e coli dna polymerase  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    Thermo Fisher e coli dna polymerase
    E Coli Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli dna polymerase/product/Thermo Fisher
    Average 95 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    e coli dna polymerase - by Bioz Stars, 2020-02
    95/100 stars

    Images

    Related Articles

    Clone Assay:

    Article Title: Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters
    Article Snippet: The ends of the fragmented DNA were repaired and blunted by using 5 µl 10× T4 DNA polymerase buffer (Life Technologies), 12.5 µl 2 mM dNTP mixture and 10 units Escherichia coli DNA polymerase (Life Technologies) at room temperature for 1 hr, followed by treatment with 5 units of T4 DNA polymerase (Life Technologies) at 37°C for 5 min and at 75°C for 20 min. A 25-µl aliquot of the blunted DNA was treated with 1 unit of calf intestine alkaline phosphatase (Promega) and 1 unit of shrimp alkaline phosphatase (Promega) at 37°C for 1 hr. .. Shotgun sequencing : Purified DNA fragments were cloned into pCR4 Blunt-TOPO (Life Technologies) according to the manufacturer’s instructions, and 1µl of the resulting plasmid was introduced into DH10B (Life Technologies).

    Article Title: DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid
    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, 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], 10 μl Yellow Tango buffer). .. The A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified by standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Amplification:

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription
    Article Snippet: Reverse transcriptase (Superscript II, Invitrogen) was added, and the mixture was incubated at 50°C for 60 min. Second-strand cDNA was synthesized with Escherichia coli DNA polymerase (Invitrogen), E.coli DNA ligase (Invitrogen) and RNaseH (Invitrogen) at 16°C for 2 h. Double-stranded cDNA was digested with Dpn II, and the resultant fragments were extracted from the digest by using a silicon-membrane-based purification kit (Gene Elute Purification Kit; Sigma–Aldrich). .. Linker-derived amplification of DNA fragments and selective amplification steps of cDNA RDA were performed according to the method described by Hubank and Schatz ( ).

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer. .. After the cRNA was linearly amplified with T7 polymerase, the biotinylated cRNA was cleaned with an RNeasy mini column (QIAGEN), fragmented to 50 to 200 nucleotides, and then hybridized to mouse genome U74A v. 2 arrays (Affymetrix).

    Article Title: Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters
    Article Snippet: A 10-µl aliquot of the 20 µl amplified DNA product was digested with 1 unit S1 nuclease (Promega, Tokyo, Japan) in 50 µl of 1× S1 nuclease buffer at 37°C for 30 min. .. The ends of the fragmented DNA were repaired and blunted by using 5 µl 10× T4 DNA polymerase buffer (Life Technologies), 12.5 µl 2 mM dNTP mixture and 10 units Escherichia coli DNA polymerase (Life Technologies) at room temperature for 1 hr, followed by treatment with 5 units of T4 DNA polymerase (Life Technologies) at 37°C for 5 min and at 75°C for 20 min. A 25-µl aliquot of the blunted DNA was treated with 1 unit of calf intestine alkaline phosphatase (Promega) and 1 unit of shrimp alkaline phosphatase (Promega) at 37°C for 1 hr.

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: Paragraph title: Brain Samples, RNA Preparation, and Amplification. ... After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel).

    Article Title: Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade
    Article Snippet: The RNA was converted to cDNA with the primer BSL-18E (5'-AAAGCGGCCGCATGCTTTTTTTTTTTTTTTTTT-3') and Superscript III reverse transcriptase (Invitrogen), after which double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Invitrogen–Thermo Fisher Scientific), was rendered blunt-ended with T4 DNA polymerase (Invitrogen–Thermo Fisher Scientific), was ligated at the 5' end to P20EA (5'-TAATACGACTCCGAATTCCC-3') and P10EA (5'-GGGAATTCGG-3') adapters, and was then cut with the NotI restriction enzyme. .. The second set of PCR products was then amplified with P22EA-ST1-R (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTAATACGACTCCGAATTCCC-3') and CB-ST1-R (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTCAAACACAGCGACCTC-3'), and index (barcode) sequences were added to the amlicons with the use of a Nextera XT index kit v2 setA (Illumina).

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: Preparation of the labeled samples for microarray hybridization was performed following the linear amplification protocol ( ) with modifications. .. The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen).

    Article Title: Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein
    Article Snippet: .. To generate pcDNA3-GST-Paip2, the Paip2 coding region was PCR amplified from pBluescriptKS-Paip2 and digested with Bam HI/ Xba I, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase (MBI Fermentas), and ligated into pcDNA3-GST, cut with Xba I, and blunt ended with the Klenow fragment of E. coli DNA polymerase (MBI Fermentas). .. For construction of FLAG-HMK-Paip2, the Paip2 coding region was PCR amplified using pcDNA3-Paip2 as a template.

    Nucleic Acid Electrophoresis:

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: RNA preparations from each group were pooled, their quantity was assessed by gel electrophoresis, and their OD260 /OD280 ratios were determined. .. After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel).

    Synthesized:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: First, cDNA was synthesized from ∼10 μg of total RNA in first-strand buffer containing 25 ng/mL engineered random primers containing T7 sequence (Ambion), 10 mM DTT, 0.5 mM dNTPs (Invitrogen), and 200 U/μL SuperScript III RT (Invitrogen). .. Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen).

