Reaction:

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: Paragraph title: Quantitative PCR ... Briefly, 100 nM of the genomic Alu forward primer, Alu-F (5'-GCCTCAATAAAGCTTGCCTTGA-3'), 600 nM of HIV-1 gag reverse primer, Gag-R (5'-GCTCTCGCACCCATCTCTCTCC-3'), and 100 ng of cellular genomic DNA were mixed with 1.5 mM MgCl2 , 0.25 mM dNTPs, 0.05 U of Platinum Taq DNA polymerase (Invitrogen) and Taq polymerase reaction buffer (Invitrogen).

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: Library amplification was performed on one half of the Pippin eluate in 1× Phusion GC buffer with 0.2 mM dNTPs, 0.1 μM forward primer (IDT), 0.1 μM reverse primer, 1 unit of Phusion Hot Start II Polymerase (Thermo Fisher Scientific). .. The amplified library was cleaned using a 1× volume of AMPure XP beads and QC was run with the Agilent Bioanalyzer DNA 1000 kit, followed by concentration determination by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems).

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: .. At the end of all these protocols, cDNA synthesis was performed using all of the MeRIP material in a 30μl reaction containing 500ng random primers, 0.5mM dNTPs, 20U RNaseOUT, and 200U of SuperScript II (all from Invitrogen), all according to manufacturer’s instructions. cDNA was diluted 10-fold, and 5μl were used for qPCR using KAPA Sybr Fast Low Rox (KAPA Biosystems). ..

Incubation:

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: After capture, we added 0.5 µl Proteinase K (1 mg/ml) to the samples and incubated them at 60 ˚C for 20 min, followed by 3 min at 98 ˚C. .. In the first PCR, we used 10 µM shDNA-specific primer pair with a universal tag sequence, 1 × PCR buffer, 2.0 mM MgCl2 , 2.5 mM dNTPs, and 1 unit of AmpliTaq Gold DNA Polymerase (Thermo Fisher, Cat. No. 8080241).

Expressing:

Article Title: An essential role for the Zn2+ transporter ZIP7 in B cell development
Article Snippet: 100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 ×107 dilution of ERCC spike in control, comprising a pre-formulated blend of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5 mM dNTPs (Thermo-Fisher), 2.5 μM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice. .. DESeq2 was used for statistical analysis of the differential expression for each gene between WT and ZIP7P198A/P198A mice.

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: At the end of all these protocols, cDNA synthesis was performed using all of the MeRIP material in a 30μl reaction containing 500ng random primers, 0.5mM dNTPs, 20U RNaseOUT, and 200U of SuperScript II (all from Invitrogen), all according to manufacturer’s instructions. cDNA was diluted 10-fold, and 5μl were used for qPCR using KAPA Sybr Fast Low Rox (KAPA Biosystems). .. Results of MeRIP-qPCR for each gene were then calculated using the ΔΔCt approach by using the negative region to normalize both for the expression level of the transcript of interest and for background binding.

Derivative Assay:

Article Title: An essential role for the Zn2+ transporter ZIP7 in B cell development
Article Snippet: .. 100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 ×107 dilution of ERCC spike in control, comprising a pre-formulated blend of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5 mM dNTPs (Thermo-Fisher), 2.5 μM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice. ..

Concentration Assay:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: Library amplification was performed on one half of the Pippin eluate in 1× Phusion GC buffer with 0.2 mM dNTPs, 0.1 μM forward primer (IDT), 0.1 μM reverse primer, 1 unit of Phusion Hot Start II Polymerase (Thermo Fisher Scientific). .. The amplified library was cleaned using a 1× volume of AMPure XP beads and QC was run with the Agilent Bioanalyzer DNA 1000 kit, followed by concentration determination by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems).

