t4 dna ligase  (Thermo Fisher)


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    Name:
    E coli DNA Ligase
    Description:
    E coli DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of β NAD between double stranded DNAs with 3 hydroxyl and 5 phosphate cohesive termini Single stranded nucleic acids are not substrates for this enzyme Applications Second strand cDNA synthesis 1 T4 DNA Ligase alternative when blunt end ligation is not required 2 Source Purified from E coli 594 Su bearing λ lysogen gt4lop 11 lig S7 3 Performance and Quality Testing Endodeoxyribonuclease and 3 and 5 exodeoxyribonuclease assays ligation efficiency tested Unit Definition One unit is the amount of enzyme required to give 50 ligation of Hind III digested λ DNA in 30 min at 16°C in a final volume of 20 µl containing a 5 termini concentration of 0 12 M 300 µg ml Unit Reaction Conditions 18 8 mM Tris HCl pH 8 3 90 6 mM KCl 4 6 mM MgCl2 3 8 mM DTT 0 15 mM λ NAD 10 mM NH4 2 SO4 in 20 µl for 1 h at 16°C
    Catalog Number:
    18052019
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher t4 dna ligase
    E coli DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of β NAD between double stranded DNAs with 3 hydroxyl and 5 phosphate cohesive termini Single stranded nucleic acids are not substrates for this enzyme Applications Second strand cDNA synthesis 1 T4 DNA Ligase alternative when blunt end ligation is not required 2 Source Purified from E coli 594 Su bearing λ lysogen gt4lop 11 lig S7 3 Performance and Quality Testing Endodeoxyribonuclease and 3 and 5 exodeoxyribonuclease assays ligation efficiency tested Unit Definition One unit is the amount of enzyme required to give 50 ligation of Hind III digested λ DNA in 30 min at 16°C in a final volume of 20 µl containing a 5 termini concentration of 0 12 M 300 µg ml Unit Reaction Conditions 18 8 mM Tris HCl pH 8 3 90 6 mM KCl 4 6 mM MgCl2 3 8 mM DTT 0 15 mM λ NAD 10 mM NH4 2 SO4 in 20 µl for 1 h at 16°C
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 90 stars, based on 387 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-02
    90/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae
    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen). .. The gels were blotted onto Hybond N+ nitrocellulose membranes (Amersham Biosciences) and hybridized with probes generated from the differentially expressed cDNA clones by the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals).

    Luciferase:

    Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation
    Article Snippet: RNA-Seq and analyses RNA-seq was performed on three biological replicates of IMR90 cells with shRNA targeting macroH2A1 or Luciferase grown to 90% confluence in 6-wells dishes. .. The second strand was generated using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 μmol each, Promega), in the Second Strand Buffer (Invitrogen).

    Synthesized:

    Article Title: A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains
    Article Snippet: .. After cDNA synthesis, double strand (ds)-cDNA was synthesized with E. coli DNA polymerase I (Invitrogen), E. coli DNA Ligase (Invitrogen), and RNase H (Invitrogen). ds-cDNAs were blunted with T4 DNA polymerase (Invitrogen). ..

    Article Title: Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis
    Article Snippet: .. Following cDNA synthesis, double-stranded (ds)-cDNA was synthesized with E. coli DNA polymerase I (Invitrogen), E. coli DNA ligase (Invitrogen), and RNase H (Invitrogen), after which the ds-cDNA was blunted with T4 DNA polymerase (Invitrogen). ..

    Microarray:

    Article Title: Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome
    Article Snippet: Paragraph title: Microarray and data analysis ... Total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), by using a T7-oligo-d(T)24 as a primer, 1× first-strand buffer and 1 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed using 40 U DNA polymerase I, 10 U E.coli DNA ligase, 2 U RNAse H (Invitrogen, Burlington, ON), 1× reaction buffer and 0.2 mM dNTPs at 16°C for 2 h. The addition of 10 U of T4 polynucleotide kinase (Invitrogen) stops the reaction, and cDNAs were incubated for 10 min at 16°C. cDNAs were isolated by phenol–chloroform extraction.

    Incubation:

    Article Title: Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome
    Article Snippet: .. Total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), by using a T7-oligo-d(T)24 as a primer, 1× first-strand buffer and 1 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed using 40 U DNA polymerase I, 10 U E.coli DNA ligase, 2 U RNAse H (Invitrogen, Burlington, ON), 1× reaction buffer and 0.2 mM dNTPs at 16°C for 2 h. The addition of 10 U of T4 polynucleotide kinase (Invitrogen) stops the reaction, and cDNAs were incubated for 10 min at 16°C. cDNAs were isolated by phenol–chloroform extraction. .. Then, cDNAs were transcribed in vitro by using the T7 BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) to produce biotinylated cRNA.