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription
    Article Snippet: .. Reverse transcriptase (Superscript II, Invitrogen) was added, and the mixture was incubated at 50°C for 60 min. Second-strand cDNA was synthesized with Escherichia coli DNA polymerase (Invitrogen), E.coli DNA ligase (Invitrogen) and RNaseH (Invitrogen) at 16°C for 2 h. Double-stranded cDNA was digested with Dpn II, and the resultant fragments were extracted from the digest by using a silicon-membrane-based purification kit (Gene Elute Purification Kit; Sigma–Aldrich). .. Linker-derived amplification of DNA fragments and selective amplification steps of cDNA RDA were performed according to the method described by Hubank and Schatz ( ).

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: .. After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer. .. After the cRNA was linearly amplified with T7 polymerase, the biotinylated cRNA was cleaned with an RNeasy mini column (QIAGEN), fragmented to 50 to 200 nucleotides, and then hybridized to mouse genome U74A v. 2 arrays (Affymetrix).

    Article Title: Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade
    Article Snippet: .. The RNA was converted to cDNA with the primer BSL-18E (5'-AAAGCGGCCGCATGCTTTTTTTTTTTTTTTTTT-3') and Superscript III reverse transcriptase (Invitrogen), after which double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Invitrogen–Thermo Fisher Scientific), was rendered blunt-ended with T4 DNA polymerase (Invitrogen–Thermo Fisher Scientific), was ligated at the 5' end to P20EA (5'-TAATACGACTCCGAATTCCC-3') and P10EA (5'-GGGAATTCGG-3') adapters, and was then cut with the NotI restriction enzyme. .. The cDNA was then subjected to the polymerase chain reaction (PCR) with KAPA HiFi DNA Polymerase (Kapa Biosystems) and both a TCRβ-chain constant region–specific primer (CB1, 5'-GAACTGGACTTGACAGCGGAACT-3') and P20EA.

    Article Title: Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing
    Article Snippet: .. Double-stranded cDNA was synthesized by addition of 30 U of Escherichia coli DNA polymerase I, and 1 U of E. coli ribonuclease H to the first-strand synthesis reaction, following Fermentas’s suggested protocol for second-strand cDNA synthesis. ..

    Construct:

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid
    Article Snippet: To construct Endo VIII mutants, MV1161 and MV1571 were infected with P1 phage carrying the Δ nei::Km R allele derived from NKJ1003 ( ) and were selected for the kanamycin (Km)-resistant phenotype. .. Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) and T4 polynucleotide kinase were obtained from Life Technologies.

    Article Title: Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein
    Article Snippet: The annealed products were ligated into pGEX-GTH linearized with Eco RI. pGEX-GTH-Paip2(105–120) was constructed by annealing the forward primers (5′-GAT CCA TGC TTG TGG TCA AGA GCA ATC TGA ATC CAA ATG CAA AGG AGT TTG TTC CTT GAG GG-3′) to the reverse primers (5′-CCC TCA AGG AAC AAA CTC CTT TGC ATT TGG ATT CAG ATT GCT CTT GAC CAC AAG CAT G-3′) (Dalton Chemical laboratories); the resulting annealed product was ligated into pGEX-GTH linearized with Bam HI/ Sma I. .. To generate pcDNA3-GST-Paip2, the Paip2 coding region was PCR amplified from pBluescriptKS-Paip2 and digested with Bam HI/ Xba I, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase (MBI Fermentas), and ligated into pcDNA3-GST, cut with Xba I, and blunt ended with the Klenow fragment of E. coli DNA polymerase (MBI Fermentas).

    Electrophoresis:

    Article Title: DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid
    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, 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], 10 μl Yellow Tango buffer). .. The A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified by standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Microarray:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen). .. The biotinylated cRNA was fragmented and subjected to microarray analysis by using Affymetrix zebrafish GeneChip genome arrays containing ∼15,617 transcripts.

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: Paragraph title: Oligonucleotide microarray. ... After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer.

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: Paragraph title: Microarray Experiments ... The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen).

    Incubation:

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium
    Article Snippet: .. After washing briefly with PBS, cells were incubated for 30 min at room temperature with the in situ nick translation reaction (50 mM Tris-HCl, pH 7.9, 5 mM MgCl2 , 10 mM β-mercaptoethanol [Sigma-Aldrich], 50 μg/ml RIA grade bovine serum albumin [Sigma-Aldrich], 100 U/ml Escherichia coli DNA polymerase I [MBI Fermentas, Hanover, MD], 100 μM of each dATP, dCTP, and dGTP [MBI Fermentas], and 10 μM biotin-16-dUTP [Roche Diagnostics]) without DNase I or containing 33 ng/ml DNase I (Worthington Biochemical). ..

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: .. Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen). .. Labeled antisense cRNA was synthesized from dsDNA by using T7 RNA polymerase (Ambion) with biotin-dUTP (Sigma).

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription
    Article Snippet: .. Reverse transcriptase (Superscript II, Invitrogen) was added, and the mixture was incubated at 50°C for 60 min. Second-strand cDNA was synthesized with Escherichia coli DNA polymerase (Invitrogen), E.coli DNA ligase (Invitrogen) and RNaseH (Invitrogen) at 16°C for 2 h. Double-stranded cDNA was digested with Dpn II, and the resultant fragments were extracted from the digest by using a silicon-membrane-based purification kit (Gene Elute Purification Kit; Sigma–Aldrich). .. Linker-derived amplification of DNA fragments and selective amplification steps of cDNA RDA were performed according to the method described by Hubank and Schatz ( ).