Article Title: Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms
Article Snippet: .. Linear amplification reactions were set up using approximately 4 ng of genomic DNA, and final concentrations of 1× Buffer with 15 mM MgCl2 (Qiagen Inc., Valencia, California), 0.125 mM dNTPs (Fermentas Inc., Geln Burnie, Maryland), 0.04 Units/µl HotStar Taq DNA polymerase (Qiagen Inc., Valencia, California), and GC-rich primers at a final concentration of 0.6 µM, or 1× Buffer (Qiagen Inc., Valencia, California), 0.1 mM dNTPs (Fermentas Inc., Geln Burnie, Maryland), 100 ng/µl BSA (New England Biolabs, Ipswich, Massachusetts), 2 µl of Pfu Ultra II Fusion HS DNA polymerase (Qiagen Inc., Valencia, California), 0.2 µM GC-rich primers (Sigma Genosys, The Woodlands, Texas), and nuclease free water in a final reaction volume of 50 µl. ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †
Article Snippet: Paragraph title: PCR and RT-PCR analysis. ... Total viral RNA was annealed to an oligonucleotide (5′-GACTGCGAATCGTTCTAG-3′, antisense) complementary to sequences in the gag open reading frame at 75°C for 10 min and placed on ice, and cDNA was made by using the supplied buffers, 0.2 mM dNTPs, and M-MLV RT (Life Technologies) at 37°C for 60 min. Each cDNA reaction was assayed by PCR for the internal control (IC) cDNA (reverse transcribed from IC RNA) by using a 32 P-labeled oligonucleotide specific for pGem4z sequences (5′-GGGAGACAAGCTTGCATGCCTG, sense) and an unlabeled HIV-1-specific oligonucleotide (5′-GCAGTGGGTTCCCTAGTTAGC, antisense) for 25 cycles at 93°C for 1 min and 65°C for 2 min.

Digital PCR:

Article Title: Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type
Article Snippet: Paragraph title: Single cell reverse transcription and digital PCR ... : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No.

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: Paragraph title: Single-cell reverse transcription and dPCR of rat cortical neurons ... : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No.

Sequencing:

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: These were used to target the ISEcp 1 insertion sequence present upstream of the bla CMY -42 . .. The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase.

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: Paragraph title: Library prep and sequencing ... Library amplification was performed on one half of the Pippin eluate in 1× Phusion GC buffer with 0.2 mM dNTPs, 0.1 μM forward primer (IDT), 0.1 μM reverse primer, 1 unit of Phusion Hot Start II Polymerase (Thermo Fisher Scientific).

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: These were used to target the IS Ecp 1 insertion sequence present upstream of the bla CMY -42 . .. The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase.

Article Title: Gene expression variability across cells and species shapes innate immunity
Article Snippet: .. Library preparation from full-length RNA from single cells and sequencing Sorted plates were processed according to the Smart-seq2 protocol : Oligo-dT primer (IDT), dNTPs (ThermoFisher, Cat Number 10319879) and ERCC RNA Spike-In Mix (1:25,000,000 final dilution, Ambion, Cat Number 4456740) were added to each well, and Reverse Transcription (using 50U SmartScribe, Clontech Cat Number 639538) and PCR were performed following the original protocol with 25 PCR cycles. cDNA libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, Cat Number FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1). .. Quality Checks on cDNA were done using a Bioanalyser 2100 (Agilent Technologies).

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: .. In the first PCR, we used 10 µM shDNA-specific primer pair with a universal tag sequence, 1 × PCR buffer, 2.0 mM MgCl2 , 2.5 mM dNTPs, and 1 unit of AmpliTaq Gold DNA Polymerase (Thermo Fisher, Cat. No. 8080241). .. In the second PCR, 1 µl from the first amplification reaction was used as a template with primers complementary to the universal tag sequence.

Binding Assay:

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: At the end of all these protocols, cDNA synthesis was performed using all of the MeRIP material in a 30μl reaction containing 500ng random primers, 0.5mM dNTPs, 20U RNaseOUT, and 200U of SuperScript II (all from Invitrogen), all according to manufacturer’s instructions. cDNA was diluted 10-fold, and 5μl were used for qPCR using KAPA Sybr Fast Low Rox (KAPA Biosystems). .. Results of MeRIP-qPCR for each gene were then calculated using the ΔΔCt approach by using the negative region to normalize both for the expression level of the transcript of interest and for background binding.

Transmission Electron Microscopy:

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: Paragraph title: Characterization of Genetic Environment of bla TEM-1 , bla CTX-M-15 , bla CMY -42 , Integrons, and Flanking Regions ... The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase.

