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Thermo Fisher dntp
Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp <t>DNA</t> size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each <t>dNTP,</t> 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.
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Images

1) Product Images from "Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline"

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

Journal: AoB Plants

doi: 10.1093/aobpla/pls033

Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.
Figure Legend Snippet: Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.

Techniques Used: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.
Figure Legend Snippet: Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.

Techniques Used: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

2) Product Images from "Direct Cell Lysis for Single-Cell Gene Expression Profiling"

Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2013.00274

Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
Figure Legend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

Techniques Used: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
Figure Legend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

Techniques Used: Lysis, Polymerase Chain Reaction

Related Articles

Amplification:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: Paragraph title: PCR amplification and sequence analysis ... All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: Paragraph title: Amplification of Plasmodium DNA ... This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step.

Article Title: Development of a Sensitive Induction-Based Magnetic Nanoparticle Biodetection Method
Article Snippet: .. The RCA process was conducted as follows: 5 nM of padlock probes (Biomers, Ulm, Germany) were hybridized and ligated, templated by 15 nM synthetic Vibrio cholerae target DNA in a solution consisting of 1× Phi29 buffer, 0.25 mM ATP and 5 mU/μL T4 ligase at 37 °C for 15 min. Circular padlock probes were amplified in 1× Phi29 buffer, 0.1 mM dNTP, 0.12 μg/μL BSA, 4 mU/μL Phi29 polymerase at 37 °C for 1 h, followed by enzymatic inactivation at 65 °C for 5 min (Phi29 buffer, ATP, T4 ligase, dNTP, BSA and Phi29 polymerase from Thermo Fisher Scientific, Waltham, MA, USA). .. A hybridization mix consisting of 100 mM Tris-HCl (pH 8), 100 mM EDTA, 0.5% Tween-20, 2.5 M NaCl was added to the RCA coil solution.

Article Title: Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
Article Snippet: .. A mixture of 1 µL SMARTscribe reverse transcriptase (Clontech Laboratories, Inc), 1 µL custom designed TS oligo (12 µM, Integrated DNA Technologies ( )), 0.3 µL MgCl2 (200 mM, Sigma), 0.5 µL RNase inhibitor (Neb), 1 µL dNTP (10 mM each, Thermo), 0.25 µL DTT (100 mM, Invitrogen) were incubated at 42°C for 90 min, which was followed by enzyme inactivation at 70°C for 10 min. A mixture of 29 µL water, 5 µL Advantage2 taq polymerase buffer, 2 µL dNTP (10 mM each, Thermo), 2 µL custom-designed PCR primer (12 µM, Integrated DNA Technologies ( )), and 2 µL Advantage2 taq polymerase was directly added to the reverse transcription product and the amplification was performed for 19 cycles. .. The amplification product was purified using Ampure XP beads (Beckman-Coulter).

Article Title: Tissue-specific mutation accumulation in human adult stem cells during life
Article Snippet: We evaluated our mutation filtering procedure by independent validations of 374 pre-selected positions that were either discarded or passed during filtering using amplicon-based next-generation sequencing. .. These regions were PCR-amplified for both the organoid cultures and reference samples of donor 5 and 6, using 5 ng genomic DNA, 1× PCR Gold Buffer (Life Technologies), 1.5 mM MgCl2 , 0.2 mM of each dNTP and 1 unit of AmpliTaq Gold (Life Technologies) in a final volume of 10 μl.

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA). .. The templates were subjected to an amplification program with a peqSTAR thermal cycler (PEQLAB GmbH, Erlangen, Germany) consisting of an initial step at 96°C for 15 minutes, 35 amplification cycles (94°C for 45 seconds, 55°C at primary PCR and 58°C at secondary PCR for 45 seconds and 72°C for 60 seconds) followed by one cycle of 10 minutes at 72°C according to .

Synthesized:

Article Title: Multiplex single-molecule interaction profiling of DNA barcoded proteins
Article Snippet: .. To generate mRNA–cDNA hybrids, cDNAs were synthesized by incubating ~0.10 μM mRNA, 1 μM 5’-acrydite and desthiobiotin-modified primer, 0.5 mM each dNTP, 10 U/μL SuperScript III, 2 U/μL RNaseOUT (Invitrogen) and 5 mM dithiothreitol (DTT) in a buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 5 mM MgCl2 ) at 50°C for 30 min. .. Synthesized mRNA–cDNA hybrids serve as templates for ribosome display using a PURExpress Δ ribosome kit (New England Biolabs).

Quantitative RT-PCR:

Article Title: Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis
Article Snippet: Paragraph title: RNA isolation and QRT-PCR ... RNA (250 ng) was reverse-transcribed using SuperScript II Reverse Transcriptase, 5x First-Strand buffer, DTT, RNaseOUT Recombinant Ribonuclease Inhibitor, Oligo(dT)12–18, and dNTP (Life Technologies, Burlington, ON).