    Activity Assay:

    Article Title: Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog
    Article Snippet: .. The Klenow fragment of Escherichia coli DNA polymerase I deficient in 3′→5′ proofreading exonuclease activity (KF exo−) was obtained from MBI fermentas. ..

    Expressing:

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: For gene expression analysis, total RNA was purified from each group (25–25 mg tissue from eight rats) with a NucleoSpin RNA purification kit (Macherey & Nagel) according to the manufacturer's instructions. .. After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel).

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: Genes with a significant expression change between this reference sample and the experimental Col-0 BA0 sample were excluded from the genotype comparison. .. The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen).

    Activated Clotting Time Assay:

    Article Title: Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein
    Article Snippet: The resulting fragments were ligated to pGEX-GTH digested with Bam HI/ Sma I. pGEX-GTH-Paip2 fragments 106–127, 112–127, and 121–127 were constructed by annealing the forward primers (5′-AAT TAT GCT TGT GGT CAA GAG CAA TCT GAA TCC AAA TGC AAA GGA GTT TGT TCC-3′, 5′-AAT TAT GAA TCC AAA TGC AAA GGA GTT TGT TCC TGG GGT GAA GTA CGG AAA TAT-3′, and 5′-AAT TAT GGG GGT GAA GTA CGG AAA TAT TGG-3′) to the reverse primers (5′-AAT TTC AAA TAT TCC CGT ACT TCA CCC CAG GAA CAA ACT CCT TTG CAT TTG GAT-3′, 5′-AAT TTC AAA TAT TTC CGT ACT TCA CCC CAG GAA CAA ACT CCT TTG CAT TTG GAT-3′, and 5′-AAT TTC AAA TAT TTC CGT ACT TCA CCC CCA-3′) (Dalton Chemical Laboratories), respectively. .. To generate pcDNA3-GST-Paip2, the Paip2 coding region was PCR amplified from pBluescriptKS-Paip2 and digested with Bam HI/ Xba I, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase (MBI Fermentas), and ligated into pcDNA3-GST, cut with Xba I, and blunt ended with the Klenow fragment of E. coli DNA polymerase (MBI Fermentas).

    Derivative Assay:

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid
    Article Snippet: To construct Endo VIII mutants, MV1161 and MV1571 were infected with P1 phage carrying the Δ nei::Km R allele derived from NKJ1003 ( ) and were selected for the kanamycin (Km)-resistant phenotype. .. Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) and T4 polynucleotide kinase were obtained from Life Technologies.

    Hybridization:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen). .. Hybridization, washing, and scanning of gene chips was performed at Case Comprehensive Cancer Center Microarray core facility ( ).

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: Preparation of the labeled samples for microarray hybridization was performed following the linear amplification protocol ( ) with modifications. .. The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen).

    Countercurrent Chromatography:

    Article Title: Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein
    Article Snippet: The resulting fragments were ligated to pGEX-GTH digested with Bam HI/ Sma I. pGEX-GTH-Paip2 fragments 106–127, 112–127, and 121–127 were constructed by annealing the forward primers (5′-AAT TAT GCT TGT GGT CAA GAG CAA TCT GAA TCC AAA TGC AAA GGA GTT TGT TCC-3′, 5′-AAT TAT GAA TCC AAA TGC AAA GGA GTT TGT TCC TGG GGT GAA GTA CGG AAA TAT-3′, and 5′-AAT TAT GGG GGT GAA GTA CGG AAA TAT TGG-3′) to the reverse primers (5′-AAT TTC AAA TAT TCC CGT ACT TCA CCC CAG GAA CAA ACT CCT TTG CAT TTG GAT-3′, 5′-AAT TTC AAA TAT TTC CGT ACT TCA CCC CAG GAA CAA ACT CCT TTG CAT TTG GAT-3′, and 5′-AAT TTC AAA TAT TTC CGT ACT TCA CCC CCA-3′) (Dalton Chemical Laboratories), respectively. .. To generate pcDNA3-GST-Paip2, the Paip2 coding region was PCR amplified from pBluescriptKS-Paip2 and digested with Bam HI/ Xba I, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase (MBI Fermentas), and ligated into pcDNA3-GST, cut with Xba I, and blunt ended with the Klenow fragment of E. coli DNA polymerase (MBI Fermentas).

    Ligation:

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription
    Article Snippet: Reverse transcriptase (Superscript II, Invitrogen) was added, and the mixture was incubated at 50°C for 60 min. Second-strand cDNA was synthesized with Escherichia coli DNA polymerase (Invitrogen), E.coli DNA ligase (Invitrogen) and RNaseH (Invitrogen) at 16°C for 2 h. Double-stranded cDNA was digested with Dpn II, and the resultant fragments were extracted from the digest by using a silicon-membrane-based purification kit (Gene Elute Purification Kit; Sigma–Aldrich). .. An aliquot of 1 μl of the ligation solution was diluted with Taq mixture (10 μl of 10× Taq buffer, including 15 mM MgCl2 , and 0.2 mM each of dNTPs).