Multiplexing:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: These adaptors contain barcodes to facilitate sample multiplexing during sequencing. .. Library amplification was performed on one half of the Pippin eluate in 1× Phusion GC buffer with 0.2 mM dNTPs, 0.1 μM forward primer (IDT), 0.1 μM reverse primer, 1 unit of Phusion Hot Start II Polymerase (Thermo Fisher Scientific).

RNA Sequencing Assay:

Article Title: An essential role for the Zn2+ transporter ZIP7 in B cell development
Article Snippet: Paragraph title: RNA sequencing ... 100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 ×107 dilution of ERCC spike in control, comprising a pre-formulated blend of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5 mM dNTPs (Thermo-Fisher), 2.5 μM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice.

Methylation:

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: Paragraph title: m6A nuclear-enriched methylated RNA immunoprecipitation ... At the end of all these protocols, cDNA synthesis was performed using all of the MeRIP material in a 30μl reaction containing 500ng random primers, 0.5mM dNTPs, 20U RNaseOUT, and 200U of SuperScript II (all from Invitrogen), all according to manufacturer’s instructions. cDNA was diluted 10-fold, and 5μl were used for qPCR using KAPA Sybr Fast Low Rox (KAPA Biosystems).

Isolation:

Article Title: An essential role for the Zn2+ transporter ZIP7 in B cell development
Article Snippet: Cell suspensions from freshly isolated BM were obtained from straight chimeras, 5 WT and 5 P198A/P198A mutants, 8 weeks after reconstitution, and sorted using a FacsARIA III (BD), using the following gating strategy: viable, B220+ CD43+ CD45.1− CD45.2+ CD24+ BP1− (FrB/pro-B); B220+ CD43− CD45.1− CD45.2+IgM− IgD− (FrD/late preB) and viable, B220+ CD43− CD45.1− CD45.2+ IgM+ IgD− (FrE/Immature). .. 100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 ×107 dilution of ERCC spike in control, comprising a pre-formulated blend of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5 mM dNTPs (Thermo-Fisher), 2.5 μM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice.

Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †
Article Snippet: Supernatant containing 60 ng of p24 Ag was treated with TriPure reagent (Roche Diagnostics), 0.5 pg of in vitro-synthesized HIV-1 RNA was added, and total virion RNA was isolated according to the manufacturer’s recommendations. .. Total viral RNA was annealed to an oligonucleotide (5′-GACTGCGAATCGTTCTAG-3′, antisense) complementary to sequences in the gag open reading frame at 75°C for 10 min and placed on ice, and cDNA was made by using the supplied buffers, 0.2 mM dNTPs, and M-MLV RT (Life Technologies) at 37°C for 60 min. Each cDNA reaction was assayed by PCR for the internal control (IC) cDNA (reverse transcribed from IC RNA) by using a 32 P-labeled oligonucleotide specific for pGem4z sequences (5′-GGGAGACAAGCTTGCATGCCTG, sense) and an unlabeled HIV-1-specific oligonucleotide (5′-GCAGTGGGTTCCCTAGTTAGC, antisense) for 25 cycles at 93°C for 1 min and 65°C for 2 min.

Mouse Assay:

Article Title: An essential role for the Zn2+ transporter ZIP7 in B cell development
Article Snippet: 100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 ×107 dilution of ERCC spike in control, comprising a pre-formulated blend of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5 mM dNTPs (Thermo-Fisher), 2.5 μM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice. .. DESeq2 was used for statistical analysis of the differential expression for each gene between WT and ZIP7P198A/P198A mice.

Polymerase Chain Reaction:

Article Title: Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type
Article Snippet: : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No. .. The reverse transcription reaction mix was stored at -20°C until PCR amplification.

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No. .. The reverse transcription reaction mix was stored at −20 °C until PCR amplification.

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: The first round PCR was performed in a 25 μl reaction mix as described previously [ ]. .. Briefly, 100 nM of the genomic Alu forward primer, Alu-F (5'-GCCTCAATAAAGCTTGCCTTGA-3'), 600 nM of HIV-1 gag reverse primer, Gag-R (5'-GCTCTCGCACCCATCTCTCTCC-3'), and 100 ng of cellular genomic DNA were mixed with 1.5 mM MgCl2 , 0.25 mM dNTPs, 0.05 U of Platinum Taq DNA polymerase (Invitrogen) and Taq polymerase reaction buffer (Invitrogen).