Real-time Polymerase Chain Reaction:

Article Title: Association between Polymorphisms in Inflammatory Response-Related Genes and the Susceptibility, Progression and Prognosis of the Diffuse Histological Subtype of Gastric Cancer
Article Snippet: PCR was carried out in 25 µL of reaction volume containing 100 ng of genomic DNA using 50 mmol/L of KCl, 10 mmol/L of TrisHCl (pH = 8.5), 1.0 mmol/L of MgCl2 , 0.4 mmol/L of dNTP, 0.16 µmol/L of each primer and 1 U of recombinant Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA). .. The remaining SNPs were genotyped by TaqMan® assays with Real-Time PCR method: rs699947, rs833061, rs2010963 and rs3025039 ( VEGFA ); rs689466 and rs5275 ( COX-2 ); rs6917 ( PHB ), and p.R377H ( TP53 ).

Article Title: Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis
Article Snippet: RNA (250 ng) was reverse-transcribed using SuperScript II Reverse Transcriptase, 5x First-Strand buffer, DTT, RNaseOUT Recombinant Ribonuclease Inhibitor, Oligo(dT)12–18, and dNTP (Life Technologies, Burlington, ON). .. Gene expression was determined using qPCR reaction with Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies, Burlington, ON) and performed on the CFX96 Real-time PCR Detection System (Bio-rad Laboratories, Mississauga, ON).

Incubation:

Article Title: Development of a Sensitive Induction-Based Magnetic Nanoparticle Biodetection Method
Article Snippet: The MNPs were thereafter resuspended in 1× Wtw buffer and incubated with 10 µM biotin-conjugated oligonucleotides for 30 min at room temperature. .. The RCA process was conducted as follows: 5 nM of padlock probes (Biomers, Ulm, Germany) were hybridized and ligated, templated by 15 nM synthetic Vibrio cholerae target DNA in a solution consisting of 1× Phi29 buffer, 0.25 mM ATP and 5 mU/μL T4 ligase at 37 °C for 15 min. Circular padlock probes were amplified in 1× Phi29 buffer, 0.1 mM dNTP, 0.12 μg/μL BSA, 4 mU/μL Phi29 polymerase at 37 °C for 1 h, followed by enzymatic inactivation at 65 °C for 5 min (Phi29 buffer, ATP, T4 ligase, dNTP, BSA and Phi29 polymerase from Thermo Fisher Scientific, Waltham, MA, USA).

Article Title: Multiplex single-molecule interaction profiling of DNA barcoded proteins
Article Snippet: To generate mRNA–cDNA hybrids, cDNAs were synthesized by incubating ~0.10 μM mRNA, 1 μM 5’-acrydite and desthiobiotin-modified primer, 0.5 mM each dNTP, 10 U/μL SuperScript III, 2 U/μL RNaseOUT (Invitrogen) and 5 mM dithiothreitol (DTT) in a buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 5 mM MgCl2 ) at 50°C for 30 min. .. Typically, a 100 μL IVT reaction with ~0.40 μM mRNA–cDNA hybrids and ~0.30 μM ribosome was incubated at 37°C for 30 min, quenched by addition of 100 μL ice-cold buffer HKM (50 mM HEPES, pH 7.0, 250 mM KOAc, 25 mM Mg(OAc)2 , 0.25 U/mL RNasin (Promega), 0.5 mg/mL chloramphenicol, 5 mM 2-mercaptoethanol and 0.1% (v/v) Tween 20) and centrifuged (14,000 g, 4°C) for 10 min to remove insoluble components.

Article Title: Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
Article Snippet: .. A mixture of 1 µL SMARTscribe reverse transcriptase (Clontech Laboratories, Inc), 1 µL custom designed TS oligo (12 µM, Integrated DNA Technologies ( )), 0.3 µL MgCl2 (200 mM, Sigma), 0.5 µL RNase inhibitor (Neb), 1 µL dNTP (10 mM each, Thermo), 0.25 µL DTT (100 mM, Invitrogen) were incubated at 42°C for 90 min, which was followed by enzyme inactivation at 70°C for 10 min. A mixture of 29 µL water, 5 µL Advantage2 taq polymerase buffer, 2 µL dNTP (10 mM each, Thermo), 2 µL custom-designed PCR primer (12 µM, Integrated DNA Technologies ( )), and 2 µL Advantage2 taq polymerase was directly added to the reverse transcription product and the amplification was performed for 19 cycles. .. The amplification product was purified using Ampure XP beads (Beckman-Coulter).

Expressing:

Article Title: Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis
Article Snippet: RNA (250 ng) was reverse-transcribed using SuperScript II Reverse Transcriptase, 5x First-Strand buffer, DTT, RNaseOUT Recombinant Ribonuclease Inhibitor, Oligo(dT)12–18, and dNTP (Life Technologies, Burlington, ON). .. Gene expression was determined using qPCR reaction with Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies, Burlington, ON) and performed on the CFX96 Real-time PCR Detection System (Bio-rad Laboratories, Mississauga, ON).