    Article Title: Probabilistic estimation of microarray data reliability and underlying gene expression
    Article Snippet: RNA was prepared using Trizol (GIBCO) and 7.5 = B5g of total RNA was annealed to a T7-oligo T primer by denaturation at 70 = B0C for 10 minutes followed by 10 minutes of incubation of the samples on ice. .. This was followed by a second strand synthesis for 2 hours at 16 = B0C, using RNAseH, E coli DNA polymerase I and E coli DNA ligase (all from GIBCO), according to the manufacturers instructions.

    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: Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing
    Article Snippet: .. Briefly, each mRNA sample was mixed with 100 pmol of random hexamers (Integrated DNA Technologies), incubated at 70 °C for 10 min, chilled on ice, mixed with 5 μL of First-Strand Reaction Buffer (Invitrogen), 2.5 μL of 0.1 M DTT, 1.25 μL of 10 mM RNase-free dNTP mix, 3 μL of SuperScript III reverse transcriptase, and incubated at 50 °C for 2 h. To generate the second strand, the following Invitrogen reagents were added: 86 μL of RNase-free water, 30 μL of second-strand reaction buffer, 3 μL of 10 mM RNase-free dNTP mix, 10 units Escherichia coli DNA Ligase, 40 U E. coli DNA Polymerase, 2 U E. coli RNase H, and incubated at 16 °C for 2 h. Ten units of T4 DNA Polymerase (Invitrogen) were added and the reactions were incubated for an additional 5 min at 16 °C. .. Reactions were stopped by adding 10 μL of 0.5 M RNase-free EDTA and purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: A new strategy to amplify degraded RNA from small tissue samples for microarray studies
    Article Snippet: For second strand cDNA synthesis, 43 µl of RNase-free water, 20 µl of 5× second-strand buffer (Invitrogen, Carlsbad, CA), 2 µl of 10 mM dNTPs, 1 µl of Escherichia coli DNA ligase (Invitrogen, Carlsbad, CA), 3 µl of E.coli DNA polymerse I (Invitrogen, Carlsbad, CA), and 1 µl of RNase H (Invitrogen, Carlsbad, CA) were added to the RT reaction mix. .. The resulting solution was incubated at 16°C for 2 h. At the end of this time, 2 µl of T4 DNA polymerase (Invitrogen, Carlsbad, CA) were added and the samples were incubated at 16°C for 5 min.

    Amplification:

    Article Title: A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains
    Article Snippet: Paragraph title: Unbiased amplification of TCR genes ... After cDNA synthesis, double strand (ds)-cDNA was synthesized with E. coli DNA polymerase I (Invitrogen), E. coli DNA Ligase (Invitrogen), and RNase H (Invitrogen). ds-cDNAs were blunted with T4 DNA polymerase (Invitrogen).

    Article Title: Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis
    Article Snippet: Paragraph title: Unbiased Amplification of Antibody Genes with AL-PCR ... Following cDNA synthesis, double-stranded (ds)-cDNA was synthesized with E. coli DNA polymerase I (Invitrogen), E. coli DNA ligase (Invitrogen), and RNase H (Invitrogen), after which the ds-cDNA was blunted with T4 DNA polymerase (Invitrogen).

    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: Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system
    Article Snippet: .. The SPIA™ amplified single strand cDNA (2.5 ug) was then taken through a second strand cDNA synthesis, using the following conditions: 5X 2nd strand reaction mix 30 μl (Invitrogen), dNTP, 10 mM 3 μl (Invitrogen), E. coli DNA ligase 1 μl (Invitrogen), E. coli DNA polymerase I 4 μl (Invitrogen), RNase H 1 μl (Invitrogen), RNase-free water 91 μl (Ambion). ..

    Article Title: A new strategy to amplify degraded RNA from small tissue samples for microarray studies
    Article Snippet: Paragraph title: RNA sample preparation and amplification ... For second strand cDNA synthesis, 43 µl of RNase-free water, 20 µl of 5× second-strand buffer (Invitrogen, Carlsbad, CA), 2 µl of 10 mM dNTPs, 1 µl of Escherichia coli DNA ligase (Invitrogen, Carlsbad, CA), 3 µl of E.coli DNA polymerse I (Invitrogen, Carlsbad, CA), and 1 µl of RNase H (Invitrogen, Carlsbad, CA) were added to the RT reaction mix.

