second strand buffer  (Thermo Fisher)


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    Name:
    Second Strand Buffer
    Description:
    Second Strand Buffer is used with SuperScript cDNA Synthesis Systems to generate second strand cDNA from mRNA in the first strand reaction It is supplied as a 5X concentrate and should be diluted 1 5 1 part 5X Second Strand Buffer 4 parts other components prior to use Quality controlThis product has passed quality control assays that verify the absence of detectable levels of ribonuclease activity
    Catalog Number:
    10812014
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher second strand buffer
    Second Strand Buffer is used with SuperScript cDNA Synthesis Systems to generate second strand cDNA from mRNA in the first strand reaction It is supplied as a 5X concentrate and should be diluted 1 5 1 part 5X Second Strand Buffer 4 parts other components prior to use Quality controlThis product has passed quality control assays that verify the absence of detectable levels of ribonuclease activity
    https://www.bioz.com/result/second strand buffer/product/Thermo Fisher
    Average 90 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    second strand buffer - by Bioz Stars, 2020-02
    90/100 stars

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    Related Articles

    Amplification:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: Paragraph title: RNA isolation & amplification ... Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column.

    Article Title: Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells
    Article Snippet: Second strand cDNA synthesis was performed using RNase H, DNA Polymerase I, and Invitrogen Second Strand Buffer (ThermoFisher Scientific). .. Tagmented DNA was purified using DNA Clean & Concentrator‐5 before Nextera PCR amplification.

    Article Title: Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma
    Article Snippet: Paragraph title: RNA isolation and amplification ... Second-strand synthesis was performed by adding 53 μl of DEPC-treated water, 20 μl of 5× second strand buffer (Invitrogen Life Technology), 1 mmol/l dNTP, 1 U RNase H (Invitrogen Life Technology), 10 U Escherichia coli DNA ligase, and 40 U E. coli DNA polymerase I (Invitrogen Life Technology) to a final volume of 100 μl.

    Article Title: Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
    Article Snippet: Paragraph title: RNA amplification ... Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 μl first-strand product plus 80 μl of second strand mix containing 63 μl water, 10 μl second-strand buffer, 4 μl dNTPs, 2 μl DNA polymerase and 1 μl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according to manufacturer’s instructions.

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions. .. The gel-purified cDNA libraries were amplified by PCR (TruSeq v2 LT sample prep kit PCR, Illumina), quantified, and checked for quality on an Agilent 2100 Bioanalyzer, and sequenced as single-end 50 bp reads on an Illumina Genome Analyzer GX platform.

    Synthesized:

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB). .. After 2 h at 16 °C, 1 μl of T4 DNA polymerase (10 U/μl Promega) was added and the reaction was incubated at 16 °C for 10 min.

    Article Title: Neurons That Underlie Drosophila melanogaster Reproductive Behaviors: Detection of a Large Male-Bias in Gene Expression in fruitless-Expressing Neurons
    Article Snippet: .. Following first strand synthesis, the second strand of the cDNA was synthesized by addition of DNA polymerase I (Invitrogen), RNase H (New England Biolabs Inc.), dNTPs and second strand buffer (Invitrogen). .. The DNA from this and all subsequent reactions was purified using Zymo Research DNA Clean & Concentrator-5 kit.

    Article Title: Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
    Article Snippet: .. Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 μl first-strand product plus 80 μl of second strand mix containing 63 μl water, 10 μl second-strand buffer, 4 μl dNTPs, 2 μl DNA polymerase and 1 μl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according to manufacturer’s instructions. .. Eluted cDNA was adjusted to 16 μl and used as template for amplification reaction in a total volume of 40 μl containing 16 μl NTPs, 4 μl amplification buffer and 4 μl of T7 RNA polymerase; in vitro transcription took 14 h at 37 °C.

    Quantitative RT-PCR:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: One of these and two additional biological replicates were used for quantitative RT-PCR analysis. .. Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column.

    Electrophoresis:

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: cDNA libraries preparation and RNA-seq Total RNA was extracted from wild-type and Δseb-1 heat-shocked mycelial pads as previously described , quantified in a NanoDrop ND1000 spectrophotometer (Thermo Scientific), and checked for integrity of rRNA by electrophoresis on a 1.2% formaldehyde agarose gel. .. The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions.

    Microarray:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: For each microarray sample, 50–500 ng of total RNA was amplified using a modified Eberwine procedure [ , ]. .. Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column.