Modification:

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: Total DNA, representing 50% of the sucrose purified water concentrates, was extracted by the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the modified protocol of with additional processing of 10 freeze–thaw cycles after the resuspension step in lysis buffer, supplied from the manufacturer. .. Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA).

Hybridization:

Article Title: Development of a Sensitive Induction-Based Magnetic Nanoparticle Biodetection Method
Article Snippet: The RCA process was conducted as follows: 5 nM of padlock probes (Biomers, Ulm, Germany) were hybridized and ligated, templated by 15 nM synthetic Vibrio cholerae target DNA in a solution consisting of 1× Phi29 buffer, 0.25 mM ATP and 5 mU/μL T4 ligase at 37 °C for 15 min. Circular padlock probes were amplified in 1× Phi29 buffer, 0.1 mM dNTP, 0.12 μg/μL BSA, 4 mU/μL Phi29 polymerase at 37 °C for 1 h, followed by enzymatic inactivation at 65 °C for 5 min (Phi29 buffer, ATP, T4 ligase, dNTP, BSA and Phi29 polymerase from Thermo Fisher Scientific, Waltham, MA, USA). .. A hybridization mix consisting of 100 mM Tris-HCl (pH 8), 100 mM EDTA, 0.5% Tween-20, 2.5 M NaCl was added to the RCA coil solution.

Countercurrent Chromatography:

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step. .. The primers used were: P1UP (F): 5′ TCC ATT AAT CAA GAA CGA AAG TTA AG 3′ P2 (R): 5′ GAA CCC AAA GAC TTT GAT TTC TCA T 3′ P1 (F): 5′ ACG ATC AGA TAC CGT CGT AAT CTT 3′ V1 (R): 5′ CAA TCT AAG AAT AAA CTC CGA AGA GAA A 3′ F2 (R): 5′ CAA TCT AAA AGT CAC CTC GAA AGA TG 3′ M1 (R): 5′ GGA AGC TAT CTA AAA GAA ACA CTC ATA T 3′ The amplified product was run on 2% agarose gel at 80 V for 40 min.

Ligation:

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: To 15 μl ligation reaction, an additional 0.5 μl 10 μM MPX_RT primer were added, reactions were denatured at to 70°C for 1 minute, then placed on ice. .. RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C.

Serial Dilution:

Article Title: Facile profiling of molecular heterogeneity by microfluidic digital melt
Article Snippet: All MethyLight assays were performed using 10× Master Mix to yield a final volume of 25 μl and final working concentrations of 16.6 mM (NH4 )2 SO4 , 67 mM tris (pH 8.8), 6.7 mM MgCl2 10 mM β-mercaptoethanol, 200 μM of each dNTP, and Platinum Taq polymerase (0.04 U/μl) (Thermo Fisher Scientific). .. Each assay was validated by serial dilution to create a percent methylated reference standard curve ranging from 0.1 to 100% of bisulfite-converted methylated to unmethylated control genomic DNA (Promega).

Article Title: Facile profiling of molecular heterogeneity by microfluidic digital melt
Article Snippet: MSP/MethyLight and ddPCR-MPS/MethyLight All MethyLight assays were performed using 10× Master Mix to yield a final volume of 25 μl and final working concentrations of 16.6 mM (NH4 )2 SO4 , 67 mM tris (pH 8.8), 6.7 mM MgCl2 10 mM β-mercaptoethanol, 200 μM of each dNTP, and Platinum Taq polymerase (0.04 U/μl) (Thermo Fisher Scientific). .. Each assay was validated by serial dilution to create a percent methylated reference standard curve ranging from 0.1 to 100% of bisulfite-converted methylated to unmethylated control genomic DNA (Promega).

Sequencing:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: Paragraph title: PCR amplification and sequence analysis ... All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: To prepare sequencing libraries, 0.5 μl 9 μM of a 5’-adenylated sRNA3'MPX adapter /5Phos/AG ATC GGA AGA GCA CAC GTC TGA /3AmMO/ (IDT, desalted; adenylated with Mth ligase (NEB) according to the manufacturer's instructions, phenol-chloroform/chloroform-extracted, ethanol-precipitated with glycogen and dissolved in water at 9 μM) were heat-denatured together with the RNA for 2 minutes at 70°C, and ligated with 100 U truncated T4RNA ligase 2 K227Q (NEB) in 10 μl 1× T4 RNA ligase buffer without ATP, containing 10 U SUPERase-In and 15% PEG8000 for 2 hours at 16°C. .. RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C.

