e coli dna polymerase i  (Thermo Fisher)


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    Name:
    DNA Polymerase I
    Description:
    DNA Polymerase I is a DNA polymerase with 5 →3 and 3 →5 exodeoxyribonuclease activities DNA Polymerase I also incorporates biotinylated nucleotides • Applications DNase I dependent nick translation second strand synthesis in cDNA cloning fill in of 5 overhangs• Source purified from E coli expressing the DNA Polymerase I gene on a plasmidPerformance and quality testing Endodeoxyribonuclease assay efficiency of DNase I dependent nick translation determined Unit definition One unit incorporates 10 nmol of total deoxyribonucleotide into acid precipitable material in 30 min at 37°C using template primer Unit reaction conditions 50 mM potassium phosphate pH 7 0 6 7 mM MgCl2 1 mM 2 mercaptoethanol 80 µg mL template primer 32 µM dTTP 69 nM 3H dTTP and enzyme in 100 µL for 30 min at 37°C
    Catalog Number:
    18010017
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher e coli dna polymerase i
    DNA Polymerase I is a DNA polymerase with 5 →3 and 3 →5 exodeoxyribonuclease activities DNA Polymerase I also incorporates biotinylated nucleotides • Applications DNase I dependent nick translation second strand synthesis in cDNA cloning fill in of 5 overhangs• Source purified from E coli expressing the DNA Polymerase I gene on a plasmidPerformance and quality testing Endodeoxyribonuclease assay efficiency of DNase I dependent nick translation determined Unit definition One unit incorporates 10 nmol of total deoxyribonucleotide into acid precipitable material in 30 min at 37°C using template primer Unit reaction conditions 50 mM potassium phosphate pH 7 0 6 7 mM MgCl2 1 mM 2 mercaptoethanol 80 µg mL template primer 32 µM dTTP 69 nM 3H dTTP and enzyme in 100 µL for 30 min at 37°C
    https://www.bioz.com/result/e coli dna polymerase i/product/Thermo Fisher
    Average 90 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    e coli dna polymerase i - by Bioz Stars, 2020-02
    90/100 stars

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    Related Articles

    Functional Assay:

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014). .. Sequencing was performed on an Illumina NextSeq 500 by Stanford Functional Genomics Facility.

    Synthesized:

    Article Title: Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat
    Article Snippet: Second strand cDNA was synthesized by incubating first strand cDNA with second strand buffer, RNase Out and dNTP from the Illumina RNA-seq kit on ice for 5 min. .. The reaction mix was then treated with DNA Pol I and RNaseH at 16°C for 2.5 h (Invitrogen).

    Article Title: Large-scale transcriptome sequencing and gene analyses in the crab-eating macaque (Macaca fascicularis) for biomedical research
    Article Snippet: .. Second strand cDNA was synthesized by DNA pol I and RNase H (Fermentas), and the poly(A) tail was removed using a specific enzyme (Gsul). .. Library preparation for GS FLX sequencing The first step of library preparation involves the fragmentation of the high molecular weight DNA sample into smaller molecular species appropriate for sequencing using GS FLX Titanium chemistry.

    Article Title: H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress
    Article Snippet: .. Second-strand cDNA was synthesized using DNA Pol I, DNA ligase (both Invitrogen), and RNase H (NEB) with random hexamers (Applied Biosystems) in the presence of dUTP. .. The libraries were prepared by the Vienna Biocenter CSF NGS unit using the NEBNext Library Prep Reagent Set for Illumina (NEB), multiplexed (2 samples/lane), and sequenced on HiSeq 2000 (Illumina) at the CSF NGS unit (50 bp single-end reads).

    Article Title: Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts
    Article Snippet: .. Second strand cDNA was synthesized using RNase H and DNA pol I (Gibco-BRL). .. The double-stranded cDNA then was blunt-ended, and ligated first to EcoR I linkers and then to λgtll phage arms (Promega).