    Article Title: Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines
    Article Snippet: Construction of “standard” libraries initially required 2–50 μg of DNase-treated total RNA. mRNA was captured using oligo(dT) magnetic beads followed by synthesis of double-stranded cDNA using SuperScript II reverse transcriptase (Invitrogen), RNAseH, and Escherichia coli DNA polymerase (Invitrogen). .. The resulting bead-bound cDNA was digested with the tagging enzyme NlaIII (Invitrogen), and the product was divided into two fractions for separate ligation of two adapters with 4-bp overhangs complementary to NlaIII digestion products.

    Nick Translation:

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium
    Article Snippet: .. After washing briefly with PBS, cells were incubated for 30 min at room temperature with the in situ nick translation reaction (50 mM Tris-HCl, pH 7.9, 5 mM MgCl2 , 10 mM β-mercaptoethanol [Sigma-Aldrich], 50 μg/ml RIA grade bovine serum albumin [Sigma-Aldrich], 100 U/ml Escherichia coli DNA polymerase I [MBI Fermentas, Hanover, MD], 100 μM of each dATP, dCTP, and dGTP [MBI Fermentas], and 10 μM biotin-16-dUTP [Roche Diagnostics]) without DNase I or containing 33 ng/ml DNase I (Worthington Biochemical). ..

    Infection:

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid
    Article Snippet: To construct Endo VIII mutants, MV1161 and MV1571 were infected with P1 phage carrying the Δ nei::Km R allele derived from NKJ1003 ( ) and were selected for the kanamycin (Km)-resistant phenotype. .. Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) and T4 polynucleotide kinase were obtained from Life Technologies.

    Generated:

    Article Title: Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing
    Article Snippet: First-strand cDNA was generated from ∼5 mg of total RNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) according to manufacturer’s instructions. .. Double-stranded cDNA was synthesized by addition of 30 U of Escherichia coli DNA polymerase I, and 1 U of E. coli ribonuclease H to the first-strand synthesis reaction, following Fermentas’s suggested protocol for second-strand cDNA synthesis.

    Sequencing:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: First, cDNA was synthesized from ∼10 μg of total RNA in first-strand buffer containing 25 ng/mL engineered random primers containing T7 sequence (Ambion), 10 mM DTT, 0.5 mM dNTPs (Invitrogen), and 200 U/μL SuperScript III RT (Invitrogen). .. Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen).

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: Briefly, 20 μg total RNA was reverse-transcribed in the presence of 2 μg HPLC-purified, anchored oligo(dT) primer having T7 promoter sequence at its 5′ end. .. After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel).

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

    Article Title: DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid
    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, 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], 10 μl Yellow Tango buffer).

    Article Title: Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing
    Article Snippet: Paragraph title: Library preparation and sequencing ... Double-stranded cDNA was synthesized by addition of 30 U of Escherichia coli DNA polymerase I, and 1 U of E. coli ribonuclease H to the first-strand synthesis reaction, following Fermentas’s suggested protocol for second-strand cDNA synthesis.

    Injection:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: Total RNA from erythrocytes isolated from embryos injected with control or indicated gene-specific MOs was used to identify differentially regulated genes. .. Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen).

    Cellular Antioxidant Activity Assay:

    Article Title: Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein
    Article Snippet: The annealed products were ligated into pGEX-GTH linearized with Eco RI. pGEX-GTH-Paip2(105–120) was constructed by annealing the forward primers (5′-GAT CCA TGC TTG TGG TCA AGA GCA ATC TGA ATC CAA ATG CAA AGG AGT TTG TTC CTT GAG GG-3′) to the reverse primers (5′-CCC TCA AGG AAC AAA CTC CTT TGC ATT TGG ATT CAG ATT GCT CTT GAC CAC AAG CAT G-3′) (Dalton Chemical laboratories); the resulting annealed product was ligated into pGEX-GTH linearized with Bam HI/ Sma I. .. To generate pcDNA3-GST-Paip2, the Paip2 coding region was PCR amplified from pBluescriptKS-Paip2 and digested with Bam HI/ Xba I, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase (MBI Fermentas), and ligated into pcDNA3-GST, cut with Xba I, and blunt ended with the Klenow fragment of E. coli DNA polymerase (MBI Fermentas).

    High Performance Liquid Chromatography:

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: Briefly, 20 μg total RNA was reverse-transcribed in the presence of 2 μg HPLC-purified, anchored oligo(dT) primer having T7 promoter sequence at its 5′ end. .. After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel).

    Shotgun Sequencing:

    Article Title: Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters
    Article Snippet: The ends of the fragmented DNA were repaired and blunted by using 5 µl 10× T4 DNA polymerase buffer (Life Technologies), 12.5 µl 2 mM dNTP mixture and 10 units Escherichia coli DNA polymerase (Life Technologies) at room temperature for 1 hr, followed by treatment with 5 units of T4 DNA polymerase (Life Technologies) at 37°C for 5 min and at 75°C for 20 min. A 25-µl aliquot of the blunted DNA was treated with 1 unit of calf intestine alkaline phosphatase (Promega) and 1 unit of shrimp alkaline phosphatase (Promega) at 37°C for 1 hr. .. Shotgun sequencing : Purified DNA fragments were cloned into pCR4 Blunt-TOPO (Life Technologies) according to the manufacturer’s instructions, and 1µl of the resulting plasmid was introduced into DH10B (Life Technologies).

