phusion 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 phusion 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/phusion dna polymerase i/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion dna polymerase i - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains
    Article Snippet: DNA was end repaired (30 min, 15°C; 100-μl reaction mixture consisting of 2 μg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 μM each deoxynucleoside triphosphate, and 10 μl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min, 50°C; 100 μl reaction mixture consisting of 2 μg sheared DNA, 50 μM each dCTP, dGTP, and dTTP, 2 mM dATP, 20 U Taq polymerase [MBI Fermentas], and 10 μl Yellow Tango buffer). .. A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified using standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Amplification:

    Article Title: Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease
    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: Transcriptome sequencing and analysis of zinc-uptake-related genes in Trichophyton mentagrophytes
    Article Snippet: Use DNA polymerase I (Thermo Fisher Scientific, USA), RNase H, dNTP, and buffer to synthesize second-strand cDNA. .. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq™ 2000 by Gene De novo Biotechnology Co (Guangzhou, China).

    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: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
    Article Snippet: The dl VAR vectors were generated as previously described , in brief the amplicon was digested with EcoRI and NcoI (both restriction sites added by PCR) and ligated, using a triple ligation strategy, with the 5248 bp-EcoRI/XbaI and 1656 bp -NcoI/XbaI fragments of the previously digested dl HIV-1 IRES . .. To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    Stable Transfection:

    Article Title: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: Gene expression analysis NIH3T3 cells stably expressing dominant negative MKL1 (a.a. 1-630)(DN-MKL1) or containing the vector, pBabePuro, were previously described [ ]. .. This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen).

    Synthesized:

    Article Title: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii
    Article Snippet: .. Second Strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen). .. The fragmented cDNA was treated with end-pair, A-tailing, adapter ligation using the TruSeq Sample Preparation Kit (Illumina).

    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). ..

    Construct:

    Article Title: A functional assay in Escherichia coli to detect non-assisted interaction between galactose repressor dimers
    Article Snippet: .. The insertion of 8 bp between the operators of the ← 0e ← 0i construct was performed by filling in of the Bgl II restriction site (see Fig. B) by the Klenow fragment of E.coli DNA polymerase I (Life Technologies). .. Insertion of 14 bp between the operators of the same ← Oe ← Oi construct was performed by introducing the self-complementary oligonucleotide 5′-GATCTGTCTAGACA-3′ into the Bgl II site.

    Electrophoresis:

    Article Title: DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains
    Article Snippet: DNA was end repaired (30 min, 15°C; 100-μl reaction mixture consisting of 2 μg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 μM each deoxynucleoside triphosphate, and 10 μl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min, 50°C; 100 μl reaction mixture consisting of 2 μg sheared DNA, 50 μM each dCTP, dGTP, and dTTP, 2 mM dATP, 20 U Taq polymerase [MBI Fermentas], and 10 μl Yellow Tango buffer). .. A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified using standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Article Title: Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks
    Article Snippet: The quantity and quality of RNA was confirmed by spectrophotometry (A260/A280 ratio) and capillary electrophoresis (2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA) by using an RNA 6000 Picochip kit. .. The single-stranded cDNA was then converted to double-stranded cDNA by using Escherichia coli DNA polymerase I (Affymetrix) for the first cycle.

    Microarray:

    Article Title: Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks
    Article Snippet: Target preparation for microarray analysis was performed according to the manufacturer's established protocol (www.affymetrix.com). .. The single-stranded cDNA was then converted to double-stranded cDNA by using Escherichia coli DNA polymerase I (Affymetrix) for the first cycle.