    Microarray:

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: RNA-seq Extraction of RNA using bam testes or S2 cells was performed using similar methods as described for microarray experiments. .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Random Hexamer Labeling:

    Article Title: Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA
    Article Snippet: The resulting RNA preparations were used in reverse transcription reactions using random hexamer primers and Superscript II Reverse Transcriptase (Invitrogen) following the protocol provided by the manufacturer. .. Second strand synthesis was carried out using Dna Pol I and RnaseH (both Invitrogen) following the protocol provided by the manufacturer.

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: First strand synthesis was performed using Random Hexamer Primers (Invitrogen, #48190-011) and SuperScript II (Invitrogen, #18064-014). .. Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014).

    Expressing:

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014). .. Reads were mapped to hg19 annotation using Tophat2 (version 2.0.13) and transcript expression was quantified against RefSeq gene annotations using featureCounts .

    Article Title: H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress
    Article Snippet: Second-strand cDNA was synthesized using DNA Pol I, DNA ligase (both Invitrogen), and RNase H (NEB) with random hexamers (Applied Biosystems) in the presence of dUTP. .. Differential expression was performed using htseq-count script ( ) with the “union” model and the Bioconductor package edgeR ( ; ).

    Flow Cytometry:

    Article Title: Mapping segmental and sequence variations among laboratory mice using BAC array CGH
    Article Snippet: We denatured the DNA in a thermal cycler at 100°C for 15 min and cooled it to 4°C before adding 12 units of Klenow fragment (Bioprime DNA labeling system; Invitrogen). .. We removed unincorporated nucleotides from the labeling reaction using a Sephadex G-50 spin column and visually assessed the labeling efficiency by the intensity of the color of the flow-through.

    Chromatography:

    Article Title: Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts
    Article Snippet: Poly-A+ RNA was purified by poly-U Sephadex chromatography according to the manufacturer’s instructions (Gibco-BRL). cDNA was synthesized using 5 μg Poly-A+ RNA by the procedure of , with modifications. .. Second strand cDNA was synthesized using RNase H and DNA pol I (Gibco-BRL).

    Ligation:

    Article Title: Frequent miRNA-convergent fusion gene events in breast cancer
    Article Snippet: Second strand synthesis was performed in 100 µl reactions with purified first strand cDNA (~16 µl) and 84 µl of second strand master mix containing 1.3 µl 5× First Strand buffer, 20 µl 5× Second Strand buffer, 3 µl 10 mM dNTPs with dUTP instead of dTTP, 1 µl 0.1 M DTT, 5 µl DNA Pol I (10 U/µl), 0.2 µl RNase H (10 U/µl), and 53.5 µl H2 O (all reagents from Invitrogen/Thermo Fisher Scientific). .. Adapter ligation was then performed in 50 µl reactions containing 40 µl of end-repaired and A-tailed double-stranded cDNA, 1 µl of a 1:5 dilution of adapters from TruSeq DNA LT Sample Prep Kit A and B (Illumina, San Diego, CA, USA), 3 µl T4 DNA ligase (5 U/µl), 1 µl 10× T4 DNA ligase buffer, and 5 µl H2 O by incubation at 22 °C 30 min, 4 °C hold.

    Article Title: A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs
    Article Snippet: RNA–cDNA hybrids were nicked with RNase H (2 U/µL, Invitrogen, 18021-014) and second strand cDNA was polymerized with DNA Pol I (10 U/µL, Invitrogen, 18010-025). .. The double-stranded cDNA library was sent to the UNC genomics core facility for size selection and subsequent ligation of the Illumina Genomic DNA oligonucleotide Adapters.

    Article Title: Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape
    Article Snippet: Second strand synthesis was conducted with DNA Pol I in the presence of RNase H (Invitrogen). .. To prepare samples for ligation of adapters, adenosine overhangs were added to library fragments with Klenow (3′to5′exo-) polymerase (New England Biolabs).

    Generated:

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). ..

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). ..