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: .. The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase. ..

Article Title: Effect of Nicotine on CYP2B1 Expression in a Glioma Animal Model and Analysis of CYP2B6 Expression in Pediatric Gliomas
Article Snippet: .. All reactions were performed in a final volume of 50 μL using PCR buffer (1×; BioTecMol, Mexico City, Mexico), magnesium chloride (liver: 1.5 mM; brain: 4 mM; BioTecMol), dNTPs (liver: 200 μM; brain: 400 μM; Thermo Fisher Scientific), oligonucleotides for Cyp2b1/2 (liver: 300 nM; brain: 600 nM), Gapdh (liver: 120 nM; brain: 200 nM) and 18s (200 nM), Taq polymerase (2.5 U; BioTecMol) and 1 μg of cDNA for each sample. ..

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: .. The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase. ..

Article Title: Gene expression variability across cells and species shapes innate immunity
Article Snippet: .. Library preparation from full-length RNA from single cells and sequencing Sorted plates were processed according to the Smart-seq2 protocol : Oligo-dT primer (IDT), dNTPs (ThermoFisher, Cat Number 10319879) and ERCC RNA Spike-In Mix (1:25,000,000 final dilution, Ambion, Cat Number 4456740) were added to each well, and Reverse Transcription (using 50U SmartScribe, Clontech Cat Number 639538) and PCR were performed following the original protocol with 25 PCR cycles. cDNA libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, Cat Number FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1). .. Quality Checks on cDNA were done using a Bioanalyser 2100 (Agilent Technologies).

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: .. In the first PCR, we used 10 µM shDNA-specific primer pair with a universal tag sequence, 1 × PCR buffer, 2.0 mM MgCl2 , 2.5 mM dNTPs, and 1 unit of AmpliTaq Gold DNA Polymerase (Thermo Fisher, Cat. No. 8080241). .. In the second PCR, 1 µl from the first amplification reaction was used as a template with primers complementary to the universal tag sequence.

Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †
Article Snippet: .. Total viral RNA was annealed to an oligonucleotide (5′-GACTGCGAATCGTTCTAG-3′, antisense) complementary to sequences in the gag open reading frame at 75°C for 10 min and placed on ice, and cDNA was made by using the supplied buffers, 0.2 mM dNTPs, and M-MLV RT (Life Technologies) at 37°C for 60 min. Each cDNA reaction was assayed by PCR for the internal control (IC) cDNA (reverse transcribed from IC RNA) by using a 32 P-labeled oligonucleotide specific for pGem4z sequences (5′-GGGAGACAAGCTTGCATGCCTG, sense) and an unlabeled HIV-1-specific oligonucleotide (5′-GCAGTGGGTTCCCTAGTTAGC, antisense) for 25 cycles at 93°C for 1 min and 65°C for 2 min. ..

Lysis:

Article Title: An essential role for the Zn2+ transporter ZIP7 in B cell development
Article Snippet: .. 100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 ×107 dilution of ERCC spike in control, comprising a pre-formulated blend of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5 mM dNTPs (Thermo-Fisher), 2.5 μM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice. ..

Nested PCR:

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: Two-step nested PCR assays were used for quantitative HIV-1 DNA integration analysis. .. Briefly, 100 nM of the genomic Alu forward primer, Alu-F (5'-GCCTCAATAAAGCTTGCCTTGA-3'), 600 nM of HIV-1 gag reverse primer, Gag-R (5'-GCTCTCGCACCCATCTCTCTCC-3'), and 100 ng of cellular genomic DNA were mixed with 1.5 mM MgCl2 , 0.25 mM dNTPs, 0.05 U of Platinum Taq DNA polymerase (Invitrogen) and Taq polymerase reaction buffer (Invitrogen).

Chloramphenicol Acetyltransferase Assay:

Article Title: Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type
Article Snippet: .. : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No. .. : 10297018, 1.5 μl 5X first-strand buffer, 0.45 μl 0.1 mol/L DTT, 0.45 μl RNase inhibitor (Thermo Fisher, Cat.No.

Article Title: Intelligent image-based in situ single-cell isolation
Article Snippet: .. : SCP-250), 0.45 μl TaqMan Assays (Thermo Fisher), 0.45 μl 10 mM dNTPs (Thermo Fisher, Cat.No. .. : 10297018, 1.5 μl 5× first-strand buffer, 0.45 μl 0.1 mol/l DTT, 0.45 μl RNase inhibitor (Thermo Fisher, Cat.No.