Article Title: Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
Article Snippet: Paragraph title: Library Preparation and Sequencing ... A mixture of 1 µL SMARTscribe reverse transcriptase (Clontech Laboratories, Inc), 1 µL custom designed TS oligo (12 µM, Integrated DNA Technologies ( )), 0.3 µL MgCl2 (200 mM, Sigma), 0.5 µL RNase inhibitor (Neb), 1 µL dNTP (10 mM each, Thermo), 0.25 µL DTT (100 mM, Invitrogen) were incubated at 42°C for 90 min, which was followed by enzyme inactivation at 70°C for 10 min. A mixture of 29 µL water, 5 µL Advantage2 taq polymerase buffer, 2 µL dNTP (10 mM each, Thermo), 2 µL custom-designed PCR primer (12 µM, Integrated DNA Technologies ( )), and 2 µL Advantage2 taq polymerase was directly added to the reverse transcription product and the amplification was performed for 19 cycles.

Affinity Purification:

Article Title: Multiplex single-molecule interaction profiling of DNA barcoded proteins
Article Snippet: To generate mRNA–cDNA hybrids, cDNAs were synthesized by incubating ~0.10 μM mRNA, 1 μM 5’-acrydite and desthiobiotin-modified primer, 0.5 mM each dNTP, 10 U/μL SuperScript III, 2 U/μL RNaseOUT (Invitrogen) and 5 mM dithiothreitol (DTT) in a buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 5 mM MgCl2 ) at 50°C for 30 min. .. PRMC complexes, always kept on ice or in cold room, were subjected to two-step Flag tag and desthiobiotin tag affinity purification to enrich full-length and barcoded target proteins.

Binding Assay:

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. .. Complementary DNA was isolated by adding 35 μl AMPure XL beads (Beckman), binding and washing according to manufacturer's instructions and dissolved in 40 μl TET (0.1 % Tween 20/TE).

Cellular Antioxidant Activity Assay:

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step. .. The primers used were: P1UP (F): 5′ TCC ATT AAT CAA GAA CGA AAG TTA AG 3′ P2 (R): 5′ GAA CCC AAA GAC TTT GAT TTC TCA T 3′ P1 (F): 5′ ACG ATC AGA TAC CGT CGT AAT CTT 3′ V1 (R): 5′ CAA TCT AAG AAT AAA CTC CGA AGA GAA A 3′ F2 (R): 5′ CAA TCT AAA AGT CAC CTC GAA AGA TG 3′ M1 (R): 5′ GGA AGC TAT CTA AAA GAA ACA CTC ATA T 3′ The amplified product was run on 2% agarose gel at 80 V for 40 min.

Staining:

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step. .. The gel was stained using ethidium bromide, and the bands were visualized under a UV transilluminator.

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA). .. PCR products were analyzed in 1.5% agarose gel stained with ethidium bromide solution (1 μg/ml) and visualized under UV light.

DNA Extraction:

Article Title: Association between Polymorphisms in Inflammatory Response-Related Genes and the Susceptibility, Progression and Prognosis of the Diffuse Histological Subtype of Gastric Cancer
Article Snippet: Paragraph title: 2.3. Blood Sample Collection and Processing, DNA Extraction and Genotyping Analyses ... PCR was carried out in 25 µL of reaction volume containing 100 ng of genomic DNA using 50 mmol/L of KCl, 10 mmol/L of TrisHCl (pH = 8.5), 1.0 mmol/L of MgCl2 , 0.4 mmol/L of dNTP, 0.16 µmol/L of each primer and 1 U of recombinant Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA).

RNA Sequencing Assay:

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: Paragraph title: PolyA RNA-Seq ... RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C.

Methylation:

Article Title: Facile profiling of molecular heterogeneity by microfluidic digital melt
Article Snippet: All MethyLight assays were performed using 10× Master Mix to yield a final volume of 25 μl and final working concentrations of 16.6 mM (NH4 )2 SO4 , 67 mM tris (pH 8.8), 6.7 mM MgCl2 10 mM β-mercaptoethanol, 200 μM of each dNTP, and Platinum Taq polymerase (0.04 U/μl) (Thermo Fisher Scientific). .. Each assay was validated by serial dilution to create a percent methylated reference standard curve ranging from 0.1 to 100% of bisulfite-converted methylated to unmethylated control genomic DNA (Promega).

Article Title: Facile profiling of molecular heterogeneity by microfluidic digital melt
Article Snippet: MSP/MethyLight and ddPCR-MPS/MethyLight All MethyLight assays were performed using 10× Master Mix to yield a final volume of 25 μl and final working concentrations of 16.6 mM (NH4 )2 SO4 , 67 mM tris (pH 8.8), 6.7 mM MgCl2 10 mM β-mercaptoethanol, 200 μM of each dNTP, and Platinum Taq polymerase (0.04 U/μl) (Thermo Fisher Scientific). .. Each assay was validated by serial dilution to create a percent methylated reference standard curve ranging from 0.1 to 100% of bisulfite-converted methylated to unmethylated control genomic DNA (Promega).