    Incubation:

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication
    Article Snippet: The labeled oligonucleotides were incubated with 20 ng of the purified DBP40 in 20 mM HEPES-NaOH (pH 7.9)–100 mM NaCl–5 mM MgCl2 –1 mM dithiothreitol–10% glycerol–1 μg of poly(dI-dC)–1 μg of BSA and then electrophoresed in 6% nondenaturing polyacrylamide gels, which were dried and exposed to X-ray film ( ). .. An FPV infectious clone in plasmid pGEM3Z (Promega, Madison, Wis.) was digested with Dde I and Hin fI, and the 3′ ends were 32 P labeled with the Klenow fragment of E. coli DNA polymerase I (Gibco/BRL, Gaithersburg, Md.) and [α-32 P]dATP.

    Article Title: Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease
    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: Relationship between 3?-Azido-3?-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
    Article Snippet: .. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (0.3 U; USB Corp.) and all four dNTPs (100 μM each). ..

    Article Title: NicE-seq: high resolution open chromatin profiling
    Article Snippet: Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1× NEB buffer 2. .. The labeling reaction was carried out at 37 °C in a thermo-mixer at 800 RPM for 2 h. We added 20 μL of 0.5 M EDTA and 2 μg of RNase A to the labeling reaction and incubated it at 37 °C for 0.5 h to stop the reaction and digest RNA.

    Article Title: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen). .. The obtained double-stranded cDNA was then blunted by the addition of 20 units T4 DNA polymerase and incubation for 5 min at 16°C.

    Expressing:

    Article Title: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: Paragraph title: Gene expression analysis ... This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen).

    Modification:

    Article Title: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: The cells were serum starved in Dulbecco's modified Eagle's medium (DMEM) with 0.2% new born calf serum (NCS) for 24 hours and then induced with 20% NCS for 30, 60 and 120 minutes. .. This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen).

    Ligation:

    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: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii
    Article Snippet: The fragments were treated with end-repair, A- tailing, and ligation of Illumina-compatible adapters (IDT, Inc.) using the KAPA-Illumina library creation kit (KAPA biosystems). qPCR was used to determine the concentration of the libraries to be sequenced on the Illumina Hiseq. .. Second Strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen).

    Article Title: Transcriptome sequencing and analysis of zinc-uptake-related genes in Trichophyton mentagrophytes
    Article Snippet: Use DNA polymerase I (Thermo Fisher Scientific, USA), RNase H, dNTP, and buffer to synthesize second-strand cDNA. .. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq™ 2000 by Gene De novo Biotechnology Co (Guangzhou, China).

    Article Title: Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair
    Article Snippet: .. This was accomplished by first digesting the plasmid with NotI, then using the Klenow fragment of E. coli DNA polymerase (Invitrogen) to fill in the single-stranded NotI overhang prior to ligation. .. We next eliminated a unique SacI restriction site, by digesting the plasmid with SacI, then using the T4 DNA polymerase (Invitrogen) to remove the single-stranded SacI overhang prior to ligation.

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
    Article Snippet: The dl VAR vectors were generated as previously described , in brief the amplicon was digested with EcoRI and NcoI (both restriction sites added by PCR) and ligated, using a triple ligation strategy, with the 5248 bp-EcoRI/XbaI and 1656 bp -NcoI/XbaI fragments of the previously digested dl HIV-1 IRES . .. To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    Generated:

    Article Title: Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair
    Article Snippet: The pEJ5-GFP-tel plasmid was generated from the pEJ5-GFP plasmid [ ] by the insertion of telomeric repeat sequences ( ). .. This was accomplished by first digesting the plasmid with NotI, then using the Klenow fragment of E. coli DNA polymerase (Invitrogen) to fill in the single-stranded NotI overhang prior to ligation.

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
    Article Snippet: The dl VAR vectors were generated as previously described , in brief the amplicon was digested with EcoRI and NcoI (both restriction sites added by PCR) and ligated, using a triple ligation strategy, with the 5248 bp-EcoRI/XbaI and 1656 bp -NcoI/XbaI fragments of the previously digested dl HIV-1 IRES . .. To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    Polymerase Chain Reaction:

    Article Title: A functional assay in Escherichia coli to detect non-assisted interaction between galactose repressor dimers
    Article Snippet: Presence of the expected PCR fragment for either the sense or antisense operator primer led to the orientation assignment. .. The insertion of 8 bp between the operators of the ← 0e ← 0i construct was performed by filling in of the Bgl II restriction site (see Fig. B) by the Klenow fragment of E.coli DNA polymerase I (Life Technologies).