    Modification:

    Article Title: A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection
    Article Snippet: For second-strand cDNA synthesis, the 40 μL first-strand reaction was mixed with 60 μL of 5× second-strand reaction buffer, 6 μL of 10 mM dNTP mix, 20 units of Escherichia coli DNA ligase, 80 units of E. coli DNA polymerase I (Invitrogen), and 4 units of E. coli RNase H (Epicentre) in a total volume of 300 μL for 2 h at 16°C. .. Double-stranded cDNA was further purified using Qiaquick PCR purification kit (Qiagen), and then labeled using BioPrime DNA labeling system (Invitrogen) with conditions modified as previously described ( ); 95 μL labeling reaction from each of four cDNA samples was subjected to hybridization to AtTILE1 forward chips (Affymetrix), using hybridization protocol for eukaryotic cRNA target (Affymetrix).

    Hybridization:

    Article Title: A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection
    Article Snippet: Paragraph title: Tiling array sample, hybridization, data acquisition, and analysis ... For second-strand cDNA synthesis, the 40 μL first-strand reaction was mixed with 60 μL of 5× second-strand reaction buffer, 6 μL of 10 mM dNTP mix, 20 units of Escherichia coli DNA ligase, 80 units of E. coli DNA polymerase I (Invitrogen), and 4 units of E. coli RNase H (Epicentre) in a total volume of 300 μL for 2 h at 16°C.

    High Performance Liquid Chromatography:

    Article Title: Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method
    Article Snippet: .. Materials The following materials were obtained from the sources indicated: HPLC purified T7-(dT)24 primer [5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGC GG(T)24 ] from GE Healthcare UK Ltd. (Buckinghamshire, England), RNase-free water, dNTP, SusperScript II, Escherichia coli (E. coli ) RNase H, E. coli DNA polymerase I, E. coli DNA ligase, T4 DNA polymerase and random hexamers from Invitrogen (San Diego, CA), RNase inhibitor, glycogen, and MEGAscript T7 kit from Ambion (Austin, TX). .. Balb/c mice were obtained from JapanClea (Hamamatsu, Japan).

    Ligation:

    Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation
    Article Snippet: The second strand was generated using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 μmol each, Promega), in the Second Strand Buffer (Invitrogen). .. Adapter ligation was performed using indexed adapters and Quick Ligase (NEB).

    Generated:

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae
    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen). .. The gels were blotted onto Hybond N+ nitrocellulose membranes (Amersham Biosciences) and hybridized with probes generated from the differentially expressed cDNA clones by the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals).

    Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation
    Article Snippet: .. The second strand was generated using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 μmol each, Promega), in the Second Strand Buffer (Invitrogen). .. Then the cDNA were sheared with Covaris to roughly 300 bp and purified with MinElute columns (Qiagen).

    Article Title: Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing
    Article Snippet: Samples were resuspended in 15 μL of RNase-free water. cDNA was generated according to instructions given in Invitrogen's SuperScript Double-Stranded cDNA Synthesis Kit. .. Briefly, each mRNA sample was mixed with 100 pmol of random hexamers (Integrated DNA Technologies), incubated at 70 °C for 10 min, chilled on ice, mixed with 5 μL of First-Strand Reaction Buffer (Invitrogen), 2.5 μL of 0.1 M DTT, 1.25 μL of 10 mM RNase-free dNTP mix, 3 μL of SuperScript III reverse transcriptase, and incubated at 50 °C for 2 h. To generate the second strand, the following Invitrogen reagents were added: 86 μL of RNase-free water, 30 μL of second-strand reaction buffer, 3 μL of 10 mM RNase-free dNTP mix, 10 units Escherichia coli DNA Ligase, 40 U E. coli DNA Polymerase, 2 U E. coli RNase H, and incubated at 16 °C for 2 h. Ten units of T4 DNA Polymerase (Invitrogen) were added and the reactions were incubated for an additional 5 min at 16 °C.

    DNA Labeling:

    Article Title: A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection
    Article Snippet: For second-strand cDNA synthesis, the 40 μL first-strand reaction was mixed with 60 μL of 5× second-strand reaction buffer, 6 μL of 10 mM dNTP mix, 20 units of Escherichia coli DNA ligase, 80 units of E. coli DNA polymerase I (Invitrogen), and 4 units of E. coli RNase H (Epicentre) in a total volume of 300 μL for 2 h at 16°C. .. Double-stranded cDNA was further purified using Qiaquick PCR purification kit (Qiagen), and then labeled using BioPrime DNA labeling system (Invitrogen) with conditions modified as previously described ( ); 95 μL labeling reaction from each of four cDNA samples was subjected to hybridization to AtTILE1 forward chips (Affymetrix), using hybridization protocol for eukaryotic cRNA target (Affymetrix).