    DNA Labeling:

    Article Title: Mapping segmental and sequence variations among laboratory mice using BAC array CGH
    Article Snippet: .. We denatured the DNA in a thermal cycler at 100°C for 15 min and cooled it to 4°C before adding 12 units of Klenow fragment (Bioprime DNA labeling system; Invitrogen). .. We removed unincorporated nucleotides from the labeling reaction using a Sephadex G-50 spin column and visually assessed the labeling efficiency by the intensity of the color of the flow-through.

    Polymerase Chain Reaction:

    Article Title: Transcriptome sequencing and analysis of the entomopathogenic fungus Hirsutella sinensis isolated from Ophiocordyceps sinensis
    Article Snippet: The cleaned mRNA fragments primed by random primers were converted to double-stranded cDNA using SuperScript II (Invitrogen), RNase H (Invitrogen) and DNA Pol I (Invitrogen). .. The resulting cDNA was purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). cDNA was then subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK, and subsequent purification was performed using QIAquick PCR Purification Kit (Qiagen).

    Article Title: Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs
    Article Snippet: Following precipitation of fragmented RNA, first strand cDNA synthesis was performed using random N6 primers and Superscript II Reverse Transcriptase, followed by second strand cDNA synthesis using RNaseH and DNA pol I (Invitrogen, CA). .. Double-stranded cDNA was purified using Qiaquick PCR spin columns according to the manufacturer's protocol (Qiagen).

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. The double-stranded DNA (dsDNA) was purified with a QIAquick PCR purification kit (Qiagen, #28106) and the concentration was quantified using a Qubit fluorometer (Invitrogen).

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. The double-stranded DNA (dsDNA) was purified with QIAquick PCR purification kit (Qiagen, #28106) and the concentration was quantified by a Qubit fluorometer (Invitrogen).

    Sonication:

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. To generate sequencing libraries, about 300 ng dsDNA from each sample was fragmented by sonication using Bioruptor (Diagenode, UCD-200-TM-EX, Sparta, NJ, USA) using medium power output for 30 minutes in ice water.

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. For generating libraries for sequencing, about 300 ng dsDNA of each sample was fragmented by sonication using Bioruptor (Diagenode, UCD-200-TM-EX) under the following conditions: medium power output for 30 mins in ice water.

    Affinity Purification:

    Article Title: Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts
    Article Snippet: Second strand cDNA was synthesized using RNase H and DNA pol I (Gibco-BRL). .. Screening of the library was performed by standard procedures with an affinity-purified antibody to intestinal epithelial cell brush border myosin , followed by a goat anti-rabbit IgG-alkaline phosphatase conjugated secondary antibody (Boehringer Mannheim Biochemicals).

    Copurification:

    Article Title: Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA
    Article Snippet: Paragraph title: RNA-RsmA-H6 co-purification ... Second strand synthesis was carried out using Dna Pol I and RnaseH (both Invitrogen) following the protocol provided by the manufacturer.

    Nucleic Acid Electrophoresis:

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: The integrity of RNA was checked by gel electrophoresis (1% agarose). .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    RNA Sequencing Assay:

    Article Title: Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat
    Article Snippet: Second strand cDNA was synthesized by incubating first strand cDNA with second strand buffer, RNase Out and dNTP from the Illumina RNA-seq kit on ice for 5 min. .. The reaction mix was then treated with DNA Pol I and RNaseH at 16°C for 2.5 h (Invitrogen).

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Paragraph title: RNA Sequencing ... Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014).

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: Paragraph title: RNA-seq ... The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: Paragraph title: Library preparation for RNA-seq ... The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Magnetic Beads:

    Article Title: Transcriptome sequencing and analysis of the entomopathogenic fungus Hirsutella sinensis isolated from Ophiocordyceps sinensis
    Article Snippet: 10 mM Tris–HCl was used to elute the mRNA from the magnetic beads. .. The cleaned mRNA fragments primed by random primers were converted to double-stranded cDNA using SuperScript II (Invitrogen), RNase H (Invitrogen) and DNA Pol I (Invitrogen).