    Article Title: Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6)
    Article Snippet: Paragraph title: Microarray experiments ... Next, the first-strand buffer mix (4 μL of 5 × the first-strand buffer, 2 μL 0.1 M dithiothreitol (DTT), and 1 μL 10 mM dNTPs) was preincubated at 42 °C for 2 min. After addition of 2 μL (200 units) Superscript II (Life Technologies, Karlsruhe, Germany), incubation was continued at 42 °C for 1 h. For second-strand synthesis, 30 μL 5 × the second-strand buffer, 91 μL RNase-free water, 3 μL 10 mM dNTPs, 4 μL (40 U) Escherichia coli DNA polymerase I (Life Technologies), 1 μL (12 U) E. coli DNAligase (TaKaRa, Gennevilliers, France), and 1 μL (2 U) RNase H (TaKaRa) were added, and the mix was incubated at 16 °C for 2 h. Then 2.5 μL (10 U) T4 DNA polymerase I (TaKaRa) were added at 16 °C for 5 min.

    Article Title: Massive and parallel expression profiling using microarrayed single-cell sequencing
    Article Snippet: To remove cells, proteinase K (Qiagen, Limburg, Netherlands) was mixed with proteinase K digestion (PKD) buffer 1:4 then added to the microarray surface with the attached ArrayIT hybridization cassette. .. A probe release mix was prepared by mixing 6.4 U of USER enzyme and 1.5 × BSA (both from NEB, Ipswich, MA, USA), 0.8 × second strand buffer (Invitrogen, Life Technologies, Paisley, UK), 70 μM dNTP mix (Thermo Fisher Scientific, Life Technologies, Paisley, UK) and water to a final volume of 70 μl, and heated to 37 °C.

    Incubation:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™). .. Reactions were incubated at 16°C for 2.5 h. The double stranded cDNA was subsequently purified using a QIAquick PCR Purification kit (Qiagen) according to the manufacturer’s instructions and eluted in 30 μl of the provided elution buffer.

    Article Title: Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum
    Article Snippet: To digest RNA and release the first-strand cDNA, 2 μl 2 U/μl Escherichia coli RNase H (Life Technologies) was added, followed by a 20 min incubation at 37°C. .. The beads then were removed using a magnet and first-strand cDNA was used for second-strand cDNA synthesis by adding 70 μl 5× nuclease-free water (Life Technologies), 30 μl second-strand buffer (Life Technologies), 3 μl 10 mM dNTP mix (Life Technologies), 4 μl 10 U/μl E. coli DNA Polymerase (NEB) and 1 μl 10 U/μl E. coli DNA ligase (NEB).

    Article Title: Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6)
    Article Snippet: .. Next, the first-strand buffer mix (4 μL of 5 × the first-strand buffer, 2 μL 0.1 M dithiothreitol (DTT), and 1 μL 10 mM dNTPs) was preincubated at 42 °C for 2 min. After addition of 2 μL (200 units) Superscript II (Life Technologies, Karlsruhe, Germany), incubation was continued at 42 °C for 1 h. For second-strand synthesis, 30 μL 5 × the second-strand buffer, 91 μL RNase-free water, 3 μL 10 mM dNTPs, 4 μL (40 U) Escherichia coli DNA polymerase I (Life Technologies), 1 μL (12 U) E. coli DNAligase (TaKaRa, Gennevilliers, France), and 1 μL (2 U) RNase H (TaKaRa) were added, and the mix was incubated at 16 °C for 2 h. Then 2.5 μL (10 U) T4 DNA polymerase I (TaKaRa) were added at 16 °C for 5 min. .. The reaction was stopped by the addition of 10 μL 0.5 M EDTA, double-stranded (ds) cDNA was extracted with phenol/chloroform, and the aqueous phase was recovered by phase-lock gel separation (Eppendorf, Hamburg, Germany).

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: The first strand reaction was incubated at 25 °C for 10 min, and 50 °C for 90 min, followed by deactivation at 70 °C for 15 min, after which the MinElute Reaction Cleanup Kit (Qiagen) was used according to the manufacturer's protocol. .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Article Title: Nuclear envelope proteomics: Novel integral membrane proteins of the inner nuclear membrane
    Article Snippet: .. After 1 h incubation at 42°C, the reaction was supplemented with 6 μl of Second Strand Buffer (GIBCO) and 5 units of RnaseH (GIBCO) and incubated for another 30 min at 37°C. .. The obtained cDNA was used as template in a PCR reaction with complementary sense (5′-GCCACAATGGCCGCGAATTATTCCAGTACC) and antisense (5′-CTCCAACTTTTTGGCTGGCACCCGTGTCCG) primers, which resulted in amplification of a 1200-bp DNA fragment [corresponding to the cDNA encoding the putative protein PID (protein identifier) 12836214].