Mutagenesis:

Article Title: Tissue-specific mutation accumulation in human adult stem cells during life
Article Snippet: We evaluated our mutation filtering procedure by independent validations of 374 pre-selected positions that were either discarded or passed during filtering using amplicon-based next-generation sequencing. .. These regions were PCR-amplified for both the organoid cultures and reference samples of donor 5 and 6, using 5 ng genomic DNA, 1× PCR Gold Buffer (Life Technologies), 1.5 mM MgCl2 , 0.2 mM of each dNTP and 1 unit of AmpliTaq Gold (Life Technologies) in a final volume of 10 μl.

Isolation:

Article Title: Association between Polymorphisms in Inflammatory Response-Related Genes and the Susceptibility, Progression and Prognosis of the Diffuse Histological Subtype of Gastric Cancer
Article Snippet: Blood samples were collected in EDTA and genomic DNA was isolated from peripheral blood lymphocytes using PureLink® Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA), following the procedures described by the manufacturer and stored at -20 °C until use for genotyping. .. PCR was carried out in 25 µL of reaction volume containing 100 ng of genomic DNA using 50 mmol/L of KCl, 10 mmol/L of TrisHCl (pH = 8.5), 1.0 mmol/L of MgCl2 , 0.4 mmol/L of dNTP, 0.16 µmol/L of each primer and 1 U of recombinant Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA).

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: RNA fragments were isolated using Trizol LS, precipitated in the presence of 300 mM NaAc and 2 μl Glycoblue (Ambion), washed twice with 80% ethanol and dissolved in 4.5 μl water. .. RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C.

Article Title: Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis
Article Snippet: Paragraph title: RNA isolation and QRT-PCR ... RNA (250 ng) was reverse-transcribed using SuperScript II Reverse Transcriptase, 5x First-Strand buffer, DTT, RNaseOUT Recombinant Ribonuclease Inhibitor, Oligo(dT)12–18, and dNTP (Life Technologies, Burlington, ON).

Microscopy:

Article Title: Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
Article Snippet: Single CMs (αMHC-GFP) were either FACS sorted (SH800, Sony Technologies) or manually picked under the microscope into 96-plates containing water (2.4 µL) with RNase-free DNase I (0.2 µL; NEB) and RNase inhibitor (0.25 µL; NEB). .. A mixture of 1 µL SMARTscribe reverse transcriptase (Clontech Laboratories, Inc), 1 µL custom designed TS oligo (12 µM, Integrated DNA Technologies ( )), 0.3 µL MgCl2 (200 mM, Sigma), 0.5 µL RNase inhibitor (Neb), 1 µL dNTP (10 mM each, Thermo), 0.25 µL DTT (100 mM, Invitrogen) were incubated at 42°C for 90 min, which was followed by enzyme inactivation at 70°C for 10 min. A mixture of 29 µL water, 5 µL Advantage2 taq polymerase buffer, 2 µL dNTP (10 mM each, Thermo), 2 µL custom-designed PCR primer (12 µM, Integrated DNA Technologies ( )), and 2 µL Advantage2 taq polymerase was directly added to the reverse transcription product and the amplification was performed for 19 cycles.

Purification:

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: Amplification of Plasmodium DNA Genomic DNA was extracted from the samples with a NucleoSpin Tissue purification kit (Macherey–Nagel) following the manufacturer’s instructions, and amplified following Win et al. [ ]. .. This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step.

Article Title: Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
Article Snippet: A mixture of 1 µL SMARTscribe reverse transcriptase (Clontech Laboratories, Inc), 1 µL custom designed TS oligo (12 µM, Integrated DNA Technologies ( )), 0.3 µL MgCl2 (200 mM, Sigma), 0.5 µL RNase inhibitor (Neb), 1 µL dNTP (10 mM each, Thermo), 0.25 µL DTT (100 mM, Invitrogen) were incubated at 42°C for 90 min, which was followed by enzyme inactivation at 70°C for 10 min. A mixture of 29 µL water, 5 µL Advantage2 taq polymerase buffer, 2 µL dNTP (10 mM each, Thermo), 2 µL custom-designed PCR primer (12 µM, Integrated DNA Technologies ( )), and 2 µL Advantage2 taq polymerase was directly added to the reverse transcription product and the amplification was performed for 19 cycles. .. The amplification product was purified using Ampure XP beads (Beckman-Coulter).

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: Total DNA, representing 50% of the sucrose purified water concentrates, was extracted by the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the modified protocol of with additional processing of 10 freeze–thaw cycles after the resuspension step in lysis buffer, supplied from the manufacturer. .. Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA).

Polymerase Chain Reaction:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: .. All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume. .. Polymerase chain reaction primer sequences for LSU rDNA and optimized annealing temperatures are specified in Table .