Purification:

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase. .. The PCR amplicons were purified using Hi-YieldTM extraction kit (RBC Bioscience, New Taipei City, Taiwan) following manufacturer’s instructions and sequenced at a commercial facility using Sanger sequencing (Invitrogen BioServices India Pvt.

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase. .. The PCR amplicons were purified using Hi-YieldTM extraction kit (RBC Bioscience, New Taipei City, Taiwan) following manufacturer’s instructions and sequenced at a commercial facility using Sanger sequencing (Invitrogen BioServices India Pvt.

Article Title: Gene expression variability across cells and species shapes innate immunity
Article Snippet: Library preparation from full-length RNA from single cells and sequencing Sorted plates were processed according to the Smart-seq2 protocol : Oligo-dT primer (IDT), dNTPs (ThermoFisher, Cat Number 10319879) and ERCC RNA Spike-In Mix (1:25,000,000 final dilution, Ambion, Cat Number 4456740) were added to each well, and Reverse Transcription (using 50U SmartScribe, Clontech Cat Number 639538) and PCR were performed following the original protocol with 25 PCR cycles. cDNA libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, Cat Number FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1). .. Libraries were quantified using the LightCycler 480 (Roche), pooled and purified using AMPure XP beads (Beckman Coulter) with Hamilton 384 head robot (Hamilton Robotics).

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: For m6A MeRIP on mRNA, poly-A RNA was purified from 75μg of total RNA using the Dynabeads mRNA Purification Kit, and 2.5μg of the resulting mRNA were used for chemical fragmentation and subsequent MeRIP with 1μg of anti-m6A antibody. .. At the end of all these protocols, cDNA synthesis was performed using all of the MeRIP material in a 30μl reaction containing 500ng random primers, 0.5mM dNTPs, 20U RNaseOUT, and 200U of SuperScript II (all from Invitrogen), all according to manufacturer’s instructions. cDNA was diluted 10-fold, and 5μl were used for qPCR using KAPA Sybr Fast Low Rox (KAPA Biosystems).

Software:

Article Title: An essential role for the Zn2+ transporter ZIP7 in B cell development
Article Snippet: 100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 ×107 dilution of ERCC spike in control, comprising a pre-formulated blend of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5 mM dNTPs (Thermo-Fisher), 2.5 μM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice. .. Briefly, reads were aligned to the mm10 mouse genome using hisat2 version 2.1.0 , and reads were quantified over feature annotations (ensemble81) using featureCounts program version 1.4.6 within the Subread software package .

Electrophoresis:

Article Title: Effect of Nicotine on CYP2B1 Expression in a Glioma Animal Model and Analysis of CYP2B6 Expression in Pediatric Gliomas
Article Snippet: All reactions were performed in a final volume of 50 μL using PCR buffer (1×; BioTecMol, Mexico City, Mexico), magnesium chloride (liver: 1.5 mM; brain: 4 mM; BioTecMol), dNTPs (liver: 200 μM; brain: 400 μM; Thermo Fisher Scientific), oligonucleotides for Cyp2b1/2 (liver: 300 nM; brain: 600 nM), Gapdh (liver: 120 nM; brain: 200 nM) and 18s (200 nM), Taq polymerase (2.5 U; BioTecMol) and 1 μg of cDNA for each sample. .. The amplicons were analyzed by electrophoresis in 2.5% agarose gels, which were stained with 1 μg/mL ethidium bromide and visualized with a UV Transilluminator (BioDoc-It Imagen System UVP M-20, Upland, CA, USA).

Multiplex Assay:

Article Title: Effect of Nicotine on CYP2B1 Expression in a Glioma Animal Model and Analysis of CYP2B6 Expression in Pediatric Gliomas
Article Snippet: Paragraph title: 4.7. Multiplex PCR ... All reactions were performed in a final volume of 50 μL using PCR buffer (1×; BioTecMol, Mexico City, Mexico), magnesium chloride (liver: 1.5 mM; brain: 4 mM; BioTecMol), dNTPs (liver: 200 μM; brain: 400 μM; Thermo Fisher Scientific), oligonucleotides for Cyp2b1/2 (liver: 300 nM; brain: 600 nM), Gapdh (liver: 120 nM; brain: 200 nM) and 18s (200 nM), Taq polymerase (2.5 U; BioTecMol) and 1 μg of cDNA for each sample.