    Article Title: Massive and parallel expression profiling using microarrayed single-cell sequencing
    Article Snippet: After incubation for 1 h at 56 °C, the glass slide was sequentially washed in 2 × SSC supplemented with 0.1% sodium dodecyl sulphate at 50 °C for 600 s, 0.2 × SSC for 60 s and 0.1 × SSC for 60 s at room temperature, then spin-dried. .. A probe release mix was prepared by mixing 6.4 U of USER enzyme and 1.5 × BSA (both from NEB, Ipswich, MA, USA), 0.8 × second strand buffer (Invitrogen, Life Technologies, Paisley, UK), 70 μM dNTP mix (Thermo Fisher Scientific, Life Technologies, Paisley, UK) and water to a final volume of 70 μl, and heated to 37 °C.

    Article Title: Analysis of transcript and protein overlap in a human osteosarcoma cell line
    Article Snippet: The first strand cDNA synthesis reaction was incubated at 20°C for 10 minutes followed by 37°C for 10 minutes and 42°C for 45 minutes. .. This consisted of (per sample) 79 μl nuclease-free water, 30 μl 5x second-strand buffer (Invitrogen), 15 μl dNTP mixture (2 mM/dNTP), 1 μl 10 U/μl E. coli DNA Ligase (Invitrogen), 4 μl 10 U/μl E. coli DNA polymerase I (Invitrogen) and 1 μl 2 U/μl RNase H (Invitrogen).

    Article Title: Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma
    Article Snippet: Second-strand synthesis was performed by adding 53 μl of DEPC-treated water, 20 μl of 5× second strand buffer (Invitrogen Life Technology), 1 mmol/l dNTP, 1 U RNase H (Invitrogen Life Technology), 10 U Escherichia coli DNA ligase, and 40 U E. coli DNA polymerase I (Invitrogen Life Technology) to a final volume of 100 μl. .. Ten units of T4 DNA polymerase I (Invitrogen Life Technology) were added and incubated again at 16°C for 5 minutes.

    Article Title: A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain
    Article Snippet: To make cDNA, 1 μl of oligo(dT) primers (500ng/μl; Ambion) and 1 μl of 10 mM dNTP (Promega) was added to 10 μl of polyA-selected mRNA; incubated at 65°C for 5 min and placed on ice. .. The second strand of cDNA was filled in by adding 35 μl of water, 15 μl of 5x second strand buffer (Invitrogen), 1.5 μl of 10 mM dNTP, 0.5 μl of 10 U/μl E Coli DNA ligase (Bioke), 2 μl of 10 U/μl E Coli DNA polymerase I (Bioke), 1 μl of 2 U/μl E Coli RNaseH and then incubating at 16°C for 2 hours.

    Article Title: Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
    Article Snippet: First, 100 ng of total RNA (in 11 μl) were mixed with first-strand cDNA reaction mix containing 1 μl of T7oligo(dT) primer, 2 μl first-strand buffer, 4 μl dNTPs, 1 μl RNase inhibitor and 1 μl ArrayScript Reverse Transcriptase and incubated for 2 h at 42 °C. .. Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 μl first-strand product plus 80 μl of second strand mix containing 63 μl water, 10 μl second-strand buffer, 4 μl dNTPs, 2 μl DNA polymerase and 1 μl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according to manufacturer’s instructions.

    Article Title: Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer
    Article Snippet: First-strand cDNA was synthesised from 5 µg of mRNA in a reaction buffer containing 1× M-MLVRT reaction buffer (Promega, Madison, WI), 250 ng random hexanucleotides (Invitrogen), 5 mM dithiothreitol (Invitrogen), 0.25 mM dNTPs (Invitrogen), 5 µl of M-MLVRT(H-) enzyme (Promega) in DEPC-treated H2 O; the reaction was incubated for 1 h at 37°C. .. Second-strand synthesis was performed on the first-strand reaction product in a buffer containing 1× Second Strand Buffer (Invitrogen), 0.2 mM dNTPs, 5 U Escherichia coli T4 DNA ligase (MBI Fermentas, Vilnius, Lithuania), 5 U Klenow fragment (MBI Fermentas), 2 U E.coli RNase H (Invitrogen) in DEPC-treated H2 O.

    Modification:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: For each microarray sample, 50–500 ng of total RNA was amplified using a modified Eberwine procedure [ , ]. .. Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column.

    Hybridization:

    Article Title: Massive and parallel expression profiling using microarrayed single-cell sequencing
    Article Snippet: To remove cells, proteinase K (Qiagen, Limburg, Netherlands) was mixed with proteinase K digestion (PKD) buffer 1:4 then added to the microarray surface with the attached ArrayIT hybridization cassette. .. A probe release mix was prepared by mixing 6.4 U of USER enzyme and 1.5 × BSA (both from NEB, Ipswich, MA, USA), 0.8 × second strand buffer (Invitrogen, Life Technologies, Paisley, UK), 70 μM dNTP mix (Thermo Fisher Scientific, Life Technologies, Paisley, UK) and water to a final volume of 70 μl, and heated to 37 °C.