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: .. This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step. .. The second amplification was carried out using the same equipment as the first, and the cycling parameters were 92 °C for 2 min, followed by 18 cycles of 92 °C for 30 s and 60 °C for 1 min, and one cycle of 60 °C for 5 min.

Article Title: Association between Polymorphisms in Inflammatory Response-Related Genes and the Susceptibility, Progression and Prognosis of the Diffuse Histological Subtype of Gastric Cancer
Article Snippet: .. PCR was carried out in 25 µL of reaction volume containing 100 ng of genomic DNA using 50 mmol/L of KCl, 10 mmol/L of TrisHCl (pH = 8.5), 1.0 mmol/L of MgCl2 , 0.4 mmol/L of dNTP, 0.16 µmol/L of each primer and 1 U of recombinant Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA). .. The mixture was denaturated for 5 min at 94 °C, passed through 40 cycles of denaturing at 94 °C for 30 s, annealing for 30 s (specific annealing temperatures were described in ) and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min. RFLP reactions were performed using specific enzymes by New England Biolabs (NEB, Ipswich, MA, USA), with conditions also described in .

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. .. Libraries were PCR-amplified for 9 (polyA RNA-Seq), 11 (5’-GRO-Seq), 12 (rRNA- -5'-RNA-Seq) or 13 (polyA-5’-RNA-Seq) cycles with 0.75 μM primers oNTI201 and TruSeq-compatible indexed primers (e.g. 5’-CAA GCA GAA GAC GGC ATA CGA GAT nnn nnn GTG ACT GGA GTT CAG ACG TGT GCT CTT-3’ (desalted, IDT, index in lowercase letters) using Phusion Hot Start II (Thermo Scientific) in HF buffer containing 0.5 M betaine (98°C, 30s/12x(98°C, 10s/57°C, 25s/72°C, 20s)/ 72°C, 1min/4°C, ∞), and 175-225 bp fragments were size-selected on 10% PAGE gels.

Article Title: Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
Article Snippet: .. A mixture of 1 µL SMARTscribe reverse transcriptase (Clontech Laboratories, Inc), 1 µL custom designed TS oligo (12 µM, Integrated DNA Technologies ( )), 0.3 µL MgCl2 (200 mM, Sigma), 0.5 µL RNase inhibitor (Neb), 1 µL dNTP (10 mM each, Thermo), 0.25 µL DTT (100 mM, Invitrogen) were incubated at 42°C for 90 min, which was followed by enzyme inactivation at 70°C for 10 min. A mixture of 29 µL water, 5 µL Advantage2 taq polymerase buffer, 2 µL dNTP (10 mM each, Thermo), 2 µL custom-designed PCR primer (12 µM, Integrated DNA Technologies ( )), and 2 µL Advantage2 taq polymerase was directly added to the reverse transcription product and the amplification was performed for 19 cycles. .. The amplification product was purified using Ampure XP beads (Beckman-Coulter).

Article Title: Tissue-specific mutation accumulation in human adult stem cells during life
Article Snippet: .. These regions were PCR-amplified for both the organoid cultures and reference samples of donor 5 and 6, using 5 ng genomic DNA, 1× PCR Gold Buffer (Life Technologies), 1.5 mM MgCl2 , 0.2 mM of each dNTP and 1 unit of AmpliTaq Gold (Life Technologies) in a final volume of 10 μl. ..

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: .. Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA). .. The templates were subjected to an amplification program with a peqSTAR thermal cycler (PEQLAB GmbH, Erlangen, Germany) consisting of an initial step at 96°C for 15 minutes, 35 amplification cycles (94°C for 45 seconds, 55°C at primary PCR and 58°C at secondary PCR for 45 seconds and 72°C for 60 seconds) followed by one cycle of 10 minutes at 72°C according to .

Polyacrylamide Gel Electrophoresis:

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. .. Libraries were PCR-amplified for 9 (polyA RNA-Seq), 11 (5’-GRO-Seq), 12 (rRNA- -5'-RNA-Seq) or 13 (polyA-5’-RNA-Seq) cycles with 0.75 μM primers oNTI201 and TruSeq-compatible indexed primers (e.g. 5’-CAA GCA GAA GAC GGC ATA CGA GAT nnn nnn GTG ACT GGA GTT CAG ACG TGT GCT CTT-3’ (desalted, IDT, index in lowercase letters) using Phusion Hot Start II (Thermo Scientific) in HF buffer containing 0.5 M betaine (98°C, 30s/12x(98°C, 10s/57°C, 25s/72°C, 20s)/ 72°C, 1min/4°C, ∞), and 175-225 bp fragments were size-selected on 10% PAGE gels.