    Article Title: Transcriptome sequencing and analysis of zinc-uptake-related genes in Trichophyton mentagrophytes
    Article Snippet: Use DNA polymerase I (Thermo Fisher Scientific, USA), RNase H, dNTP, and buffer to synthesize second-strand cDNA. .. Then using QiaQuick PCR extraction kit (Qiagen, German) to purify the cDNA fragments, and the cDNA fragments were ligated to Illumina sequencing adapters.

    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: SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair
    Article Snippet: .. PCR reactions were performed using two different DNA polymerase enzymes to exclude any enzyme-specific effects, and the products were subsequently subjected to fragment analysis using the ABI3130XL Genetic Analyzer (Life Technologies Ltd, Paisley, UK) .. Thymidine block Cells were treated with 2 mM thymidine for 18 h, washed with phosphate-buffered saline and released into complete media for 9 h. Cells were subsequently treated with a second thymidine block for a further 18 h. Cells were washed, released into complete media and harvested at the indicated time points.

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
    Article Snippet: The dl VAR vectors were generated as previously described , in brief the amplicon was digested with EcoRI and NcoI (both restriction sites added by PCR) and ligated, using a triple ligation strategy, with the 5248 bp-EcoRI/XbaI and 1656 bp -NcoI/XbaI fragments of the previously digested dl HIV-1 IRES . .. To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    Sequencing:

    Article Title: DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains
    Article Snippet: Paragraph title: Shotgun library construction and sequencing. ... DNA was end repaired (30 min, 15°C; 100-μl reaction mixture consisting of 2 μg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 μM each deoxynucleoside triphosphate, and 10 μl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min, 50°C; 100 μl reaction mixture consisting of 2 μg sheared DNA, 50 μM each dCTP, dGTP, and dTTP, 2 mM dATP, 20 U Taq polymerase [MBI Fermentas], and 10 μl Yellow Tango buffer).

    Article Title: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii
    Article Snippet: Paragraph title: 2.2 Genome and transcriptome sequencing ... Second Strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen).

    Article Title: A functional assay in Escherichia coli to detect non-assisted interaction between galactose repressor dimers
    Article Snippet: When required, the 20 bp Oi operator sequence was inserted into the Xba I restriction site and the 20 bp Oe sequence into the Spe I restriction site. .. The insertion of 8 bp between the operators of the ← 0e ← 0i construct was performed by filling in of the Bgl II restriction site (see Fig. B) by the Klenow fragment of E.coli DNA polymerase I (Life Technologies).

    Article Title: Transcriptome sequencing and analysis of zinc-uptake-related genes in Trichophyton mentagrophytes
    Article Snippet: Paragraph title: cDNA library construction and Illumina sequencing ... Use DNA polymerase I (Thermo Fisher Scientific, USA), RNase H, dNTP, and buffer to synthesize second-strand cDNA.

    Binding Assay:

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication
    Article Snippet: Paragraph title: ssDNA binding and in vitro fill-in assays. ... An FPV infectious clone in plasmid pGEM3Z (Promega, Madison, Wis.) was digested with Dde I and Hin fI, and the 3′ ends were 32 P labeled with the Klenow fragment of E. coli DNA polymerase I (Gibco/BRL, Gaithersburg, Md.) and [α-32 P]dATP.