    Generated:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: Thirty-five strand-specific Illumina® RNA-seq libraries were generated (seven libraries for the MAP-infected and control groups at the 2 and 6 hpi time points and seven 0 h time point control samples) using 150–200 ng of total RNA. .. Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™).

    Sequencing:

    Article Title: A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain
    Article Snippet: The second strand of cDNA was filled in by adding 35 μl of water, 15 μl of 5x second strand buffer (Invitrogen), 1.5 μl of 10 mM dNTP, 0.5 μl of 10 U/μl E Coli DNA ligase (Bioke), 2 μl of 10 U/μl E Coli DNA polymerase I (Bioke), 1 μl of 2 U/μl E Coli RNaseH and then incubating at 16°C for 2 hours. .. To incorporate sequencing adapters, we combined the purified cDNA with 4 μl of Nextera TD buffer (Illumina) and 1 μl of Nextera Tn5 enzyme (Illumina) on ice and incubated at 55°C for 5 min.

    Article Title: Neurons That Underlie Drosophila melanogaster Reproductive Behaviors: Detection of a Large Male-Bias in Gene Expression in fruitless-Expressing Neurons
    Article Snippet: Paragraph title: Illumina sequencing library preparation ... Following first strand synthesis, the second strand of the cDNA was synthesized by addition of DNA polymerase I (Invitrogen), RNase H (New England Biolabs Inc.), dNTPs and second strand buffer (Invitrogen).

    Recombinant:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: The RNA pellets obtained were then washed with 80% ethanol, air-dried for 10 min at room temperature and then re-suspended in 10.5 μl of DNase- and RNase-free molecular biology-grade H2 O. Synthesis of first strand cDNA was performed by incubating fragmented RNA with 261 mM Random Hexamer Primers (Invitrogen™), 1× first strand buffer (Invitrogen™); 10 mM DTT (Invitrogen™); 0.5 mM dNTPs; 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor; and 200 U SuperScript® II Reverse Transcriptase (Invitrogen™) using the following temperature profile: 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. First strand synthesis reaction mixtures were then purified using MicroSpin™ G-50 columns according to the manufacturer’s instructions (GE Healthcare UK Ltd., Buckinghamshire, UK). .. Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™).

    Magnetic Cell Separation:

    Article Title: Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells
    Article Snippet: These cells were differentiated to day 40 on Matrigel and purified using MACS. .. Second strand cDNA synthesis was performed using RNase H, DNA Polymerase I, and Invitrogen Second Strand Buffer (ThermoFisher Scientific).

    Nucleic Acid Electrophoresis:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column. .. RNA was quantitated by spectrophotometer and gel electrophoresis.

    RNA Sequencing Assay:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: Paragraph title: Strand-specific RNA-seq library preparation ... Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™).

    Article Title: Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells
    Article Snippet: Paragraph title: RNA‐Sequencing Analysis ... Second strand cDNA synthesis was performed using RNase H, DNA Polymerase I, and Invitrogen Second Strand Buffer (ThermoFisher Scientific).

    Article Title: A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain
    Article Snippet: Paragraph title: Bulk RNA-seq ... The second strand of cDNA was filled in by adding 35 μl of water, 15 μl of 5x second strand buffer (Invitrogen), 1.5 μl of 10 mM dNTP, 0.5 μl of 10 U/μl E Coli DNA ligase (Bioke), 2 μl of 10 U/μl E Coli DNA polymerase I (Bioke), 1 μl of 2 U/μl E Coli RNaseH and then incubating at 16°C for 2 hours.

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: Paragraph title: cDNA libraries preparation and RNA-seq ... The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions.

    Isolation:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: .. Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column. .. RNA was quantitated by spectrophotometer and gel electrophoresis.

    Article Title: Nuclear envelope proteomics: Novel integral membrane proteins of the inner nuclear membrane
    Article Snippet: Paragraph title: RNA Isolation and cDNA Synthesis. ... After 1 h incubation at 42°C, the reaction was supplemented with 6 μl of Second Strand Buffer (GIBCO) and 5 units of RnaseH (GIBCO) and incubated for another 30 min at 37°C.