    Polymerase Chain Reaction:

    Article Title: A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains
    Article Snippet: After cDNA synthesis, double strand (ds)-cDNA was synthesized with E. coli DNA polymerase I (Invitrogen), E. coli DNA Ligase (Invitrogen), and RNase H (Invitrogen). ds-cDNAs were blunted with T4 DNA polymerase (Invitrogen). .. After removal of adaptor and primer with MinElute Reaction Cleanup kit (Qiagen), PCR was performed using either TCR α-chain constant region-specific (CA1) or TCR β-chain constant region-specific primers (CB1) and P20EA (Table ).

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae
    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen). .. The gels were blotted onto Hybond N+ nitrocellulose membranes (Amersham Biosciences) and hybridized with probes generated from the differentially expressed cDNA clones by the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals).

    Article Title: A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection
    Article Snippet: For second-strand cDNA synthesis, the 40 μL first-strand reaction was mixed with 60 μL of 5× second-strand reaction buffer, 6 μL of 10 mM dNTP mix, 20 units of Escherichia coli DNA ligase, 80 units of E. coli DNA polymerase I (Invitrogen), and 4 units of E. coli RNase H (Epicentre) in a total volume of 300 μL for 2 h at 16°C. .. Double-stranded cDNA was further purified using Qiaquick PCR purification kit (Qiagen), and then labeled using BioPrime DNA labeling system (Invitrogen) with conditions modified as previously described ( ); 95 μL labeling reaction from each of four cDNA samples was subjected to hybridization to AtTILE1 forward chips (Affymetrix), using hybridization protocol for eukaryotic cRNA target (Affymetrix).

    Article Title: Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis
    Article Snippet: Following cDNA synthesis, double-stranded (ds)-cDNA was synthesized with E. coli DNA polymerase I (Invitrogen), E. coli DNA ligase (Invitrogen), and RNase H (Invitrogen), after which the ds-cDNA was blunted with T4 DNA polymerase (Invitrogen). .. After the removal of adaptor and primer with the MinElute Reaction Cleanup kit (Qiagen), PCR was performed with C-region-specific primers (heavy chain: IgM, IgD, IgG, IgA, and IgE) and P20EA (Table ) with KAPA HiFi DNA Polymerase (Kapa Biosystems, Woburn, MA, USA).

    Article Title: Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system
    Article Snippet: The SPIA™ amplified single strand cDNA (2.5 ug) was then taken through a second strand cDNA synthesis, using the following conditions: 5X 2nd strand reaction mix 30 μl (Invitrogen), dNTP, 10 mM 3 μl (Invitrogen), E. coli DNA ligase 1 μl (Invitrogen), E. coli DNA polymerase I 4 μl (Invitrogen), RNase H 1 μl (Invitrogen), RNase-free water 91 μl (Ambion). .. The cDNA was then purified using the Qiagen PCR clean up kit resulting 4 ug of cDNA quantified by using the Nanodrop spectrophotometer.

    Article Title: A new strategy to amplify degraded RNA from small tissue samples for microarray studies
    Article Snippet: The RNA templates were added to a 0.2 ml PCR tube containing 1 µl of T7dT (100 pmol/µl, 5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTT TTTTTTTTTTTTTTTT-3′, Operon, Alameda, CA) or T3N9 primer (100 pmol/µl, 5′-GCGCGAAATTAACCCTCACTAAAGGGAGANNNNNNNNN-3′, Operon, Alameda, CA),1 µl of RNasin (Promega, Madison, WI), and 6 µl of RNase-free water. .. For second strand cDNA synthesis, 43 µl of RNase-free water, 20 µl of 5× second-strand buffer (Invitrogen, Carlsbad, CA), 2 µl of 10 mM dNTPs, 1 µl of Escherichia coli DNA ligase (Invitrogen, Carlsbad, CA), 3 µl of E.coli DNA polymerse I (Invitrogen, Carlsbad, CA), and 1 µl of RNase H (Invitrogen, Carlsbad, CA) were added to the RT reaction mix.

    RNA Sequencing Assay:

    Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation
    Article Snippet: Paragraph title: RNA-Seq and analyses ... The second strand was generated using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 μmol each, Promega), in the Second Strand Buffer (Invitrogen).

