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
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    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
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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: 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).

    Synthesized:

    Article Title: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: .. The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume. .. This reaction mixture was incubated at 16°C for 2 h, and fragmented DNA was recovered with PCR clean-up kit (QIAGEN) and eluted in 30 μl of nuclease-free water.

    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.

    Article Title: A genomic approach to investigate developmental cell death in woody tissues of Populus trees
    Article Snippet: Paragraph title: Microarray analysis ... Second-strand cDNA was prepared in a final volume of 150 μl at 16°C for 2 h with 10 U Escherichia coli DNA ligase (Invitrogen), 40 U E. coli DNA polymerase I (MBI Fermentas), 2 U RNase H (USB, GE Healthcare Bio-Sciences) in a buffer containing 0.2 mM of each dNTP, 19 mM Tris-HCl (pH 6.9), 91 mM KCl, 4.5 mM MgCl2 and 10 mM (NH4 )2 SO4 .

    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: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: Random primer (3 μg) and fragmented RNA in 4 μl was incubated in 5 μl total volume at 70°C for 10 min, and cDNA or the first strand was synthesized using SuperScript III first-strand synthesis protocol (Invitrogen). .. The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume.

    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: A genomic approach to investigate developmental cell death in woody tissues of Populus trees
    Article Snippet: Second-strand cDNA was prepared in a final volume of 150 μl at 16°C for 2 h with 10 U Escherichia coli DNA ligase (Invitrogen), 40 U E. coli DNA polymerase I (MBI Fermentas), 2 U RNase H (USB, GE Healthcare Bio-Sciences) in a buffer containing 0.2 mM of each dNTP, 19 mM Tris-HCl (pH 6.9), 91 mM KCl, 4.5 mM MgCl2 and 10 mM (NH4 )2 SO4 . .. A further incubation was performed at 16°C for 20 min with 2 U T4 DNA polymerase (Invitrogen).

    Expressing:

    Article Title: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: Paragraph title: RNA-seq expression profiling ... The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume.

    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: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume. .. The fragmented DNA was end-repaired with End Repair Kit (New England Biolabs), and dA-tailed with dA-Tailing Kit (New England Biolabs), and then ligated with 7.5 μg of DNA adaptor mixture with Quick Ligation Kit (New England Biolabs).

    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).

    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).

    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).

    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: 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.

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Paragraph title: Paired-end library preparation for Illumina sequencing ... Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen).

    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.

    RNA Sequencing Assay:

    Article Title: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: Paragraph title: RNA-seq expression profiling ... The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume.

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Paired-end library preparation for Illumina sequencing Paired-end transcriptome sequencing (RNA-seq) was done to discover the identity of the fusion partner of candidate fusion genes. .. Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen).

    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: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: The ribosomal RNAs were removed from 2 μg of isolated total RNA with Ribo-Zero rRNA Removal Kit (Epicentre) in accordance with the manufacturer's instruction. .. The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume.

    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.

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Briefly, mRNA was isolated and purified from 1 to 10 µg of total RNA using Sera-Mag Magnetic Oligo(dT) Beads. mRNA was subsequently fragmented at 94°C in a fragmentation buffer and converted to single stranded cDNA using SuperScript II reverse transcriptase (Invitrogen). .. Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen).

    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).

    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.

    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: A genomic approach to investigate developmental cell death in woody tissues of Populus trees
    Article Snippet: Second-strand cDNA was prepared in a final volume of 150 μl at 16°C for 2 h with 10 U Escherichia coli DNA ligase (Invitrogen), 40 U E. coli DNA polymerase I (MBI Fermentas), 2 U RNase H (USB, GE Healthcare Bio-Sciences) in a buffer containing 0.2 mM of each dNTP, 19 mM Tris-HCl (pH 6.9), 91 mM KCl, 4.5 mM MgCl2 and 10 mM (NH4 )2 SO4 . .. The precipitates were dissolved in 2 mM Tris-HCl (pH 7.5) and purified using Autoseq G-50 columns (Amersham Biosciences).

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Briefly, mRNA was isolated and purified from 1 to 10 µg of total RNA using Sera-Mag Magnetic Oligo(dT) Beads. mRNA was subsequently fragmented at 94°C in a fragmentation buffer and converted to single stranded cDNA using SuperScript II reverse transcriptase (Invitrogen). .. Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen).

    Polymerase Chain Reaction:

    Article Title: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume. .. This reaction mixture was incubated at 16°C for 2 h, and fragmented DNA was recovered with PCR clean-up kit (QIAGEN) and eluted in 30 μl of nuclease-free water.

    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).

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen). .. Purified cDNA fragments were enriched with 15 PCR cycles using Phusion DNA Polymerase and provided buffers.

    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: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume. .. The adaptor-ligated DNA was size-selected to removed un-ligated adaptors with GeneRead Size Selection Kit (QIAGEN), and treated with 1 U of USER enzyme (New England Biolabs) in 30 μl of total volume, and incubated at 37 °C for 15 min followed by 5 min at 95°C.