    Article Title: Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma
    Article Snippet: Paragraph title: RNA isolation and amplification ... Second-strand synthesis was performed by adding 53 μl of DEPC-treated water, 20 μl of 5× second strand buffer (Invitrogen Life Technology), 1 mmol/l dNTP, 1 U RNase H (Invitrogen Life Technology), 10 U Escherichia coli DNA ligase, and 40 U E. coli DNA polymerase I (Invitrogen Life Technology) to a final volume of 100 μl.

    Article Title: Neurons That Underlie Drosophila melanogaster Reproductive Behaviors: Detection of a Large Male-Bias in Gene Expression in fruitless-Expressing Neurons
    Article Snippet: Illumina sequencing library preparation Poly(A)+ transcripts were isolated using Ambion MicroPoly(A)Purist Kit. mRNA was chemically fragmented to a range of approximately 200–500 bp using the Ambion RNA Fragmentation Reagent, and the RNA was purified using Zymo Research RNA Clean & Concentrator-5. .. Following first strand synthesis, the second strand of the cDNA was synthesized by addition of DNA polymerase I (Invitrogen), RNase H (New England Biolabs Inc.), dNTPs and second strand buffer (Invitrogen).

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: The mRNA from biological triplicates was isolated from total RNA samples using Dynabeads Oligo(dT)25 (Invitrogen) and fragmented by the RNA Fragmentation Reagents kit (Ambion). .. The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions.

    Labeling:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column. .. This cRNA was used directly for cDNA labeling for spotted microarrays (see Additional file ), or processed through a second round amplification for Affymetrix arrays.

    Article Title: Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6)
    Article Snippet: Next, the first-strand buffer mix (4 μL of 5 × the first-strand buffer, 2 μL 0.1 M dithiothreitol (DTT), and 1 μL 10 mM dNTPs) was preincubated at 42 °C for 2 min. After addition of 2 μL (200 units) Superscript II (Life Technologies, Karlsruhe, Germany), incubation was continued at 42 °C for 1 h. For second-strand synthesis, 30 μL 5 × the second-strand buffer, 91 μL RNase-free water, 3 μL 10 mM dNTPs, 4 μL (40 U) Escherichia coli DNA polymerase I (Life Technologies), 1 μL (12 U) E. coli DNAligase (TaKaRa, Gennevilliers, France), and 1 μL (2 U) RNase H (TaKaRa) were added, and the mix was incubated at 16 °C for 2 h. Then 2.5 μL (10 U) T4 DNA polymerase I (TaKaRa) were added at 16 °C for 5 min. .. Five microliters ds cDNA were used to synthesize biotinylated cRNA using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, NY).

    Purification:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: The RNA pellets obtained were then washed with 80% ethanol, air-dried for 10 min at room temperature and then re-suspended in 10.5 μl of DNase- and RNase-free molecular biology-grade H2 O. Synthesis of first strand cDNA was performed by incubating fragmented RNA with 261 mM Random Hexamer Primers (Invitrogen™), 1× first strand buffer (Invitrogen™); 10 mM DTT (Invitrogen™); 0.5 mM dNTPs; 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor; and 200 U SuperScript® II Reverse Transcriptase (Invitrogen™) using the following temperature profile: 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. First strand synthesis reaction mixtures were then purified using MicroSpin™ G-50 columns according to the manufacturer’s instructions (GE Healthcare UK Ltd., Buckinghamshire, UK). .. Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™).

    Article Title: Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum
    Article Snippet: Paragraph title: Nascent RNA purification and cDNA preparation ... The beads then were removed using a magnet and first-strand cDNA was used for second-strand cDNA synthesis by adding 70 μl 5× nuclease-free water (Life Technologies), 30 μl second-strand buffer (Life Technologies), 3 μl 10 mM dNTP mix (Life Technologies), 4 μl 10 U/μl E. coli DNA Polymerase (NEB) and 1 μl 10 U/μl E. coli DNA ligase (NEB).

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB). .. After 2 h at 16 °C, 1 μl of T4 DNA polymerase (10 U/μl Promega) was added and the reaction was incubated at 16 °C for 10 min.

    Article Title: Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells
    Article Snippet: These cells were differentiated to day 40 on Matrigel and purified using MACS. .. Second strand cDNA synthesis was performed using RNase H, DNA Polymerase I, and Invitrogen Second Strand Buffer (ThermoFisher Scientific).

    Article Title: Analysis of transcript and protein overlap in a human osteosarcoma cell line
    Article Snippet: First-strand cDNA was purified by addition of 70 μl nuclease-free water using a MinElute spin column following the manufacture's instructions (Qiagen). .. This consisted of (per sample) 79 μl nuclease-free water, 30 μl 5x second-strand buffer (Invitrogen), 15 μl dNTP mixture (2 mM/dNTP), 1 μl 10 U/μl E. coli DNA Ligase (Invitrogen), 4 μl 10 U/μl E. coli DNA polymerase I (Invitrogen) and 1 μl 2 U/μl RNase H (Invitrogen).