    Article Title: Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan
    Article Snippet: For poly (A+) RNA purification, 10 μg of total RNA was bound to dynal oligo (dT) magnetic beads (Invitrogen) twice, using the manufacturer's instructions. .. Superscript II (200 U; Invitrogen) were added and the samples were incubated at 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. For second strand synthesis, 51 μL of H2 O, 20 μL of 5× second strand buffer (Invitrogen), and dNTPs (10 mM) were added to the first cDNA strand synthesis mix and incubated on ice for 5 min. RNaseH (2 U; Invitrogen), DNA pol I (50 U; Invitrogen) were then added and the mixture was incubated at 16°C for 2.5h.

    Isolation:

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: For the total RNA from bam testes (8.5 μg) and S2 cells (approximately 20 μg), we performed two rounds of mRNA isolation using Dynabeads mRNA purification kit (Invitrogen, #610-06), according to the manufacturer's instructions. .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: From ~10 μg total RNA for each sample, we performed two rounds of mRNA isolation using Dynabeads mRNA purification kit (Invitrogen, #610-06), according to the manufacturer’s instructions. .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Article Title: Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape
    Article Snippet: Briefly, RNA was isolated from approximately 2×106 purity-sor ted cells using RNA-Bee (Tel-Test), treated with Turbo DNase (Ambion) and subsequently re-isolated with the RNA Clean-Up kit (Zymo Research). .. Second strand synthesis was conducted with DNA Pol I in the presence of RNase H (Invitrogen).

    Article Title: Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts
    Article Snippet: RNA was isolated from whole embryonic small intestine and adult epithelial cells and brain by the method of . .. Second strand cDNA was synthesized using RNase H and DNA pol I (Gibco-BRL).

    Negative Control:

    Article Title: A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs
    Article Snippet: Negative control RIPs were performed with irrelevant antibody, no antibody or by peptide competition. .. RNA–cDNA hybrids were nicked with RNase H (2 U/µL, Invitrogen, 18021-014) and second strand cDNA was polymerized with DNA Pol I (10 U/µL, Invitrogen, 18010-025).

    Labeling:

    Article Title: Mapping segmental and sequence variations among laboratory mice using BAC array CGH
    Article Snippet: For each labeling reaction, we used ∼300 ng each of test or reference genomic DNA in a volume of 15 μL containing 10 mM Tris-1 mM EDTA (pH 7.5); 0.2 mM unlabeled dATP, dCTP, and dGTP; 0.1 mM unlabeled dTTP; 1× random primer (Bioprime DNA labeling system; Invitrogen); and 0.4 mM Cyanine-3 (test) or Cyanine-5 (reference) conjugated dUTP (Amersham). .. We denatured the DNA in a thermal cycler at 100°C for 15 min and cooled it to 4°C before adding 12 units of Klenow fragment (Bioprime DNA labeling system; Invitrogen).

    Purification:

    Article Title: Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat
    Article Snippet: Fragmented RNA was purified by ethanol precipitation. .. The reaction mix was then treated with DNA Pol I and RNaseH at 16°C for 2.5 h (Invitrogen).

    Article Title: Frequent miRNA-convergent fusion gene events in breast cancer
    Article Snippet: .. Second strand synthesis was performed in 100 µl reactions with purified first strand cDNA (~16 µl) and 84 µl of second strand master mix containing 1.3 µl 5× First Strand buffer, 20 µl 5× Second Strand buffer, 3 µl 10 mM dNTPs with dUTP instead of dTTP, 1 µl 0.1 M DTT, 5 µl DNA Pol I (10 U/µl), 0.2 µl RNase H (10 U/µl), and 53.5 µl H2 O (all reagents from Invitrogen/Thermo Fisher Scientific). .. Reactions were incubated for 2.5 h at 16 °C before clean-up on Zymo-Spin I-96 plates with the Oligo Clean & Concentrator Kit (Zymo Research) and low EtOH (~57%) to preserve DNA > 80 bp.