    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.

    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.

    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).

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen). .. Library fragments were then size selected (300–400 bp) on a 2% agarose gel and then purified using the QIAquick Gel Extraction Kit (Qiagen).

    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).

    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.

    Sample Prep:

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Adapter-ligated cDNA libraries were prepared using the mRNA Seq-8 Sample Prep Kit (Illumina). .. Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen).

    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).

    Ethanol Precipitation:

    Article Title: Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP
    Article Snippet: The cDNA was recovered by phenol–chloroform extraction followed by ethanol precipitation. .. The second strand was synthesized from this cDNA with 20 μl of fragmented cDNA:RNA, 4 μl of 5× first strand buffer, 30 μl of 5× second strand buffer, 4 μl of 10 mM dNTP with dUTP instead of dTTP, 2 μl of 100 mM DTT, 4 μl of E. coli DNA polymerase (Invitrogen), 1 μl of E. coli DNA ligase (Invitrogen), 1 μl of E. coli RNase H (Invitrogen) in 150 μl of total volume.

    Article Title: A genomic approach to investigate developmental cell death in woody tissues of Populus trees
    Article Snippet: Second-strand cDNA was prepared in a final volume of 150 μl at 16°C for 2 h with 10 U Escherichia coli DNA ligase (Invitrogen), 40 U E. coli DNA polymerase I (MBI Fermentas), 2 U RNase H (USB, GE Healthcare Bio-Sciences) in a buffer containing 0.2 mM of each dNTP, 19 mM Tris-HCl (pH 6.9), 91 mM KCl, 4.5 mM MgCl2 and 10 mM (NH4 )2 SO4 . .. The reaction was stopped with 10 μl 0.5 M EDTA, and the cDNA was recovered by phenol and chloroform:isoamylalcohol extraction followed by ethanol precipitation.

    Spectrophotometry:

    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.

    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).

    Gel Extraction:

    Article Title: Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types
    Article Snippet: Subsequently second-strand cDNA synthesis was performed using E. coli DNA polymerase I (Invitrogen). .. Library fragments were then size selected (300–400 bp) on a 2% agarose gel and then purified using the QIAquick Gel Extraction Kit (Qiagen).

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    Thermo Fisher rt pcr total rna
    Expression of markers of adipogenesis. <t>RNA</t> was isolated from cells differentiated in media supplemented with 10% FBS or 10% hPL, and expression of PPARG , ADIPOQ , GLUT4 , and LEP was measured by <t>RT-PCR.</t> Statistically significant differences were determined by one-way ANOVA with Tukey's multiple comparisons. Data represents the mean ± SEM of three biological replicates from three donors/group. RB represents the mean ± SEM of three biological replicates from one donor from a commercial source. Significant differences between groups are designated by alphabetical letters. Any groups with the same letter are not statistically different from each other ( p
    Rt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli bw25113
    MqsA decreases EPS production. (a) Colony morphology of strains grown on salt-free CR plates containing 1 mM IPTG for 7 days. Red color indicated curli/cellulose production and scale bars represent 1 cm. Empty vector: <t>BW25113</t> Δ mqsRA /pBS(Kan); MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA ; and Δ csgD : BW25113 Δ csgD . (b) The amount of Congo red bound to planktonic cells at various time points. Error bars denote standard deviation ( n = 2).
    E Coli Bw25113, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of markers of adipogenesis. RNA was isolated from cells differentiated in media supplemented with 10% FBS or 10% hPL, and expression of PPARG , ADIPOQ , GLUT4 , and LEP was measured by RT-PCR. Statistically significant differences were determined by one-way ANOVA with Tukey's multiple comparisons. Data represents the mean ± SEM of three biological replicates from three donors/group. RB represents the mean ± SEM of three biological replicates from one donor from a commercial source. Significant differences between groups are designated by alphabetical letters. Any groups with the same letter are not statistically different from each other ( p

    Journal: Stem Cells International

    Article Title: Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    doi: 10.1155/2017/7108458

    Figure Lengend Snippet: Expression of markers of adipogenesis. RNA was isolated from cells differentiated in media supplemented with 10% FBS or 10% hPL, and expression of PPARG , ADIPOQ , GLUT4 , and LEP was measured by RT-PCR. Statistically significant differences were determined by one-way ANOVA with Tukey's multiple comparisons. Data represents the mean ± SEM of three biological replicates from three donors/group. RB represents the mean ± SEM of three biological replicates from one donor from a commercial source. Significant differences between groups are designated by alphabetical letters. Any groups with the same letter are not statistically different from each other ( p

    Article Snippet: RNA Isolation and RT-PCR Total RNA from differentiated cells was isolated using TRIzol LS reagent (TRIzol, Thermo Fisher).