Selection:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: After AMPure XP cleanup, one half of the pooled library was run on the Pippin Size Selection Instrument (Sage Sciences) to select for 200 bp fragments. .. Library amplification was performed on one half of the Pippin eluate in 1× Phusion GC buffer with 0.2 mM dNTPs, 0.1 μM forward primer (IDT), 0.1 μM reverse primer, 1 unit of Phusion Hot Start II Polymerase (Thermo Fisher Scientific).

Sample Prep:

Article Title: Gene expression variability across cells and species shapes innate immunity
Article Snippet: .. Library preparation from full-length RNA from single cells and sequencing Sorted plates were processed according to the Smart-seq2 protocol : Oligo-dT primer (IDT), dNTPs (ThermoFisher, Cat Number 10319879) and ERCC RNA Spike-In Mix (1:25,000,000 final dilution, Ambion, Cat Number 4456740) were added to each well, and Reverse Transcription (using 50U SmartScribe, Clontech Cat Number 639538) and PCR were performed following the original protocol with 25 PCR cycles. cDNA libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, Cat Number FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1). .. Quality Checks on cDNA were done using a Bioanalyser 2100 (Agilent Technologies).

Immunoprecipitation:

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: Paragraph title: m6A nuclear-enriched methylated RNA immunoprecipitation ... At the end of all these protocols, cDNA synthesis was performed using all of the MeRIP material in a 30μl reaction containing 500ng random primers, 0.5mM dNTPs, 20U RNaseOUT, and 200U of SuperScript II (all from Invitrogen), all according to manufacturer’s instructions. cDNA was diluted 10-fold, and 5μl were used for qPCR using KAPA Sybr Fast Low Rox (KAPA Biosystems).

Fractionation:

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: For m6A MeRIP on total RNA, the protocol just described was followed exactly, with the exception that the subcellular fractionation step was bypassed, and that total RNA was extracted from 5x106 cells. .. At the end of all these protocols, cDNA synthesis was performed using all of the MeRIP material in a 30μl reaction containing 500ng random primers, 0.5mM dNTPs, 20U RNaseOUT, and 200U of SuperScript II (all from Invitrogen), all according to manufacturer’s instructions. cDNA was diluted 10-fold, and 5μl were used for qPCR using KAPA Sybr Fast Low Rox (KAPA Biosystems).

Staining:

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase. .. PCR amplicons were electrophoresed on 1% agarose gels at 80 V, stained with ethidium bromide and visualized using a UV tansilluminator.

Article Title: Effect of Nicotine on CYP2B1 Expression in a Glioma Animal Model and Analysis of CYP2B6 Expression in Pediatric Gliomas
Article Snippet: All reactions were performed in a final volume of 50 μL using PCR buffer (1×; BioTecMol, Mexico City, Mexico), magnesium chloride (liver: 1.5 mM; brain: 4 mM; BioTecMol), dNTPs (liver: 200 μM; brain: 400 μM; Thermo Fisher Scientific), oligonucleotides for Cyp2b1/2 (liver: 300 nM; brain: 600 nM), Gapdh (liver: 120 nM; brain: 200 nM) and 18s (200 nM), Taq polymerase (2.5 U; BioTecMol) and 1 μg of cDNA for each sample. .. The amplicons were analyzed by electrophoresis in 2.5% agarose gels, which were stained with 1 μg/mL ethidium bromide and visualized with a UV Transilluminator (BioDoc-It Imagen System UVP M-20, Upland, CA, USA).

Article Title: Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment
Article Snippet: The 25 μl PCR reaction mixture prepared contained 2.5 μl of 1× buffer, 200 μM of each dNTPs (Thermo Fisher Scientific, Waltham, MA, United States), 20 pmol of each forward and reverse primers, 6 μl of template DNA and 1 U of Taq DNA polymerase. .. PCR amplicons were electrophoresed on 1% agarose gels at 80 V, stained with ethidium bromide and visualized using a UV tansilluminator.

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