    Magnetic Beads:

    Article Title: Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines
    Article Snippet: .. Construction of “standard” libraries initially required 2–50 μg of DNase-treated total RNA. mRNA was captured using oligo(dT) magnetic beads followed by synthesis of double-stranded cDNA using SuperScript II reverse transcriptase (Invitrogen), RNAseH, and Escherichia coli DNA polymerase (Invitrogen). .. The resulting bead-bound cDNA was digested with the tagging enzyme NlaIII (Invitrogen), and the product was divided into two fractions for separate ligation of two adapters with 4-bp overhangs complementary to NlaIII digestion products.

    Multiple Displacement Amplification:

    Article Title: Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters
    Article Snippet: Amplification and fragmentation of viral cDNA : A 4-µl aliquot of the first-strand viral cDNA underwent multiple displacement amplification [ ] by using a REPLI-g UltraFast Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. .. The ends of the fragmented DNA were repaired and blunted by using 5 µl 10× T4 DNA polymerase buffer (Life Technologies), 12.5 µl 2 mM dNTP mixture and 10 units Escherichia coli DNA polymerase (Life Technologies) at room temperature for 1 hr, followed by treatment with 5 units of T4 DNA polymerase (Life Technologies) at 37°C for 5 min and at 75°C for 20 min. A 25-µl aliquot of the blunted DNA was treated with 1 unit of calf intestine alkaline phosphatase (Promega) and 1 unit of shrimp alkaline phosphatase (Promega) at 37°C for 1 hr.

    Isolation:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: Total RNA from erythrocytes isolated from embryos injected with control or indicated gene-specific MOs was used to identify differentially regulated genes. .. Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen).

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

    Article Title: Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade
    Article Snippet: Mononuclear cells were isolated by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare Health Sciences), and total RNA was extracted from the cells and purified with the use of an RNeasy Mini Kit (Qiagen). .. The RNA was converted to cDNA with the primer BSL-18E (5'-AAAGCGGCCGCATGCTTTTTTTTTTTTTTTTTT-3') and Superscript III reverse transcriptase (Invitrogen), after which double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Invitrogen–Thermo Fisher Scientific), was rendered blunt-ended with T4 DNA polymerase (Invitrogen–Thermo Fisher Scientific), was ligated at the 5' end to P20EA (5'-TAATACGACTCCGAATTCCC-3') and P10EA (5'-GGGAATTCGG-3') adapters, and was then cut with the NotI restriction enzyme.

    Article Title: DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid
    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, 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], 10 μl Yellow Tango buffer). .. The A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified by standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Size-exclusion Chromatography:

    Article Title: Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters
    Article Snippet: The resultant DNA was fragmented in a sonicator (duty cycle, constant; output control, 4; Sonifier 250, Branson Ultrasonics, Danbury, CT, U.S.A.) for 5 sec. .. The ends of the fragmented DNA were repaired and blunted by using 5 µl 10× T4 DNA polymerase buffer (Life Technologies), 12.5 µl 2 mM dNTP mixture and 10 units Escherichia coli DNA polymerase (Life Technologies) at room temperature for 1 hr, followed by treatment with 5 units of T4 DNA polymerase (Life Technologies) at 37°C for 5 min and at 75°C for 20 min. A 25-µl aliquot of the blunted DNA was treated with 1 unit of calf intestine alkaline phosphatase (Promega) and 1 unit of shrimp alkaline phosphatase (Promega) at 37°C for 1 hr.

    Labeling:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen). .. Labeled antisense cRNA was synthesized from dsDNA by using T7 RNA polymerase (Ambion) with biotin-dUTP (Sigma).

    Article Title: Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog
    Article Snippet: Oligonucleotides were labeled using [γ-32 P]ATP (specific activity 3000 Ci/mmol) (NEM™ Life Science) and T4 polynucleotide kinase purchased from MBI fermentas. .. The Klenow fragment of Escherichia coli DNA polymerase I deficient in 3′→5′ proofreading exonuclease activity (KF exo−) was obtained from MBI fermentas.

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

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: Preparation of the labeled samples for microarray hybridization was performed following the linear amplification protocol ( ) with modifications. .. The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen).

    Purification:

    Article Title: Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy
    Article Snippet: Purified total RNA was used to prepare fragmented and biotin-dUTP–labeled cRNA according to Affymetrix target preparation protocols available online ( ). .. Ten microliters of first-strand reaction was incubated with Escherichia coli DNA polymerase (10 U/μL), E. coli RNaseH (2 U/μL), and 10 mM dNTPs in second-strand buffer (Invitrogen).

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription
    Article Snippet: .. Reverse transcriptase (Superscript II, Invitrogen) was added, and the mixture was incubated at 50°C for 60 min. Second-strand cDNA was synthesized with Escherichia coli DNA polymerase (Invitrogen), E.coli DNA ligase (Invitrogen) and RNaseH (Invitrogen) at 16°C for 2 h. Double-stranded cDNA was digested with Dpn II, and the resultant fragments were extracted from the digest by using a silicon-membrane-based purification kit (Gene Elute Purification Kit; Sigma–Aldrich). .. Linker-derived amplification of DNA fragments and selective amplification steps of cDNA RDA were performed according to the method described by Hubank and Schatz ( ).