Lysis:

Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling
Article Snippet: .. The following chemicals were evaluated (final lysis concentrations are shown): 7-deaza-2′-deoxyguanosine-5′-triphosphate lithium salt (100 μM, Sigma-Aldrich); Betaine solution (4 M, Sigma-Aldrich); BSA (1–4 mg/ml, Fermentas); guanidine thiocyanate solution (GTC) (40–80 mM, Sigma-Aldrich); GenElute linear polyacrylamide (LPA) (50 ng/μl, Sigma-Aldrich); Igepal CA-630 (also known as Non-idet P-40, 0.5–4%, Sigma-Aldrich); polyinosinic acid potassium salt (50 ng/μl, Sigma-Aldrich); RNAseOUT (10 U/μl, Invitrogen); 2× reverse transcription buffer: 100 mM Tris-HCl (pH 8.3), 150 mM KCl, and 6 mM MgCl2 (Invitrogen); d -(+)-trehalose dihydrate (1 M, Sigma-Aldrich); yeast tRNA (50 ng/μl, Ambion); RT mix (2× RT buffer, Invitrogen, 5 μM random hexamers (Metabion), 5 μM oligo-dT (Metabion), 1 mM dNTP); RT mix with BSA (2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, 1 mM dNTP, 1 mg/ml BSA); and RNase-free water (Gibco). ..

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: Total DNA, representing 50% of the sucrose purified water concentrates, was extracted by the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the modified protocol of with additional processing of 10 freeze–thaw cycles after the resuspension step in lysis buffer, supplied from the manufacturer. .. Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA).

Nested PCR:

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: Briefly, nested-PCR, which consists of two rounds of amplification, was carried out with primers that target the Plasmodium 18S RNA gene subunit. .. This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step.

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: The nested PCR targeting the small subunit (SSU) rRNA gene was performed as described by ; and . .. Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA).

Activated Clotting Time Assay:

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: To reduce adapter dimer formation, 0.5 μl 10 μM MPX_RT primer 5’-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3’ (IDT, desalted) was added and annealed to the ligation product by incubating at 75°C for 2 minutes, then 37°C for 30 minutes, then 25°C for 15 minutes. .. RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C.

SYBR Green Assay:

Article Title: Impact of natural genetic variation on enhancer selection and function
Article Snippet: RNA was reverse-transcribed by adding 3 μl 10× first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. .. Libraries were diluted 1:105 with TET and quantified relative to samples of known cluster density by SYBR green Q-PCR with primers Solexa_1G_A 5’-AAT GAT ACG GCG ACC ACC GA-3’, Solexa_1G_B 5’-CAA GCA GAA GAC GGC ATA CGA-3’ (95°C, 15 min/25x(95°C, 10s/60°C, 60 s)) and sequenced for 51 (insert) +7 (index) cycles on a HiSeq 2000 sequencer (Illumina) with small RNA sequencing primer 5’-CGA CAG GTT CAG AGT TCT ACA GTC CGA CGA TC-3’ and TruSeq Index sequencing primer (Illumina).

Article Title: Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis
Article Snippet: RNA (250 ng) was reverse-transcribed using SuperScript II Reverse Transcriptase, 5x First-Strand buffer, DTT, RNaseOUT Recombinant Ribonuclease Inhibitor, Oligo(dT)12–18, and dNTP (Life Technologies, Burlington, ON). .. Gene expression was determined using qPCR reaction with Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies, Burlington, ON) and performed on the CFX96 Real-time PCR Detection System (Bio-rad Laboratories, Mississauga, ON).

Recombinant:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: .. All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume. .. Polymerase chain reaction primer sequences for LSU rDNA and optimized annealing temperatures are specified in Table .

Article Title: Association between Polymorphisms in Inflammatory Response-Related Genes and the Susceptibility, Progression and Prognosis of the Diffuse Histological Subtype of Gastric Cancer
Article Snippet: .. PCR was carried out in 25 µL of reaction volume containing 100 ng of genomic DNA using 50 mmol/L of KCl, 10 mmol/L of TrisHCl (pH = 8.5), 1.0 mmol/L of MgCl2 , 0.4 mmol/L of dNTP, 0.16 µmol/L of each primer and 1 U of recombinant Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA). .. The mixture was denaturated for 5 min at 94 °C, passed through 40 cycles of denaturing at 94 °C for 30 s, annealing for 30 s (specific annealing temperatures were described in ) and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min. RFLP reactions were performed using specific enzymes by New England Biolabs (NEB, Ipswich, MA, USA), with conditions also described in .

Article Title: Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis
Article Snippet: .. RNA (250 ng) was reverse-transcribed using SuperScript II Reverse Transcriptase, 5x First-Strand buffer, DTT, RNaseOUT Recombinant Ribonuclease Inhibitor, Oligo(dT)12–18, and dNTP (Life Technologies, Burlington, ON). .. Gene expression was determined using qPCR reaction with Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies, Burlington, ON) and performed on the CFX96 Real-time PCR Detection System (Bio-rad Laboratories, Mississauga, ON).