    Article Title: Transcriptome sequencing and analysis of the entomopathogenic fungus Hirsutella sinensis isolated from Ophiocordyceps sinensis
    Article Snippet: The poly(A) containing mRNA molecules was purified using Sera-mag Magnetic Oligo(dT) Beads (Illumina) from 20 mg total RNA of each sample. .. The cleaned mRNA fragments primed by random primers were converted to double-stranded cDNA using SuperScript II (Invitrogen), RNase H (Invitrogen) and DNA Pol I (Invitrogen).

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Polyadenylated RNA was selected using Ambion Dynabeads mRNA Purification Kit (Life Technologies 61006) and fragmented with Fragmentation Buffer (Ambion, #AM8740). .. Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014).

    Article Title: Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan
    Article Snippet: Purified poly (A+) RNA was fragmented by metal-ion catalysis (Ambion) followed by precipitation with 1/10 vol 3M sodium acetate and 3× vol 100% ethanol. .. Superscript II (200 U; Invitrogen) were added and the samples were incubated at 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. For second strand synthesis, 51 μL of H2 O, 20 μL of 5× second strand buffer (Invitrogen), and dNTPs (10 mM) were added to the first cDNA strand synthesis mix and incubated on ice for 5 min. RNaseH (2 U; Invitrogen), DNA pol I (50 U; Invitrogen) were then added and the mixture was incubated at 16°C for 2.5h.

    Article Title: Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs
    Article Snippet: Following precipitation of fragmented RNA, first strand cDNA synthesis was performed using random N6 primers and Superscript II Reverse Transcriptase, followed by second strand cDNA synthesis using RNaseH and DNA pol I (Invitrogen, CA). .. Double-stranded cDNA was purified using Qiaquick PCR spin columns according to the manufacturer's protocol (Qiagen).

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: For the total RNA from bam testes (8.5 μg) and S2 cells (approximately 20 μg), we performed two rounds of mRNA isolation using Dynabeads mRNA purification kit (Invitrogen, #610-06), according to the manufacturer's instructions. .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: From ~10 μg total RNA for each sample, we performed two rounds of mRNA isolation using Dynabeads mRNA purification kit (Invitrogen, #610-06), according to the manufacturer’s instructions. .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Article Title: H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress
    Article Snippet: Ten micrograms of total RNA was subjected to two rounds of poly(A) selection with the Dynabeads mRNA Purification kit (Invitrogen). mRNA was subsequently fragmented by hydrolysis (40 mM TrisOAc at pH 8.2, 100 mM KOAc, 150 mM MgOAc) at 94°C for 3 min. First-strand cDNA synthesis was performed using the SuperScript III Reverse Transcriptase kit (Invitrogen) with random hexamers priming (Applied Biosystems) in the presence of actinomycin D (5 ng/μL). .. Second-strand cDNA was synthesized using DNA Pol I, DNA ligase (both Invitrogen), and RNase H (NEB) with random hexamers (Applied Biosystems) in the presence of dUTP.

    Article Title: Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts
    Article Snippet: Poly-A+ RNA was purified by poly-U Sephadex chromatography according to the manufacturer’s instructions (Gibco-BRL). cDNA was synthesized using 5 μg Poly-A+ RNA by the procedure of , with modifications. .. Second strand cDNA was synthesized using RNase H and DNA pol I (Gibco-BRL).

    Sequencing:

    Article Title: Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat
    Article Snippet: Paragraph title: cDNA synthesis—illumina sequencing ... The reaction mix was then treated with DNA Pol I and RNaseH at 16°C for 2.5 h (Invitrogen).