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: Briefly, 3 μg purified RNA, extracted from the testes of 16-week-old Tslc1 +/+ and Tslc1 −/− mice, was reverse transcribed with Superscript II reverse transcriptase (Invitrogen), using primer T7-dT24 containing a T7 RNA polymerase promoter. .. After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer.

    Article Title: Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters
    Article Snippet: The ends of the fragmented DNA were repaired and blunted by using 5 µl 10× T4 DNA polymerase buffer (Life Technologies), 12.5 µl 2 mM dNTP mixture and 10 units Escherichia coli DNA polymerase (Life Technologies) at room temperature for 1 hr, followed by treatment with 5 units of T4 DNA polymerase (Life Technologies) at 37°C for 5 min and at 75°C for 20 min. A 25-µl aliquot of the blunted DNA was treated with 1 unit of calf intestine alkaline phosphatase (Promega) and 1 unit of shrimp alkaline phosphatase (Promega) at 37°C for 1 hr. .. The recovered DNA was purified by phenol–chloroform extraction, precipitated with ethanol [ ] and resuspended in 10µl of nuclease-free H2 O.

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: .. After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel). .. After sample concentration in vacuum, antisense RNA synthesis was performed with a RiboMax in vitro transcription kit (Promega) according to the manufacturer's manual.

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

    Article Title: Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade
    Article Snippet: Mononuclear cells were isolated by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare Health Sciences), and total RNA was extracted from the cells and purified with the use of an RNeasy Mini Kit (Qiagen). .. The RNA was converted to cDNA with the primer BSL-18E (5'-AAAGCGGCCGCATGCTTTTTTTTTTTTTTTTTT-3') and Superscript III reverse transcriptase (Invitrogen), after which double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Invitrogen–Thermo Fisher Scientific), was rendered blunt-ended with T4 DNA polymerase (Invitrogen–Thermo Fisher Scientific), was ligated at the 5' end to P20EA (5'-TAATACGACTCCGAATTCCC-3') and P10EA (5'-GGGAATTCGG-3') adapters, and was then cut with the NotI restriction enzyme.

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen). .. After purification with the QiaQuick PCR purification kit (Qiagen), the whole second-strand product was used for in vitro transcription with the MEGAScript High Yield Transcription Kit (Ambion/Applera).

    Article Title: DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid
    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, 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], 10 μl Yellow Tango buffer). .. The A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified by standard methods ( ) prior to cloning into pGEM-T (Promega, Madison, WI).

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid
    Article Snippet: Endo III, Endo VIII, Endo IV, formamidopyrimidine DNA glycosylase (Fpg/MutM) and AlkA were purified from E.coli strains that overproduced these enzymes ( , ). .. Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) and T4 polynucleotide kinase were obtained from Life Technologies.

    Polymerase Chain Reaction:

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: .. After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel). .. After sample concentration in vacuum, antisense RNA synthesis was performed with a RiboMax in vitro transcription kit (Promega) according to the manufacturer's manual.

    Article Title: Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade
    Article Snippet: The RNA was converted to cDNA with the primer BSL-18E (5'-AAAGCGGCCGCATGCTTTTTTTTTTTTTTTTTT-3') and Superscript III reverse transcriptase (Invitrogen), after which double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Invitrogen–Thermo Fisher Scientific), was rendered blunt-ended with T4 DNA polymerase (Invitrogen–Thermo Fisher Scientific), was ligated at the 5' end to P20EA (5'-TAATACGACTCCGAATTCCC-3') and P10EA (5'-GGGAATTCGG-3') adapters, and was then cut with the NotI restriction enzyme. .. The cDNA was then subjected to the polymerase chain reaction (PCR) with KAPA HiFi DNA Polymerase (Kapa Biosystems) and both a TCRβ-chain constant region–specific primer (CB1, 5'-GAACTGGACTTGACAGCGGAACT-3') and P20EA.

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen). .. After purification with the QiaQuick PCR purification kit (Qiagen), the whole second-strand product was used for in vitro transcription with the MEGAScript High Yield Transcription Kit (Ambion/Applera).

    Article Title: Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing
    Article Snippet: RNA samples were treated with DNase I (10 U/50 mg of total RNA) and the absence of contaminating genomic DNA was confirmed by PCR. .. Double-stranded cDNA was synthesized by addition of 30 U of Escherichia coli DNA polymerase I, and 1 U of E. coli ribonuclease H to the first-strand synthesis reaction, following Fermentas’s suggested protocol for second-strand cDNA synthesis.

    Article Title: Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines
    Article Snippet: Construction of “standard” libraries initially required 2–50 μg of DNase-treated total RNA. mRNA was captured using oligo(dT) magnetic beads followed by synthesis of double-stranded cDNA using SuperScript II reverse transcriptase (Invitrogen), RNAseH, and Escherichia coli DNA polymerase (Invitrogen). .. The two adapter–tag fractions were then ligated to form ∼131-bp adapter–ditag–adapter products that served as template for scale-up PCR.