    Article Title: Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma
    Article Snippet: Second-strand synthesis was performed by adding 53 μl of DEPC-treated water, 20 μl of 5× second strand buffer (Invitrogen Life Technology), 1 mmol/l dNTP, 1 U RNase H (Invitrogen Life Technology), 10 U Escherichia coli DNA ligase, and 40 U E. coli DNA polymerase I (Invitrogen Life Technology) to a final volume of 100 μl. .. UltraPure™ Phenol (Invitrogen, Carlsbad, CA, USA):chloroform:isoamyl alcohol (Merck), at a ratio of 25:24:1 and a pH of 8.0, was used for cDNA purification.

    Article Title: A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain
    Article Snippet: For the Tn5-mediated, unstranded RNA-seq library prep, mRNA was enriched using the Dynabeads mRNA purification kit (Invitrogen). .. The second strand of cDNA was filled in by adding 35 μl of water, 15 μl of 5x second strand buffer (Invitrogen), 1.5 μl of 10 mM dNTP, 0.5 μl of 10 U/μl E Coli DNA ligase (Bioke), 2 μl of 10 U/μl E Coli DNA polymerase I (Bioke), 1 μl of 2 U/μl E Coli RNaseH and then incubating at 16°C for 2 hours.

    Article Title: Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
    Article Snippet: .. Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 μl first-strand product plus 80 μl of second strand mix containing 63 μl water, 10 μl second-strand buffer, 4 μl dNTPs, 2 μl DNA polymerase and 1 μl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according to manufacturer’s instructions. .. Eluted cDNA was adjusted to 16 μl and used as template for amplification reaction in a total volume of 40 μl containing 16 μl NTPs, 4 μl amplification buffer and 4 μl of T7 RNA polymerase; in vitro transcription took 14 h at 37 °C.

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions. .. The end-labeled cDNAs of about 200 bp were purified by electrophoresis on a 2.5% low-melting point agarose gel (Qiagen, CA).

    Polymerase Chain Reaction:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™). .. Reactions were incubated at 16°C for 2.5 h. The double stranded cDNA was subsequently purified using a QIAquick PCR Purification kit (Qiagen) according to the manufacturer’s instructions and eluted in 30 μl of the provided elution buffer.

    Article Title: Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells
    Article Snippet: Second strand cDNA synthesis was performed using RNase H, DNA Polymerase I, and Invitrogen Second Strand Buffer (ThermoFisher Scientific). .. Tagmented DNA was purified using DNA Clean & Concentrator‐5 before Nextera PCR amplification.

    Article Title: Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
    Article Snippet: .. Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 μl first-strand product plus 80 μl of second strand mix containing 63 μl water, 10 μl second-strand buffer, 4 μl dNTPs, 2 μl DNA polymerase and 1 μl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according to manufacturer’s instructions. .. Eluted cDNA was adjusted to 16 μl and used as template for amplification reaction in a total volume of 40 μl containing 16 μl NTPs, 4 μl amplification buffer and 4 μl of T7 RNA polymerase; in vitro transcription took 14 h at 37 °C.

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions. .. The gel-purified cDNA libraries were amplified by PCR (TruSeq v2 LT sample prep kit PCR, Illumina), quantified, and checked for quality on an Agilent 2100 Bioanalyzer, and sequenced as single-end 50 bp reads on an Illumina Genome Analyzer GX platform.

    IA:

    Article Title: Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum
    Article Snippet: The preparation of cDNA was performed using nascent RNA captured on the beads. cDNA synthesis reaction mix (6 μg of random hexamer (integrated DNA technologies, Coralville, IA, USA), 2 μg of anchored oligo (dT)20 (Integrated DNA Technologies), 2 μl 10 mM dNTP mix (Life Technologies) and 14 μl buffer J from Click-iT Nascent RNA Capture Kit (Thermo Fisher) in a total of 20 μl volume) was added to the beads and incubated for 10 min at 70°C, and then chilled on ice for 5 min. Next, a mix of 4 μl 10X RT buffer, 8 μl 20 mM MgCl2 , 4 μl 0.1 M DTT, 2 μl 20 U/μl SuperaseIn and 2 μl 200 U/μl SuperScript III Reverse Transcriptase (all from Life Technologies) was added to the mixture and incubated for 10 min at 25°C, 50 min at 50°C, and finally 5 min at 85°C for first strand cDNA synthesis. .. The beads then were removed using a magnet and first-strand cDNA was used for second-strand cDNA synthesis by adding 70 μl 5× nuclease-free water (Life Technologies), 30 μl second-strand buffer (Life Technologies), 3 μl 10 mM dNTP mix (Life Technologies), 4 μl 10 U/μl E. coli DNA Polymerase (NEB) and 1 μl 10 U/μl E. coli DNA ligase (NEB).