    Article Title: Frequent miRNA-convergent fusion gene events in breast cancer
    Article Snippet: Paragraph title: Strand-specific mRNA sequencing ... Second strand synthesis was performed in 100 µl reactions with purified first strand cDNA (~16 µl) and 84 µl of second strand master mix containing 1.3 µl 5× First Strand buffer, 20 µl 5× Second Strand buffer, 3 µl 10 mM dNTPs with dUTP instead of dTTP, 1 µl 0.1 M DTT, 5 µl DNA Pol I (10 U/µl), 0.2 µl RNase H (10 U/µl), and 53.5 µl H2 O (all reagents from Invitrogen/Thermo Fisher Scientific).

    Article Title: Transcriptome sequencing and analysis of the entomopathogenic fungus Hirsutella sinensis isolated from Ophiocordyceps sinensis
    Article Snippet: Paragraph title: Transcriptome sequencing and analysis ... The cleaned mRNA fragments primed by random primers were converted to double-stranded cDNA using SuperScript II (Invitrogen), RNase H (Invitrogen) and DNA Pol I (Invitrogen).

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014). .. Sequencing was performed on an Illumina NextSeq 500 by Stanford Functional Genomics Facility.

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. To generate sequencing libraries, about 300 ng dsDNA from each sample was fragmented by sonication using Bioruptor (Diagenode, UCD-200-TM-EX, Sparta, NJ, USA) using medium power output for 30 minutes in ice water.

    Article Title: Large-scale transcriptome sequencing and gene analyses in the crab-eating macaque (Macaca fascicularis) for biomedical research
    Article Snippet: cDNA synthesis and poly(A) tail removal First strand cDNA synthesis was conducted using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) using oligo(dT) primers optimized for the 454 sequencing procedures (5′- GAGCTAGTTCTGGAG(T)16 VN-3′). .. Second strand cDNA was synthesized by DNA pol I and RNase H (Fermentas), and the poly(A) tail was removed using a specific enzyme (Gsul).

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. For generating libraries for sequencing, about 300 ng dsDNA of each sample was fragmented by sonication using Bioruptor (Diagenode, UCD-200-TM-EX) under the following conditions: medium power output for 30 mins in ice water.

    Article Title: Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape
    Article Snippet: Paragraph title: Preparation of cDNA Libraries and Sequencing ... Second strand synthesis was conducted with DNA Pol I in the presence of RNase H (Invitrogen).

    Article Title: H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress
    Article Snippet: Paragraph title: mRNA sequencing ... Second-strand cDNA was synthesized using DNA Pol I, DNA ligase (both Invitrogen), and RNase H (NEB) with random hexamers (Applied Biosystems) in the presence of dUTP.

    Selection:

    Article Title: A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs
    Article Snippet: RNA–cDNA hybrids were nicked with RNase H (2 U/µL, Invitrogen, 18021-014) and second strand cDNA was polymerized with DNA Pol I (10 U/µL, Invitrogen, 18010-025). .. The double-stranded cDNA library was sent to the UNC genomics core facility for size selection and subsequent ligation of the Illumina Genomic DNA oligonucleotide Adapters.

    Article Title: Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape
    Article Snippet: Nine µg of total RNA from each sample was supplemented with 108 strands each of eight polyadenylated spikes (ArrayControl, Ambion) and subjected to two sequential rounds of poly(A) selection on oligo(dT) Dynabeads (Invitrogen). mRNA was partially hydrolyzed in Fragmentation Buffer (Ambion), followed by first strand cDNA synthesis with random hexamers using Superscript II (Invitrogen). .. Second strand synthesis was conducted with DNA Pol I in the presence of RNase H (Invitrogen).