    Mutagenesis:

    Article Title: Relationship between 3?-Azido-3?-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
    Article Snippet: In brief, 200 nM WT or mutant HIV-1 RT was incubated with 5 nM AZTMP-terminated, 5′-32 P-labeled L33 primer-WL50 template and 3.2 mM ATP in RB buffer at 37°C for the times indicated for each experiment. .. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (0.3 U; USB Corp.) and all four dNTPs (100 μM each).

    Isolation:

    Article Title: DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains
    Article Snippet: DNA was end repaired (30 min, 15°C; 100-μl reaction mixture consisting of 2 μg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 μM each deoxynucleoside triphosphate, and 10 μl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min, 50°C; 100 μl reaction mixture consisting of 2 μg sheared DNA, 50 μM each dCTP, dGTP, and dTTP, 2 mM dATP, 20 U Taq polymerase [MBI Fermentas], and 10 μl Yellow Tango buffer). .. A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified using standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Article Title: Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease
    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: NicE-seq: high resolution open chromatin profiling
    Article Snippet: Nuclei were isolated by incubating the cross-linked cells in cytosolic buffer (15 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP40) for 10 min on ice with occasional agitation. .. Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1× NEB buffer 2.

    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: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication
    Article Snippet: .. An FPV infectious clone in plasmid pGEM3Z (Promega, Madison, Wis.) was digested with Dde I and Hin fI, and the 3′ ends were 32 P labeled with the Klenow fragment of E. coli DNA polymerase I (Gibco/BRL, Gaithersburg, Md.) and [α-32 P]dATP. .. After boiling for 10 min, the DNA was transferred to a dry ice-ethanol bath and incubated for 30 min with 0.1 μg of E. coli -expressed DBP40 at 30°C for 1 h; then the protein and any associated DNA were immunoprecipitated with antibody against the T7 epitope (Novagen).

    Article Title: NicE-seq: high resolution open chromatin profiling
    Article Snippet: .. Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1× NEB buffer 2. .. The labeling reaction was carried out at 37 °C in a thermo-mixer at 800 RPM for 2 h. We added 20 μL of 0.5 M EDTA and 2 μg of RNase A to the labeling reaction and incubated it at 37 °C for 0.5 h to stop the reaction and digest RNA.

    Purification:

    Article Title: DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains
    Article Snippet: DNA was end repaired (30 min, 15°C; 100-μl reaction mixture consisting of 2 μg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 μM each deoxynucleoside triphosphate, and 10 μl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min, 50°C; 100 μl reaction mixture consisting of 2 μg sheared DNA, 50 μM each dCTP, dGTP, and dTTP, 2 mM dATP, 20 U Taq polymerase [MBI Fermentas], and 10 μl Yellow Tango buffer). .. A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified using standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Article Title: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii
    Article Snippet: The transcriptome libraries of both organisms were prepared by purifying 2 μg (5 μg for C. torrendii ) of total RNA using Dynabeads® mRNA Purification Kit (Invitrogen) and chemically fragmented to 200–250 bp (Ambion). mRNA was reverse transcribed with SuperScript II using random hexamers. .. Second Strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen).

    Article Title: Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease
    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. .. UltraPureTM 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: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: Total RNA from these cells was prepared using Trizol (Invitrogen) and then purified using RNeasy columns (Qiagen). .. This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
    Article Snippet: For the generation of the bicistronic vectors dl VAR, the 5 ′ UTR of natural variants were recovered from the randomly pooled viral RNA extracts by RT-PCR using the SuperScript™III one step RT-PCR system with platinum® Taq DNA polymerase (Invitrogen, Life Technologies Corporation, Carlsbad, California, USA) using the primers HIV1-F (5 ′ -CACGAATTCGGTCTCTCTG-3′) and HIV1-R ( 5′-CCATGGTCTCTCTCCTTC-3′ ). .. To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    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.