    Sample Prep:

    Article Title: Analysis of transcript and protein overlap in a human osteosarcoma cell line
    Article Snippet: Paragraph title: RNA sample preparation and cDNA synthesis ... This consisted of (per sample) 79 μl nuclease-free water, 30 μl 5x second-strand buffer (Invitrogen), 15 μl dNTP mixture (2 mM/dNTP), 1 μl 10 U/μl E. coli DNA Ligase (Invitrogen), 4 μl 10 U/μl E. coli DNA polymerase I (Invitrogen) and 1 μl 2 U/μl RNase H (Invitrogen).

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions. .. For the RNA-seq experiments, the double-stranded cDNAs were end-labeled with different adaptors using the TruSeq DNA LT sample prep kit (Illumina), according to the manufacturer’s instructions.

    Activated Clotting Time Assay:

    Article Title: Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma
    Article Snippet: The total RNA was first denatured at 70°C for 10 minutes in presence of 200 ng oligo (dT) [ ]-T7 primer (5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T (24)-3'; 57 base pairs) and snap cooled on ice. .. Second-strand synthesis was performed by adding 53 μl of DEPC-treated water, 20 μl of 5× second strand buffer (Invitrogen Life Technology), 1 mmol/l dNTP, 1 U RNase H (Invitrogen Life Technology), 10 U Escherichia coli DNA ligase, and 40 U E. coli DNA polymerase I (Invitrogen Life Technology) to a final volume of 100 μl.

    RNA Extraction:

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: Paragraph title: RNA extraction ... The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Article Title: Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma
    Article Snippet: RNA isolation and amplification Laser capture microdissection (LCM) captured cells on CapSure™ HS LCM Caps (Arcturus Engineering) were resuspended in 10 μl of PicoPure RNA extraction buffer (Arcturus Engineering). .. Second-strand synthesis was performed by adding 53 μl of DEPC-treated water, 20 μl of 5× second strand buffer (Invitrogen Life Technology), 1 mmol/l dNTP, 1 U RNase H (Invitrogen Life Technology), 10 U Escherichia coli DNA ligase, and 40 U E. coli DNA polymerase I (Invitrogen Life Technology) to a final volume of 100 μl.

    Agarose Gel Electrophoresis:

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: cDNA libraries preparation and RNA-seq Total RNA was extracted from wild-type and Δseb-1 heat-shocked mycelial pads as previously described , quantified in a NanoDrop ND1000 spectrophotometer (Thermo Scientific), and checked for integrity of rRNA by electrophoresis on a 1.2% formaldehyde agarose gel. .. The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions.

    In Vitro:

    Article Title: Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
    Article Snippet: For classic Eberwine amplification method, first-strand cDNA was synthesized using a T7oligo(dT) primer that contains a T7 RNA polymerase promoter upstream to the poly-T tract; this promoter will further direct the in vitro mRNA synthesis (amplification). .. Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 μl first-strand product plus 80 μl of second strand mix containing 63 μl water, 10 μl second-strand buffer, 4 μl dNTPs, 2 μl DNA polymerase and 1 μl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according to manufacturer’s instructions.

    Ethanol Precipitation:

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: Each 50 μl fragmentation reaction was incubated at 95 °C for 1.5 min on a thermal cycler, and placed on ice for 10 min. Ethanol precipitation was used to purify the reactions. .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Laser Capture Microdissection:

    Article Title: Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma
    Article Snippet: RNA isolation and amplification Laser capture microdissection (LCM) captured cells on CapSure™ HS LCM Caps (Arcturus Engineering) were resuspended in 10 μl of PicoPure RNA extraction buffer (Arcturus Engineering). .. Second-strand synthesis was performed by adding 53 μl of DEPC-treated water, 20 μl of 5× second strand buffer (Invitrogen Life Technology), 1 mmol/l dNTP, 1 U RNase H (Invitrogen Life Technology), 10 U Escherichia coli DNA ligase, and 40 U E. coli DNA polymerase I (Invitrogen Life Technology) to a final volume of 100 μl.