    Article Title: H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress
    Article Snippet: Ten micrograms of total RNA was subjected to two rounds of poly(A) selection with the Dynabeads mRNA Purification kit (Invitrogen). mRNA was subsequently fragmented by hydrolysis (40 mM TrisOAc at pH 8.2, 100 mM KOAc, 150 mM MgOAc) at 94°C for 3 min. First-strand cDNA synthesis was performed using the SuperScript III Reverse Transcriptase kit (Invitrogen) with random hexamers priming (Applied Biosystems) in the presence of actinomycin D (5 ng/μL). .. Second-strand cDNA was synthesized using DNA Pol I, DNA ligase (both Invitrogen), and RNase H (NEB) with random hexamers (Applied Biosystems) in the presence of dUTP.

    cDNA Library Assay:

    Article Title: A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs
    Article Snippet: RNA–cDNA hybrids were nicked with RNase H (2 U/µL, Invitrogen, 18021-014) and second strand cDNA was polymerized with DNA Pol I (10 U/µL, Invitrogen, 18010-025). .. The double-stranded cDNA library was sent to the UNC genomics core facility for size selection and subsequent ligation of the Illumina Genomic DNA oligonucleotide Adapters.

    Article Title: Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts
    Article Snippet: Paragraph title: Preparation of RNA and colon epithelial cell cDNA library ... Second strand cDNA was synthesized using RNase H and DNA pol I (Gibco-BRL).

    RNA Extraction:

    Article Title: Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan
    Article Snippet: Paragraph title: RNA extraction and cDNA synthesis ... Superscript II (200 U; Invitrogen) were added and the samples were incubated at 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. For second strand synthesis, 51 μL of H2 O, 20 μL of 5× second strand buffer (Invitrogen), and dNTPs (10 mM) were added to the first cDNA strand synthesis mix and incubated on ice for 5 min. RNaseH (2 U; Invitrogen), DNA pol I (50 U; Invitrogen) were then added and the mixture was incubated at 16°C for 2.5h.

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: Samples used for total RNA extraction were from ~ 200 pairs of bam testes (8.5 μg), ~200 pairs of bam ovaries (6.9 μg), ~200 pairs of wt testes (19 μg) and 45 pairs of wt ovaries (15 μg). .. The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025).

    Multiplex Assay:

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014). .. Libraries were end-repaired, 3′ A-tailed, and ligated to NEBNext Multiplex Oligo Adaptors (NEB E7335S).

    Binding Assay:

    Article Title: Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA
    Article Snippet: Due to the use of random primers, the resulting cDNA fragments were shorter than the template RNA fragments and did not necessarily represent the exact location of RsmA binding. .. Second strand synthesis was carried out using Dna Pol I and RnaseH (both Invitrogen) following the protocol provided by the manufacturer.

    Sample Prep:

    Article Title: Frequent miRNA-convergent fusion gene events in breast cancer
    Article Snippet: Second strand synthesis was performed in 100 µl reactions with purified first strand cDNA (~16 µl) and 84 µl of second strand master mix containing 1.3 µl 5× First Strand buffer, 20 µl 5× Second Strand buffer, 3 µl 10 mM dNTPs with dUTP instead of dTTP, 1 µl 0.1 M DTT, 5 µl DNA Pol I (10 U/µl), 0.2 µl RNase H (10 U/µl), and 53.5 µl H2 O (all reagents from Invitrogen/Thermo Fisher Scientific). .. Adapter ligation was then performed in 50 µl reactions containing 40 µl of end-repaired and A-tailed double-stranded cDNA, 1 µl of a 1:5 dilution of adapters from TruSeq DNA LT Sample Prep Kit A and B (Illumina, San Diego, CA, USA), 3 µl T4 DNA ligase (5 U/µl), 1 µl 10× T4 DNA ligase buffer, and 5 µl H2 O by incubation at 22 °C 30 min, 4 °C hold.

    Ethanol Precipitation:

    Article Title: Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat
    Article Snippet: Fragmented RNA was purified by ethanol precipitation. .. The reaction mix was then treated with DNA Pol I and RNaseH at 16°C for 2.5 h (Invitrogen).

    Next-Generation Sequencing:

    Article Title: H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress
    Article Snippet: Second-strand cDNA was synthesized using DNA Pol I, DNA ligase (both Invitrogen), and RNase H (NEB) with random hexamers (Applied Biosystems) in the presence of dUTP. .. The libraries were prepared by the Vienna Biocenter CSF NGS unit using the NEBNext Library Prep Reagent Set for Illumina (NEB), multiplexed (2 samples/lane), and sequenced on HiSeq 2000 (Illumina) at the CSF NGS unit (50 bp single-end reads).