    Immunoprecipitation:

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication
    Article Snippet: An FPV infectious clone in plasmid pGEM3Z (Promega, Madison, Wis.) was digested with Dde I and Hin fI, and the 3′ ends were 32 P labeled with the Klenow fragment of E. coli DNA polymerase I (Gibco/BRL, Gaithersburg, Md.) and [α-32 P]dATP. .. After boiling for 10 min, the DNA was transferred to a dry ice-ethanol bath and incubated for 30 min with 0.1 μg of E. coli -expressed DBP40 at 30°C for 1 h; then the protein and any associated DNA were immunoprecipitated with antibody against the T7 epitope (Novagen).

    Electrophoretic Mobility Shift Assay:

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication
    Article Snippet: For electrophoretic mobility shift assays (EMSA), synthetic oligonucleotides representing sequences near the 3′ and 5′ ends of the viral genome (Table ) were 5′-end labeled with polynucleotide kinase and [γ-32 P]ATP. .. An FPV infectious clone in plasmid pGEM3Z (Promega, Madison, Wis.) was digested with Dde I and Hin fI, and the 3′ ends were 32 P labeled with the Klenow fragment of E. coli DNA polymerase I (Gibco/BRL, Gaithersburg, Md.) and [α-32 P]dATP.

    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: Transcriptome sequencing and analysis of zinc-uptake-related genes in Trichophyton mentagrophytes
    Article Snippet: Paragraph title: cDNA library construction and Illumina sequencing ... Use DNA polymerase I (Thermo Fisher Scientific, USA), RNase H, dNTP, and buffer to synthesize second-strand cDNA.

    Sample Prep:

    Article Title: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii
    Article Snippet: Second Strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen). .. The fragmented cDNA was treated with end-pair, A-tailing, adapter ligation using the TruSeq Sample Preparation Kit (Illumina).

    Activated Clotting Time Assay:

    Article Title: Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease
    Article Snippet: The total RNA was first denatured at 70°C for 10 minutes in presence of 200 ng oligo dT (24)-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.

    Plasmid Preparation:

    Article Title: DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains
    Article Snippet: Plasmid DNA was sheared, concentrated, and desalted using standard protocols ( ). .. DNA was end repaired (30 min, 15°C; 100-μl reaction mixture consisting of 2 μg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 μM each deoxynucleoside triphosphate, and 10 μl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min, 50°C; 100 μl reaction mixture consisting of 2 μg sheared DNA, 50 μM each dCTP, dGTP, and dTTP, 2 mM dATP, 20 U Taq polymerase [MBI Fermentas], and 10 μl Yellow Tango buffer).

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication
    Article Snippet: .. An FPV infectious clone in plasmid pGEM3Z (Promega, Madison, Wis.) was digested with Dde I and Hin fI, and the 3′ ends were 32 P labeled with the Klenow fragment of E. coli DNA polymerase I (Gibco/BRL, Gaithersburg, Md.) and [α-32 P]dATP. .. After boiling for 10 min, the DNA was transferred to a dry ice-ethanol bath and incubated for 30 min with 0.1 μg of E. coli -expressed DBP40 at 30°C for 1 h; then the protein and any associated DNA were immunoprecipitated with antibody against the T7 epitope (Novagen).

    Article Title: Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair
    Article Snippet: .. This was accomplished by first digesting the plasmid with NotI, then using the Klenow fragment of E. coli DNA polymerase (Invitrogen) to fill in the single-stranded NotI overhang prior to ligation. .. We next eliminated a unique SacI restriction site, by digesting the plasmid with SacI, then using the T4 DNA polymerase (Invitrogen) to remove the single-stranded SacI overhang prior to ligation.

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
    Article Snippet: Paragraph title: Plasmid Construction ... To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    Article Title: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: Gene expression analysis NIH3T3 cells stably expressing dominant negative MKL1 (a.a. 1-630)(DN-MKL1) or containing the vector, pBabePuro, were previously described [ ]. .. This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen).