    Random Hexamer Labeling:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: The RNA pellets obtained were then washed with 80% ethanol, air-dried for 10 min at room temperature and then re-suspended in 10.5 μl of DNase- and RNase-free molecular biology-grade H2 O. Synthesis of first strand cDNA was performed by incubating fragmented RNA with 261 mM Random Hexamer Primers (Invitrogen™), 1× first strand buffer (Invitrogen™); 10 mM DTT (Invitrogen™); 0.5 mM dNTPs; 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor; and 200 U SuperScript® II Reverse Transcriptase (Invitrogen™) using the following temperature profile: 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. First strand synthesis reaction mixtures were then purified using MicroSpin™ G-50 columns according to the manufacturer’s instructions (GE Healthcare UK Ltd., Buckinghamshire, UK). .. Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™).

    Article Title: Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum
    Article Snippet: The preparation of cDNA was performed using nascent RNA captured on the beads. cDNA synthesis reaction mix (6 μg of random hexamer (integrated DNA technologies, Coralville, IA, USA), 2 μg of anchored oligo (dT)20 (Integrated DNA Technologies), 2 μl 10 mM dNTP mix (Life Technologies) and 14 μl buffer J from Click-iT Nascent RNA Capture Kit (Thermo Fisher) in a total of 20 μl volume) was added to the beads and incubated for 10 min at 70°C, and then chilled on ice for 5 min. Next, a mix of 4 μl 10X RT buffer, 8 μl 20 mM MgCl2 , 4 μl 0.1 M DTT, 2 μl 20 U/μl SuperaseIn and 2 μl 200 U/μl SuperScript III Reverse Transcriptase (all from Life Technologies) was added to the mixture and incubated for 10 min at 25°C, 50 min at 50°C, and finally 5 min at 85°C for first strand cDNA synthesis. .. The beads then were removed using a magnet and first-strand cDNA was used for second-strand cDNA synthesis by adding 70 μl 5× nuclease-free water (Life Technologies), 30 μl second-strand buffer (Life Technologies), 3 μl 10 mM dNTP mix (Life Technologies), 4 μl 10 U/μl E. coli DNA Polymerase (NEB) and 1 μl 10 U/μl E. coli DNA ligase (NEB).

    Spectrophotometry:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column. .. RNA was quantitated by spectrophotometer and gel electrophoresis.

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: cDNA libraries preparation and RNA-seq Total RNA was extracted from wild-type and Δseb-1 heat-shocked mycelial pads as previously described , quantified in a NanoDrop ND1000 spectrophotometer (Thermo Scientific), and checked for integrity of rRNA by electrophoresis on a 1.2% formaldehyde agarose gel. .. The first cDNA strand was prepared using the SuperScript III First Strand Synthesis kit (Invitrogen) and [d(N6 )] random primers (Invitrogen), and the second cDNA strand was prepared using the Second Strand Buffer (Invitrogen), according to the manufacturer’s instructions.

    Produced:

    Article Title: Analysis of gene expression during neurite outgrowth and regeneration
    Article Snippet: .. Second strand cDNA synthesis utilized DNA ligase, DNA polymerase I holoenzyme (both New England Biolabs, Ipswich, MA), dNTPs, and second strand buffer (Invitrogen) for 2–6 hours at 16 degrees C. cDNA was isolated by phenol chloroform extraction and precipitated. cRNA was produced from cDNA with an Ambion T7 Megascript kit, and collected via Qiagen RNeasy column. .. RNA was quantitated by spectrophotometer and gel electrophoresis.

    Concentration Assay:

    Article Title: Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq
    Article Snippet: .. Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen™); 1× second strand buffer (Invitrogen™); a dNTP solution consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich), and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen™); 2 U RNase H (Invitrogen™); and 50 U E. coli DNA Polymerase I (Invitrogen™). .. Reactions were incubated at 16°C for 2.5 h. The double stranded cDNA was subsequently purified using a QIAquick PCR Purification kit (Qiagen) according to the manufacturer’s instructions and eluted in 30 μl of the provided elution buffer.

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: The RNA fragmentation reactions were performed using fragmentation buffer (5 ×; 200 mM Tris-Ac, 500 mM Potassium-Ac, 150 mM Magnesium-Ac) in a final concentration of 1 × per reaction. .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Lysis:

    Article Title: A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain
    Article Snippet: Twenty to thirty adult brains were dissected in PBS and total RNA was extracted according to the RNAqueous micro protocol (Ambion), using 200 μl lysis buffer. .. The second strand of cDNA was filled in by adding 35 μl of water, 15 μl of 5x second strand buffer (Invitrogen), 1.5 μl of 10 mM dNTP, 0.5 μl of 10 U/μl E Coli DNA ligase (Bioke), 2 μl of 10 U/μl E Coli DNA polymerase I (Bioke), 1 μl of 2 U/μl E Coli RNaseH and then incubating at 16°C for 2 hours.

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    Second Strand Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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