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

    Early and late markers of osteogenesis. RNA was isolated from cells differentiated in media supplemented with 10% FBS or 10% hPL, and expression of early and late markers was measured by RT-PCR and is presented for differentiated commercial ASCs (RB-OSTEO), undifferentiated controls (HAP-CTRL and BH-CTRL), and cells cultured under osteogenic conditions (HAP-OSTEO and BH-OSTEO). All conditions were normalized to undifferentiated controls (data not shown). Sp7 and Spp1 expression is given for FBS (a) and hPL (b) conditions at day 7. Alpl , Bglap , and Runx2 are presented for day 14 for FBS (c) and hPL (d). Statistically significant differences were determined by one-way ANOVA with Tukey's multiple comparisons. Data represent the mean ± SEM of three biological replicates from three donors/group. RB represents the mean ± SEM of three biological replicates from one donor from a commercial source. Significant differences between groups are designated by alphabetical letters. Any groups with the same letter are not statistically different from each other ( p

    Journal: Stem Cells International

    Article Title: Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    doi: 10.1155/2017/7108458

    Figure Lengend Snippet: Early and late markers of osteogenesis. RNA was isolated from cells differentiated in media supplemented with 10% FBS or 10% hPL, and expression of early and late markers was measured by RT-PCR and is presented for differentiated commercial ASCs (RB-OSTEO), undifferentiated controls (HAP-CTRL and BH-CTRL), and cells cultured under osteogenic conditions (HAP-OSTEO and BH-OSTEO). All conditions were normalized to undifferentiated controls (data not shown). Sp7 and Spp1 expression is given for FBS (a) and hPL (b) conditions at day 7. Alpl , Bglap , and Runx2 are presented for day 14 for FBS (c) and hPL (d). Statistically significant differences were determined by one-way ANOVA with Tukey's multiple comparisons. Data represent the mean ± SEM of three biological replicates from three donors/group. RB represents the mean ± SEM of three biological replicates from one donor from a commercial source. Significant differences between groups are designated by alphabetical letters. Any groups with the same letter are not statistically different from each other ( p

    Article Snippet: RNA Isolation and RT-PCR Total RNA from differentiated cells was isolated using TRIzol LS reagent (TRIzol, Thermo Fisher).

    Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    MqsA decreases EPS production. (a) Colony morphology of strains grown on salt-free CR plates containing 1 mM IPTG for 7 days. Red color indicated curli/cellulose production and scale bars represent 1 cm. Empty vector: BW25113 Δ mqsRA /pBS(Kan); MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA ; and Δ csgD : BW25113 Δ csgD . (b) The amount of Congo red bound to planktonic cells at various time points. Error bars denote standard deviation ( n = 2).

    Journal: Scientific Reports

    Article Title: Antitoxin MqsA Represses Curli Formation Through the Master Biofilm Regulator CsgD

    doi: 10.1038/srep03186

    Figure Lengend Snippet: MqsA decreases EPS production. (a) Colony morphology of strains grown on salt-free CR plates containing 1 mM IPTG for 7 days. Red color indicated curli/cellulose production and scale bars represent 1 cm. Empty vector: BW25113 Δ mqsRA /pBS(Kan); MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA ; and Δ csgD : BW25113 Δ csgD . (b) The amount of Congo red bound to planktonic cells at various time points. Error bars denote standard deviation ( n = 2).

    Article Snippet: Electrophoretic mobility shift assay (EMSA) Gene promoters were PCR-amplified from the genomic DNA of E. coli BW25113 and 3′-labeled with biotin (Biotin 3′-End DNA Labeling kit, Thermo Scientific, Waltham, MA).

    Techniques: Plasmid Preparation, Standard Deviation

    Curli and cellulose are reduced in MqsA-producing cells. Curli production was assayed from cells in 2-day old colonies on agar plates with 1 mM IPTG, and imaged using SEM. Empty vector: BW25113 Δ mqsRA /pBS(Kan) and MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA . For each strain, 3 independent colonies were examined, and an image from one representative colony is shown. Scale bars represent 2 μm (left) and 3 μm (right).

    Journal: Scientific Reports

    Article Title: Antitoxin MqsA Represses Curli Formation Through the Master Biofilm Regulator CsgD

    doi: 10.1038/srep03186

    Figure Lengend Snippet: Curli and cellulose are reduced in MqsA-producing cells. Curli production was assayed from cells in 2-day old colonies on agar plates with 1 mM IPTG, and imaged using SEM. Empty vector: BW25113 Δ mqsRA /pBS(Kan) and MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA . For each strain, 3 independent colonies were examined, and an image from one representative colony is shown. Scale bars represent 2 μm (left) and 3 μm (right).

    Article Snippet: Electrophoretic mobility shift assay (EMSA) Gene promoters were PCR-amplified from the genomic DNA of E. coli BW25113 and 3′-labeled with biotin (Biotin 3′-End DNA Labeling kit, Thermo Scientific, Waltham, MA).

    Techniques: Plasmid Preparation