    Article Title: Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein
    Article Snippet: .. To generate pcDNA3-GST-Paip2, the Paip2 coding region was PCR amplified from pBluescriptKS-Paip2 and digested with Bam HI/ Xba I, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase (MBI Fermentas), and ligated into pcDNA3-GST, cut with Xba I, and blunt ended with the Klenow fragment of E. coli DNA polymerase (MBI Fermentas). .. For construction of FLAG-HMK-Paip2, the Paip2 coding region was PCR amplified using pcDNA3-Paip2 as a template.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein
    Article Snippet: The annealed products were ligated into pGEX-GTH linearized with Eco RI. pGEX-GTH-Paip2(105–120) was constructed by annealing the forward primers (5′-GAT CCA TGC TTG TGG TCA AGA GCA ATC TGA ATC CAA ATG CAA AGG AGT TTG TTC CTT GAG GG-3′) to the reverse primers (5′-CCC TCA AGG AAC AAA CTC CTT TGC ATT TGG ATT CAG ATT GCT CTT GAC CAC AAG CAT G-3′) (Dalton Chemical laboratories); the resulting annealed product was ligated into pGEX-GTH linearized with Bam HI/ Sma I. .. To generate pcDNA3-GST-Paip2, the Paip2 coding region was PCR amplified from pBluescriptKS-Paip2 and digested with Bam HI/ Xba I, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase (MBI Fermentas), and ligated into pcDNA3-GST, cut with Xba I, and blunt ended with the Klenow fragment of E. coli DNA polymerase (MBI Fermentas).

    Mouse Assay:

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: Briefly, 3 μg purified RNA, extracted from the testes of 16-week-old Tslc1 +/+ and Tslc1 −/− mice, was reverse transcribed with Superscript II reverse transcriptase (Invitrogen), using primer T7-dT24 containing a T7 RNA polymerase promoter. .. After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer.

    Plasmid Preparation:

    Article Title: Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters
    Article Snippet: The ends of the fragmented DNA were repaired and blunted by using 5 µl 10× T4 DNA polymerase buffer (Life Technologies), 12.5 µl 2 mM dNTP mixture and 10 units Escherichia coli DNA polymerase (Life Technologies) at room temperature for 1 hr, followed by treatment with 5 units of T4 DNA polymerase (Life Technologies) at 37°C for 5 min and at 75°C for 20 min. A 25-µl aliquot of the blunted DNA was treated with 1 unit of calf intestine alkaline phosphatase (Promega) and 1 unit of shrimp alkaline phosphatase (Promega) at 37°C for 1 hr. .. Shotgun sequencing : Purified DNA fragments were cloned into pCR4 Blunt-TOPO (Life Technologies) according to the manufacturer’s instructions, and 1µl of the resulting plasmid was introduced into DH10B (Life Technologies).

    Article Title: DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid
    Article Snippet: Plasmid DNA was sheared, concentrated, and desalted by 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, 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], 10 μl Yellow Tango buffer).

    Software:

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer. .. The stained microarrays were scanned with a GeneArray scanner (Affymetrix), and the signals were calculated with the Affymetrix software Microarray Suite 5.0.

    Sample Prep:

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: The protocol used for sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA). .. After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer.

    In Vitro:

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: .. After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer. .. After the cRNA was linearly amplified with T7 polymerase, the biotinylated cRNA was cleaned with an RNeasy mini column (QIAGEN), fragmented to 50 to 200 nucleotides, and then hybridized to mouse genome U74A v. 2 arrays (Affymetrix).

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: Total RNA was amplified by using an antisense RNA amplification protocol by in vitro ; ref. ). .. After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel).

    Article Title: The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C]The Transcriptional Repressor ARR1-SRDX Suppresses Pleiotropic Cytokinin Activities in Arabidopsis 1 [C] [W]
    Article Snippet: The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen). .. After purification with the QiaQuick PCR purification kit (Qiagen), the whole second-strand product was used for in vitro transcription with the MEGAScript High Yield Transcription Kit (Ambion/Applera).

    Next-Generation Sequencing:

    Article Title: Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade
    Article Snippet: Paragraph title: Human TCR repertoire analysis by next-generation sequencing ... The RNA was converted to cDNA with the primer BSL-18E (5'-AAAGCGGCCGCATGCTTTTTTTTTTTTTTTTTT-3') and Superscript III reverse transcriptase (Invitrogen), after which double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Invitrogen–Thermo Fisher Scientific), was rendered blunt-ended with T4 DNA polymerase (Invitrogen–Thermo Fisher Scientific), was ligated at the 5' end to P20EA (5'-TAATACGACTCCGAATTCCC-3') and P10EA (5'-GGGAATTCGG-3') adapters, and was then cut with the NotI restriction enzyme.

    Concentration Assay:

    Article Title: The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
    Article Snippet: After first-strand synthesis, double-stranded DNA was prepared with Escherichia coli DNA polymerase, E. coli ligase, and RNase H (Life Technologies, Vienna, Austria) at 16°C for 5 h and purified with a PCR purification kit (Macherey & Nagel). .. After sample concentration in vacuum, antisense RNA synthesis was performed with a RiboMax in vitro transcription kit (Promega) according to the manufacturer's manual.

    In Situ:

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium
    Article Snippet: .. After washing briefly with PBS, cells were incubated for 30 min at room temperature with the in situ nick translation reaction (50 mM Tris-HCl, pH 7.9, 5 mM MgCl2 , 10 mM β-mercaptoethanol [Sigma-Aldrich], 50 μg/ml RIA grade bovine serum albumin [Sigma-Aldrich], 100 U/ml Escherichia coli DNA polymerase I [MBI Fermentas, Hanover, MD], 100 μM of each dATP, dCTP, and dGTP [MBI Fermentas], and 10 μM biotin-16-dUTP [Roche Diagnostics]) without DNase I or containing 33 ng/ml DNase I (Worthington Biochemical). ..