    Isolation:

    Article Title: Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome
    Article Snippet: .. Total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), by using a T7-oligo-d(T)24 as a primer, 1× first-strand buffer and 1 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed using 40 U DNA polymerase I, 10 U E.coli DNA ligase, 2 U RNAse H (Invitrogen, Burlington, ON), 1× reaction buffer and 0.2 mM dNTPs at 16°C for 2 h. The addition of 10 U of T4 polynucleotide kinase (Invitrogen) stops the reaction, and cDNAs were incubated for 10 min at 16°C. cDNAs were isolated by phenol–chloroform extraction. .. Then, cDNAs were transcribed in vitro by using the T7 BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) to produce biotinylated cRNA.

    Article Title: ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells
    Article Snippet: Paragraph title: RNA Isolation and cDNA Synthesis ... Second strand synthesis was carried out using Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Affymetrix 900431).

    Article Title: A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection
    Article Snippet: The seeds from each line were stratified for 5 d and grown horizontally in a growth chamber (Percival Scientific Inc., model E361) for 3 d. About 20 μg of total RNA was isolated from 120 seedlings from each line using RNeasy plant mini kit (Qiagen). .. For second-strand cDNA synthesis, the 40 μL first-strand reaction was mixed with 60 μL of 5× second-strand reaction buffer, 6 μL of 10 mM dNTP mix, 20 units of Escherichia coli DNA ligase, 80 units of E. coli DNA polymerase I (Invitrogen), and 4 units of E. coli RNase H (Epicentre) in a total volume of 300 μL for 2 h at 16°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: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation
    Article Snippet: Total RNA was extracted using Tripure Isolation Reagent (Roche). .. The second strand was generated using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 μmol each, Promega), in the Second Strand Buffer (Invitrogen).

    Labeling:

    Article Title: Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome
    Article Snippet: Total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), by using a T7-oligo-d(T)24 as a primer, 1× first-strand buffer and 1 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed using 40 U DNA polymerase I, 10 U E.coli DNA ligase, 2 U RNAse H (Invitrogen, Burlington, ON), 1× reaction buffer and 0.2 mM dNTPs at 16°C for 2 h. The addition of 10 U of T4 polynucleotide kinase (Invitrogen) stops the reaction, and cDNAs were incubated for 10 min at 16°C. cDNAs were isolated by phenol–chloroform extraction. .. Total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), by using a T7-oligo-d(T)24 as a primer, 1× first-strand buffer and 1 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed using 40 U DNA polymerase I, 10 U E.coli DNA ligase, 2 U RNAse H (Invitrogen, Burlington, ON), 1× reaction buffer and 0.2 mM dNTPs at 16°C for 2 h. The addition of 10 U of T4 polynucleotide kinase (Invitrogen) stops the reaction, and cDNAs were incubated for 10 min at 16°C. cDNAs were isolated by phenol–chloroform extraction.

    Article Title: ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells
    Article Snippet: Second strand synthesis was carried out using Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Affymetrix 900431). .. Five microliters of purified ds cDNA was used to generate biotin-labeled cRNA using the IVT labeling kit (Affymetrix 900449).

    Article Title: A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection
    Article Snippet: For second-strand cDNA synthesis, the 40 μL first-strand reaction was mixed with 60 μL of 5× second-strand reaction buffer, 6 μL of 10 mM dNTP mix, 20 units of Escherichia coli DNA ligase, 80 units of E. coli DNA polymerase I (Invitrogen), and 4 units of E. coli RNase H (Epicentre) in a total volume of 300 μL for 2 h at 16°C. .. Double-stranded cDNA was further purified using Qiaquick PCR purification kit (Qiagen), and then labeled using BioPrime DNA labeling system (Invitrogen) with conditions modified as previously described ( ); 95 μL labeling reaction from each of four cDNA samples was subjected to hybridization to AtTILE1 forward chips (Affymetrix), using hybridization protocol for eukaryotic cRNA target (Affymetrix).

    Article Title: Probabilistic estimation of microarray data reliability and underlying gene expression
    Article Snippet: This was followed by a second strand synthesis for 2 hours at 16 = B0C, using RNAseH, E coli DNA polymerase I and E coli DNA ligase (all from GIBCO), according to the manufacturers instructions. .. The cDNA was then used in an in vitro transcription reaction for 6 h at 37 = B0C using a T7 IVT kit and biotin labeled ribonucloetides.

    Article Title: A genome-wide approach reveals novel imprinted genes expressed in the human placenta
    Article Snippet: .. Genotyping arrays After validation of the RNA quality with the Agilent Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit) controlling for the absence of genomic contamination, 4 μg of total RNA were reverse transcribed using the One Cycle Target Labeling Kit (Affymetrix): briefly, a first strand cDNA synthesis was performed using Superscript II and T7oligodT followed by a second strand cDNA synthesis using E.coli DNA ligase, E.coli DNA polymerase and RNase H. The double strand cDNA was then purified using cDNA clean up spin column, eluted and quantified with the Nanodrop ND1000 UV-Vis Spectrophotometer (Nanodrop Technologies, Inc.). ..