Agarose Gel Electrophoresis:

Article Title: Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil
Article Snippet: This step was carried out in a final volume of 20 μL, containing 2 μL of each primer (10 μM), 0.5 μL of dNTP (10 mM each, Thermo Fisher Scientific), 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step. .. The primers used were: P1UP (F): 5′ TCC ATT AAT CAA GAA CGA AAG TTA AG 3′ P2 (R): 5′ GAA CCC AAA GAC TTT GAT TTC TCA T 3′ P1 (F): 5′ ACG ATC AGA TAC CGT CGT AAT CTT 3′ V1 (R): 5′ CAA TCT AAG AAT AAA CTC CGA AGA GAA A 3′ F2 (R): 5′ CAA TCT AAA AGT CAC CTC GAA AGA TG 3′ M1 (R): 5′ GGA AGC TAT CTA AAA GAA ACA CTC ATA T 3′ The amplified product was run on 2% agarose gel at 80 V for 40 min.

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA). .. PCR products were analyzed in 1.5% agarose gel stained with ethidium bromide solution (1 μg/ml) and visualized under UV light.

In Vitro:

Article Title: Multiplex single-molecule interaction profiling of DNA barcoded proteins
Article Snippet: Barcoded linear DNA templates were pooled and in vitro transcribed using a HiScribe T7 kit (New England Biolabs). .. To generate mRNA–cDNA hybrids, cDNAs were synthesized by incubating ~0.10 μM mRNA, 1 μM 5’-acrydite and desthiobiotin-modified primer, 0.5 mM each dNTP, 10 U/μL SuperScript III, 2 U/μL RNaseOUT (Invitrogen) and 5 mM dithiothreitol (DTT) in a buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 5 mM MgCl2 ) at 50°C for 30 min.

Next-Generation Sequencing:

Article Title: Tissue-specific mutation accumulation in human adult stem cells during life
Article Snippet: We evaluated our mutation filtering procedure by independent validations of 374 pre-selected positions that were either discarded or passed during filtering using amplicon-based next-generation sequencing. .. These regions were PCR-amplified for both the organoid cultures and reference samples of donor 5 and 6, using 5 ng genomic DNA, 1× PCR Gold Buffer (Life Technologies), 1.5 mM MgCl2 , 0.2 mM of each dNTP and 1 unit of AmpliTaq Gold (Life Technologies) in a final volume of 10 μl.

FLAG-tag:

Article Title: Multiplex single-molecule interaction profiling of DNA barcoded proteins
Article Snippet: To generate mRNA–cDNA hybrids, cDNAs were synthesized by incubating ~0.10 μM mRNA, 1 μM 5’-acrydite and desthiobiotin-modified primer, 0.5 mM each dNTP, 10 U/μL SuperScript III, 2 U/μL RNaseOUT (Invitrogen) and 5 mM dithiothreitol (DTT) in a buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 5 mM MgCl2 ) at 50°C for 30 min. .. PRMC complexes, always kept on ice or in cold room, were subjected to two-step Flag tag and desthiobiotin tag affinity purification to enrich full-length and barcoded target proteins.

Lamp Assay:

Article Title: Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey
Article Snippet: Both PCRs were performed in standard mixtures of 50 μl containing 200 nmoles of each primer, 200 μM dNTP of each dNTP (Finnzymes, Espoo, Finland), 1× PCR buffer containing 1.5 mM MgCl2 (Qiagen GmbH), 3 mM MgCl2 (Qiagen GmbH), 2.5 U HotstarTaq DNA polymerase (Qiagen GmbH) and 2 μl bovine serum albumin (acetylated, 10 mg/ml) (Promega, Madison, WI, USA). .. LAMP assay was performed on all DNA-extracted water samples according to and targeting the SAM gene.

FACS:

Article Title: Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
Article Snippet: Single CMs (αMHC-GFP) were either FACS sorted (SH800, Sony Technologies) or manually picked under the microscope into 96-plates containing water (2.4 µL) with RNase-free DNase I (0.2 µL; NEB) and RNase inhibitor (0.25 µL; NEB). .. A mixture of 1 µL SMARTscribe reverse transcriptase (Clontech Laboratories, Inc), 1 µL custom designed TS oligo (12 µM, Integrated DNA Technologies ( )), 0.3 µL MgCl2 (200 mM, Sigma), 0.5 µL RNase inhibitor (Neb), 1 µL dNTP (10 mM each, Thermo), 0.25 µL DTT (100 mM, Invitrogen) were incubated at 42°C for 90 min, which was followed by enzyme inactivation at 70°C for 10 min. A mixture of 29 µL water, 5 µL Advantage2 taq polymerase buffer, 2 µL dNTP (10 mM each, Thermo), 2 µL custom-designed PCR primer (12 µM, Integrated DNA Technologies ( )), and 2 µL Advantage2 taq polymerase was directly added to the reverse transcription product and the amplification was performed for 19 cycles.

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