    Staining:

    Article Title: Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule †
    Article Snippet: After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce a biotin-labeled cRNA with a MEGAscript High Yield transcription kit (Ambion, Austin, TX), as recommended by the manufacturer. .. The stained microarrays were scanned with a GeneArray scanner (Affymetrix), and the signals were calculated with the Affymetrix software Microarray Suite 5.0.

    Gradient Centrifugation:

    Article Title: Detection of identical T cell clones in peritumoral pleural effusion and pneumonitis lesions in a cancer patient during immune-checkpoint blockade
    Article Snippet: Mononuclear cells were isolated by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare Health Sciences), and total RNA was extracted from the cells and purified with the use of an RNeasy Mini Kit (Qiagen). .. The RNA was converted to cDNA with the primer BSL-18E (5'-AAAGCGGCCGCATGCTTTTTTTTTTTTTTTTTT-3') and Superscript III reverse transcriptase (Invitrogen), after which double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I, E. coli DNA ligase, and RNase H (Invitrogen–Thermo Fisher Scientific), was rendered blunt-ended with T4 DNA polymerase (Invitrogen–Thermo Fisher Scientific), was ligated at the 5' end to P20EA (5'-TAATACGACTCCGAATTCCC-3') and P10EA (5'-GGGAATTCGG-3') adapters, and was then cut with the NotI restriction enzyme.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    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
    https://www.bioz.com/result/top10 escherichia coli/product/Thermo Fisher
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    top10 escherichia coli - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    88
    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
    https://www.bioz.com/result/e coli bw25113/product/Thermo Fisher
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli bw25113 - by Bioz Stars, 2020-02
    88/100 stars
      Buy from Supplier

    79
    Thermo Fisher m chimaera genomic dna
    Phylogenetic and population structure analysis of M. chimaera . (A) Core genome maximum likelihood phylogeny of 63 M. chimaera and 33 other related mycobacterial species based on alignment of 448,878 variable nucleotide positions. The tree was inferred with FastTree using a GTR model of nucleotide substitution. All major branches had FastTree support values of > 0.9. Lineages are colored, and BAPS group designations are indicated. The scale bar indicates the number of SNPs represented by the horizontal branches. The location of the MC_ANZ045 M. chimaera ) of <t>DNA:DNA</t> whole-genome comparisons among a subset of mycobacterial genomes used to identify M. chimaera -specific regions. The MC_ANZ045 M. chimaera reference chromosome is depicted by the inner black circle. The identity of the subsequent rings is given in the legend. Annotations on the outermost ring show the locations of the six M. chimaera -specific regions, highlighting the region targeted for <t>TaqMan</t> PCR assay development.
    M Chimaera Genomic Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m chimaera genomic dna/product/Thermo Fisher
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m chimaera genomic dna - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    Image Search Results


    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

    Phylogenetic and population structure analysis of M. chimaera . (A) Core genome maximum likelihood phylogeny of 63 M. chimaera and 33 other related mycobacterial species based on alignment of 448,878 variable nucleotide positions. The tree was inferred with FastTree using a GTR model of nucleotide substitution. All major branches had FastTree support values of > 0.9. Lineages are colored, and BAPS group designations are indicated. The scale bar indicates the number of SNPs represented by the horizontal branches. The location of the MC_ANZ045 M. chimaera ) of DNA:DNA whole-genome comparisons among a subset of mycobacterial genomes used to identify M. chimaera -specific regions. The MC_ANZ045 M. chimaera reference chromosome is depicted by the inner black circle. The identity of the subsequent rings is given in the legend. Annotations on the outermost ring show the locations of the six M. chimaera -specific regions, highlighting the region targeted for TaqMan PCR assay development.

    Journal: Journal of Clinical Microbiology

    Article Title: Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

    doi: 10.1128/JCM.00197-17

    Figure Lengend Snippet: Phylogenetic and population structure analysis of M. chimaera . (A) Core genome maximum likelihood phylogeny of 63 M. chimaera and 33 other related mycobacterial species based on alignment of 448,878 variable nucleotide positions. The tree was inferred with FastTree using a GTR model of nucleotide substitution. All major branches had FastTree support values of > 0.9. Lineages are colored, and BAPS group designations are indicated. The scale bar indicates the number of SNPs represented by the horizontal branches. The location of the MC_ANZ045 M. chimaera ) of DNA:DNA whole-genome comparisons among a subset of mycobacterial genomes used to identify M. chimaera -specific regions. The MC_ANZ045 M. chimaera reference chromosome is depicted by the inner black circle. The identity of the subsequent rings is given in the legend. Annotations on the outermost ring show the locations of the six M. chimaera -specific regions, highlighting the region targeted for TaqMan PCR assay development.

    Article Snippet: Purified M. chimaera genomic DNA for TaqMan assay validation was extracted from 50 mg (wet weight) of cell pellets as described previously ( ) and measured by fluorimetry using a Qubit assay kit and a High Sensitivity DNA kit (Thermo Fisher).

    Techniques: Polymerase Chain Reaction