    Purification:

    Article Title: Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome
    Article Snippet: Total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), by using a T7-oligo-d(T)24 as a primer, 1× first-strand buffer and 1 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed using 40 U DNA polymerase I, 10 U E.coli DNA ligase, 2 U RNAse H (Invitrogen, Burlington, ON), 1× reaction buffer and 0.2 mM dNTPs at 16°C for 2 h. The addition of 10 U of T4 polynucleotide kinase (Invitrogen) stops the reaction, and cDNAs were incubated for 10 min at 16°C. cDNAs were isolated by phenol–chloroform extraction. .. The mixture (20 μl final volume) was incubated at 37°C for 5 h, with gentle mixing every 30 min. Labelled cRNAs were purified by using an RNeasy Mini Kit (Qiagen, Valencia, CA).

    Article Title: ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells
    Article Snippet: Second strand synthesis was carried out using Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Affymetrix 900431). .. Five microliters of purified ds cDNA was used to generate biotin-labeled cRNA using the IVT labeling kit (Affymetrix 900449).

    Article Title: A large number of novel coding small open reading frames in the intergenic regions of the Arabidopsis thaliana genome are transcribed and/or under purifying selection
    Article Snippet: For second-strand cDNA synthesis, the 40 μL first-strand reaction was mixed with 60 μL of 5× second-strand reaction buffer, 6 μL of 10 mM dNTP mix, 20 units of Escherichia coli DNA ligase, 80 units of E. coli DNA polymerase I (Invitrogen), and 4 units of E. coli RNase H (Epicentre) in a total volume of 300 μL for 2 h at 16°C. .. Double-stranded cDNA was further purified using Qiaquick PCR purification kit (Qiagen), and then labeled using BioPrime DNA labeling system (Invitrogen) with conditions modified as previously described ( ); 95 μL labeling reaction from each of four cDNA samples was subjected to hybridization to AtTILE1 forward chips (Affymetrix), using hybridization protocol for eukaryotic cRNA target (Affymetrix).

    Article Title: Probabilistic estimation of microarray data reliability and underlying gene expression
    Article Snippet: This was followed by a second strand synthesis for 2 hours at 16 = B0C, using RNAseH, E coli DNA polymerase I and E coli DNA ligase (all from GIBCO), according to the manufacturers instructions. .. The material was then purified by Phenol:Cloroform:Isoamyl alcohol extraction followed by precipitation with NH4Ac and Ethanol.

    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 genome-wide approach reveals novel imprinted genes expressed in the human placenta
    Article Snippet: .. Genotyping arrays After validation of the RNA quality with the Agilent Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit) controlling for the absence of genomic contamination, 4 μg of total RNA were reverse transcribed using the One Cycle Target Labeling Kit (Affymetrix): briefly, a first strand cDNA synthesis was performed using Superscript II and T7oligodT followed by a second strand cDNA synthesis using E.coli DNA ligase, E.coli DNA polymerase and RNase H. The double strand cDNA was then purified using cDNA clean up spin column, eluted and quantified with the Nanodrop ND1000 UV-Vis Spectrophotometer (Nanodrop Technologies, Inc.). ..

    Article Title: Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis
    Article Snippet: Paragraph title: Synthesis and purification of cDNA ... Second-strand synthesis was performed with E. coli DNA ligase, DNA polymerase I, RNase H, and T4 DNA polymerase in 1× Second-Strand Reaction Buffer (all supplied by Invitrogen).

    Article Title: Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system
    Article Snippet: The SPIA™ amplified single strand cDNA (2.5 ug) was then taken through a second strand cDNA synthesis, using the following conditions: 5X 2nd strand reaction mix 30 μl (Invitrogen), dNTP, 10 mM 3 μl (Invitrogen), E. coli DNA ligase 1 μl (Invitrogen), E. coli DNA polymerase I 4 μl (Invitrogen), RNase H 1 μl (Invitrogen), RNase-free water 91 μl (Ambion). .. The cDNA was then purified using the Qiagen PCR clean up kit resulting 4 ug of cDNA quantified by using the Nanodrop spectrophotometer.