    Dominant Negative Mutation:

    Article Title: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: Gene expression analysis NIH3T3 cells stably expressing dominant negative MKL1 (a.a. 1-630)(DN-MKL1) or containing the vector, pBabePuro, were previously described [ ]. .. This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen).

    Real-time Polymerase Chain Reaction:

    Article Title: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii
    Article Snippet: The fragments were treated with end-repair, A- tailing, and ligation of Illumina-compatible adapters (IDT, Inc.) using the KAPA-Illumina library creation kit (KAPA biosystems). qPCR was used to determine the concentration of the libraries to be sequenced on the Illumina Hiseq. .. Second Strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen).

    RNA Extraction:

    Article Title: Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks
    Article Snippet: Array Experiments Upon completion of pressure experiments, each leaflet was rinsed twice with sterile PBS, submerged in 1 mL of RNA later RNA Stabilization Reagent (Qiagen) to avoid changes in RNA expression and stored at −80°C until RNA extraction. .. The single-stranded cDNA was then converted to double-stranded cDNA by using Escherichia coli DNA polymerase I (Affymetrix) for the first cycle.

    RNA Expression:

    Article Title: Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks
    Article Snippet: Array Experiments Upon completion of pressure experiments, each leaflet was rinsed twice with sterile PBS, submerged in 1 mL of RNA later RNA Stabilization Reagent (Qiagen) to avoid changes in RNA expression and stored at −80°C until RNA extraction. .. The single-stranded cDNA was then converted to double-stranded cDNA by using Escherichia coli DNA polymerase I (Affymetrix) for the first cycle.

    Agarose Gel Electrophoresis:

    Article Title: Transcriptome sequencing and analysis of zinc-uptake-related genes in Trichophyton mentagrophytes
    Article Snippet: Use DNA polymerase I (Thermo Fisher Scientific, USA), RNase H, dNTP, and buffer to synthesize second-strand cDNA. .. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq™ 2000 by Gene De novo Biotechnology Co (Guangzhou, China).

    In Vitro:

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication
    Article Snippet: Paragraph title: ssDNA binding and in vitro fill-in assays. ... An FPV infectious clone in plasmid pGEM3Z (Promega, Madison, Wis.) was digested with Dde I and Hin fI, and the 3′ ends were 32 P labeled with the Klenow fragment of E. coli DNA polymerase I (Gibco/BRL, Gaithersburg, Md.) and [α-32 P]dATP.

    Article Title: Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks
    Article Snippet: The single-stranded cDNA was then converted to double-stranded cDNA by using Escherichia coli DNA polymerase I (Affymetrix) for the first cycle. .. From template cDNA, biotin-labeled cRNA was prepared in an in vitro transcription (IVT) reaction by using the MEGAscript High-Yield Transcription Kit (Ambion Inc, Austin, TX).

    Article Title: Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent
    Article Snippet: This was followed by second-strand synthesis for 2 hours at 16°C using RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen). .. The cDNA was then used in an in vitro transcription reaction for 6 hours at 37°C using a T7 in vitro transcription kit (Affymetrix) and biotin-labeled ribonucleotides.

    Spectrophotometry:

    Article Title: Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease
    Article Snippet: RNA purity and yield were determined by UV spectrophotometry for all RNA samples ( ). .. 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: Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks
    Article Snippet: The quantity and quality of RNA was confirmed by spectrophotometry (A260/A280 ratio) and capillary electrophoresis (2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA) by using an RNA 6000 Picochip kit. .. The single-stranded cDNA was then converted to double-stranded cDNA by using Escherichia coli DNA polymerase I (Affymetrix) for the first cycle.

    Concentration Assay:

    Article Title: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii
    Article Snippet: The fragments were treated with end-repair, A- tailing, and ligation of Illumina-compatible adapters (IDT, Inc.) using the KAPA-Illumina library creation kit (KAPA biosystems). qPCR was used to determine the concentration of the libraries to be sequenced on the Illumina Hiseq. .. Second Strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen).

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