    Incubation:

    Article Title: Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat
    Article Snippet: cDNA synthesis—illumina sequencing 1 μg of rRNA depleted RNA was fragmented by incubation in fragmentation buffer included in the Illumina RNA-seq kit (Illumina) for 5 min at 94°C. .. The reaction mix was then treated with DNA Pol I and RNaseH at 16°C for 2.5 h (Invitrogen).

    Article Title: Frequent miRNA-convergent fusion gene events in breast cancer
    Article Snippet: After addition of 1 µl SuperScript II reactions were incubated at 25 °C for 10 min, followed by 42 °C 50 min, 70 °C 15 min and 4 °C hold. .. Second strand synthesis was performed in 100 µl reactions with purified first strand cDNA (~16 µl) and 84 µl of second strand master mix containing 1.3 µl 5× First Strand buffer, 20 µl 5× Second Strand buffer, 3 µl 10 mM dNTPs with dUTP instead of dTTP, 1 µl 0.1 M DTT, 5 µl DNA Pol I (10 U/µl), 0.2 µl RNase H (10 U/µl), and 53.5 µl H2 O (all reagents from Invitrogen/Thermo Fisher Scientific).

    Article Title: Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA
    Article Snippet: Each section was immersed in 200 μl of dH2 O and RNA was eluted from the gel slices by overnight incubation at room temperature. .. Second strand synthesis was carried out using Dna Pol I and RnaseH (both Invitrogen) following the protocol provided by the manufacturer.

    Article Title: Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan
    Article Snippet: .. Superscript II (200 U; Invitrogen) were added and the samples were incubated at 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. For second strand synthesis, 51 μL of H2 O, 20 μL of 5× second strand buffer (Invitrogen), and dNTPs (10 mM) were added to the first cDNA strand synthesis mix and incubated on ice for 5 min. RNaseH (2 U; Invitrogen), DNA pol I (50 U; Invitrogen) were then added and the mixture was incubated at 16°C for 2.5h. .. End-repair was performed by adding 45 μL of H2 O, T4 DNA ligase buffer with 10 mM ATP (NEB; 10 μL), dNTP mix (10 mM), T4 DNA polymerase (15 U; NEB), Klenow DNA polymerase (5 U; NEB), and T4 PNK (50 U; NEB) to the sample and incubating for 30 min at 20°C.

    Concentration Assay:

    Article Title: Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan
    Article Snippet: First strand buffer (4 μL; Invitrogen), Dithiothreitol (DTT), dNTPs and RNAseOUT (Invitrogen) were added to a final concentration of 1×, 10 mM, 200 μM and 1U/μL, respectively in a final volume of 20 μl and the samples were incubated at 25°C for 2 minutes. .. Superscript II (200 U; Invitrogen) were added and the samples were incubated at 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. For second strand synthesis, 51 μL of H2 O, 20 μL of 5× second strand buffer (Invitrogen), and dNTPs (10 mM) were added to the first cDNA strand synthesis mix and incubated on ice for 5 min. RNaseH (2 U; Invitrogen), DNA pol I (50 U; Invitrogen) were then added and the mixture was incubated at 16°C for 2.5h.

    Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. The double-stranded DNA (dsDNA) was purified with a QIAquick PCR purification kit (Qiagen, #28106) and the concentration was quantified using a Qubit fluorometer (Invitrogen).

    Article Title: Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq
    Article Snippet: The second strand cDNA was generated with the following recipe: 10 μl 5× second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl2 , 10 mM DTT), 30 nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). .. The double-stranded DNA (dsDNA) was purified with QIAquick PCR purification kit (Qiagen, #28106) and the concentration was quantified by a Qubit fluorometer (Invitrogen).

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    Thermo Fisher e coli dna polymerase i
    E Coli Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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