    Article Title: Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method
    Article Snippet: .. Materials The following materials were obtained from the sources indicated: HPLC purified T7-(dT)24 primer [5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGC GG(T)24 ] from GE Healthcare UK Ltd. (Buckinghamshire, England), RNase-free water, dNTP, SusperScript II, Escherichia coli (E. coli ) RNase H, E. coli DNA polymerase I, E. coli DNA ligase, T4 DNA polymerase and random hexamers from Invitrogen (San Diego, CA), RNase inhibitor, glycogen, and MEGAscript T7 kit from Ambion (Austin, TX). .. Balb/c mice were obtained from JapanClea (Hamamatsu, Japan).

    Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation
    Article Snippet: RNA samples were treated with RNase-free DNaseI and purified using the RNeasy Mini Kit (QIAGEN). .. The second strand was generated using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 μmol each, Promega), in the Second Strand Buffer (Invitrogen).

    Article Title: Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing
    Article Snippet: Paragraph title: mRNA Purification and cDNA Synthesis. ... Briefly, each mRNA sample was mixed with 100 pmol of random hexamers (Integrated DNA Technologies), incubated at 70 °C for 10 min, chilled on ice, mixed with 5 μL of First-Strand Reaction Buffer (Invitrogen), 2.5 μL of 0.1 M DTT, 1.25 μL of 10 mM RNase-free dNTP mix, 3 μL of SuperScript III reverse transcriptase, and incubated at 50 °C for 2 h. To generate the second strand, the following Invitrogen reagents were added: 86 μL of RNase-free water, 30 μL of second-strand reaction buffer, 3 μL of 10 mM RNase-free dNTP mix, 10 units Escherichia coli DNA Ligase, 40 U E. coli DNA Polymerase, 2 U E. coli RNase H, and incubated at 16 °C for 2 h. Ten units of T4 DNA Polymerase (Invitrogen) were added and the reactions were incubated for an additional 5 min at 16 °C.

    Sequencing:

    Article Title: ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells
    Article Snippet: First-strand synthesis was initiated using Superscript II Reverse Transcriptase and the T7-(dt) primer (Affymetrix 900431), which contains the T7 promoter sequence followed by an oligo (dt) tract. .. Second strand synthesis was carried out using Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Affymetrix 900431).

    Article Title: Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system
    Article Snippet: RNA amplification and cDNA preparation To obtain enough cDNA for sequencing, the 16s and 23s rRNA depleted RNAs (sample 1 and 2) were amplified using Nugen WT-Ovation Pico RNA amplification system [ ]. .. The SPIA™ amplified single strand cDNA (2.5 ug) was then taken through a second strand cDNA synthesis, using the following conditions: 5X 2nd strand reaction mix 30 μl (Invitrogen), dNTP, 10 mM 3 μl (Invitrogen), E. coli DNA ligase 1 μl (Invitrogen), E. coli DNA polymerase I 4 μl (Invitrogen), RNase H 1 μl (Invitrogen), RNase-free water 91 μl (Ambion).

    Nested PCR:

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae
    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen). .. Primers used for probe generation were the Clontech primers, nested PCR primer 1 and nested PCR primer 2R (PCR-Select cDNA Subtraction Kit, see above).

    Agarose Gel Electrophoresis:

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae
    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen). .. For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels.

    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.

    Mouse Assay:

    Article Title: Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method
    Article Snippet: Materials The following materials were obtained from the sources indicated: HPLC purified T7-(dT)24 primer [5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGC GG(T)24 ] from GE Healthcare UK Ltd. (Buckinghamshire, England), RNase-free water, dNTP, SusperScript II, Escherichia coli (E. coli ) RNase H, E. coli DNA polymerase I, E. coli DNA ligase, T4 DNA polymerase and random hexamers from Invitrogen (San Diego, CA), RNase inhibitor, glycogen, and MEGAscript T7 kit from Ambion (Austin, TX). .. Balb/c mice were obtained from JapanClea (Hamamatsu, Japan).

    Chromatin Immunoprecipitation:

    Article Title: A genome-wide approach reveals novel imprinted genes expressed in the human placenta
    Article Snippet: .. Genotyping arrays After validation of the RNA quality with the Agilent Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit) controlling for the absence of genomic contamination, 4 μg of total RNA were reverse transcribed using the One Cycle Target Labeling Kit (Affymetrix): briefly, a first strand cDNA synthesis was performed using Superscript II and T7oligodT followed by a second strand cDNA synthesis using E.coli DNA ligase, E.coli DNA polymerase and RNase H. The double strand cDNA was then purified using cDNA clean up spin column, eluted and quantified with the Nanodrop ND1000 UV-Vis Spectrophotometer (Nanodrop Technologies, Inc.). ..

    RNA Extraction:

    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.

    shRNA:

    Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation
    Article Snippet: RNA-Seq and analyses RNA-seq was performed on three biological replicates of IMR90 cells with shRNA targeting macroH2A1 or Luciferase grown to 90% confluence in 6-wells dishes. .. The second strand was generated using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 μmol each, Promega), in the Second Strand Buffer (Invitrogen).

    Sample Prep:

    Article Title: A new strategy to amplify degraded RNA from small tissue samples for microarray studies
    Article Snippet: Paragraph title: RNA sample preparation and amplification ... For second strand cDNA synthesis, 43 µl of RNase-free water, 20 µl of 5× second-strand buffer (Invitrogen, Carlsbad, CA), 2 µl of 10 mM dNTPs, 1 µl of Escherichia coli DNA ligase (Invitrogen, Carlsbad, CA), 3 µl of E.coli DNA polymerse I (Invitrogen, Carlsbad, CA), and 1 µl of RNase H (Invitrogen, Carlsbad, CA) were added to the RT reaction mix.

    In Vitro:

    Article Title: Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome
    Article Snippet: Total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), by using a T7-oligo-d(T)24 as a primer, 1× first-strand buffer and 1 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed using 40 U DNA polymerase I, 10 U E.coli DNA ligase, 2 U RNAse H (Invitrogen, Burlington, ON), 1× reaction buffer and 0.2 mM dNTPs at 16°C for 2 h. The addition of 10 U of T4 polynucleotide kinase (Invitrogen) stops the reaction, and cDNAs were incubated for 10 min at 16°C. cDNAs were isolated by phenol–chloroform extraction. .. Then, cDNAs were transcribed in vitro by using the T7 BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) to produce biotinylated cRNA.

    Article Title: Probabilistic estimation of microarray data reliability and underlying gene expression
    Article Snippet: This was followed by a second strand synthesis for 2 hours at 16 = B0C, using RNAseH, E coli DNA polymerase I and E coli DNA ligase (all from GIBCO), according to the manufacturers instructions. .. The cDNA was then used in an in vitro transcription reaction for 6 h at 37 = B0C using a T7 IVT kit and biotin labeled ribonucloetides.

    Ethanol Precipitation:

    Article Title: Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing
    Article Snippet: Briefly, each mRNA sample was mixed with 100 pmol of random hexamers (Integrated DNA Technologies), incubated at 70 °C for 10 min, chilled on ice, mixed with 5 μL of First-Strand Reaction Buffer (Invitrogen), 2.5 μL of 0.1 M DTT, 1.25 μL of 10 mM RNase-free dNTP mix, 3 μL of SuperScript III reverse transcriptase, and incubated at 50 °C for 2 h. To generate the second strand, the following Invitrogen reagents were added: 86 μL of RNase-free water, 30 μL of second-strand reaction buffer, 3 μL of 10 mM RNase-free dNTP mix, 10 units Escherichia coli DNA Ligase, 40 U E. coli DNA Polymerase, 2 U E. coli RNase H, and incubated at 16 °C for 2 h. Ten units of T4 DNA Polymerase (Invitrogen) were added and the reactions were incubated for an additional 5 min at 16 °C. .. Reactions were stopped by adding 10 μL of 0.5 M RNase-free EDTA and purified by phenol-chloroform extraction and ethanol precipitation.

    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.

    Spectrophotometry:

    Article Title: A genome-wide approach reveals novel imprinted genes expressed in the human placenta
    Article Snippet: .. Genotyping arrays After validation of the RNA quality with the Agilent Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit) controlling for the absence of genomic contamination, 4 μg of total RNA were reverse transcribed using the One Cycle Target Labeling Kit (Affymetrix): briefly, a first strand cDNA synthesis was performed using Superscript II and T7oligodT followed by a second strand cDNA synthesis using E.coli DNA ligase, E.coli DNA polymerase and RNase H. The double strand cDNA was then purified using cDNA clean up spin column, eluted and quantified with the Nanodrop ND1000 UV-Vis Spectrophotometer (Nanodrop Technologies, Inc.). ..

    Article Title: Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system
    Article Snippet: The SPIA™ amplified single strand cDNA (2.5 ug) was then taken through a second strand cDNA synthesis, using the following conditions: 5X 2nd strand reaction mix 30 μl (Invitrogen), dNTP, 10 mM 3 μl (Invitrogen), E. coli DNA ligase 1 μl (Invitrogen), E. coli DNA polymerase I 4 μl (Invitrogen), RNase H 1 μl (Invitrogen), RNase-free water 91 μl (Ambion). .. The cDNA was then purified using the Qiagen PCR clean up kit resulting 4 ug of cDNA quantified by using the Nanodrop spectrophotometer.

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