e coli dna polymerase  (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
    The full length <t>bicistronic</t> RNA is expressed from the dl VAR vectors. (A) Schematic representation of the experimental procedure to detect the full length bicistronic mRNA showing the primers and the size of the expected amplicons [23] . (B and D) HeLa cells were transfected with 200 ng of the dl HIV-1, dl ΔEMCV, or the different dl VAR plasmids. Total RNA was extracted from transfected cells and quantified. (B) Extracted RNA (3 µg) was used as template in a one-step RT-PCR designed to specifically detect the bicistronic RNAs (lanes 4, 6, 8, 10, 14, and 16). To assay for <t>DNA</t> contamination the same reaction was conducted in the absence of reverse transcriptase (lanes 5, 7, 9, 11, 13, 15, 17). In vitro transcribed dl HIV-1 IRES RNAs (lane 2) and water (lane 3) were included as RT-PCR controls. (C) Schematic representation of the experimental procedure to detect the RLuc-RNA and FLuc-RNA showing the primers and the size of the expected amplicons (D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmol)/RNA-RLuc (pmol) ratio was calculated. Values are the means +/- SEM from three independent experiments (each RNA sample was amplified in three independent reactions).
    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/product/Thermo Fisher
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1"

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035031

    The full length bicistronic RNA is expressed from the dl VAR vectors. (A) Schematic representation of the experimental procedure to detect the full length bicistronic mRNA showing the primers and the size of the expected amplicons [23] . (B and D) HeLa cells were transfected with 200 ng of the dl HIV-1, dl ΔEMCV, or the different dl VAR plasmids. Total RNA was extracted from transfected cells and quantified. (B) Extracted RNA (3 µg) was used as template in a one-step RT-PCR designed to specifically detect the bicistronic RNAs (lanes 4, 6, 8, 10, 14, and 16). To assay for DNA contamination the same reaction was conducted in the absence of reverse transcriptase (lanes 5, 7, 9, 11, 13, 15, 17). In vitro transcribed dl HIV-1 IRES RNAs (lane 2) and water (lane 3) were included as RT-PCR controls. (C) Schematic representation of the experimental procedure to detect the RLuc-RNA and FLuc-RNA showing the primers and the size of the expected amplicons (D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmol)/RNA-RLuc (pmol) ratio was calculated. Values are the means +/- SEM from three independent experiments (each RNA sample was amplified in three independent reactions).
    Figure Legend Snippet: The full length bicistronic RNA is expressed from the dl VAR vectors. (A) Schematic representation of the experimental procedure to detect the full length bicistronic mRNA showing the primers and the size of the expected amplicons [23] . (B and D) HeLa cells were transfected with 200 ng of the dl HIV-1, dl ΔEMCV, or the different dl VAR plasmids. Total RNA was extracted from transfected cells and quantified. (B) Extracted RNA (3 µg) was used as template in a one-step RT-PCR designed to specifically detect the bicistronic RNAs (lanes 4, 6, 8, 10, 14, and 16). To assay for DNA contamination the same reaction was conducted in the absence of reverse transcriptase (lanes 5, 7, 9, 11, 13, 15, 17). In vitro transcribed dl HIV-1 IRES RNAs (lane 2) and water (lane 3) were included as RT-PCR controls. (C) Schematic representation of the experimental procedure to detect the RLuc-RNA and FLuc-RNA showing the primers and the size of the expected amplicons (D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmol)/RNA-RLuc (pmol) ratio was calculated. Values are the means +/- SEM from three independent experiments (each RNA sample was amplified in three independent reactions).

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Concentration Assay, Amplification

    Analysis of a promoterless bicistronic construct containing the HIV-1 5′UTR sequences recovered from clinical samples. ( A ) Schematic representation of the bicistronic constructs. The SV40 promoter from dl ΔEMCV (lane 4), dl HIV-1 IRES, or dl VAR was removed to generate the equivalent promoterless (ΔSV40) vectors. ( B ) HeLa cells were transfected with DNA (200 ng) corresponding to the vectors depicted in (A) as previously described [23] . Total DNA was extracted from transfected cells and the presence of the transfected plasmids was confirmed by PCR (upper panel). The dl HIV-1 IRES plasmid (100 ng) was used as an amplification control. Total RNA was extracted from transfected cells and the presence of transcripts for the ΔSV40-dl ΔEMCV, ΔSV40-dl HIV-1 IRES, or the ΔSV40-dl VAR plasmids was evaluated by a one step RT-PCR designed to detect the bicistronic RNA (depicted in Fig. 2B ) [23] . In vitro transcribed RNA (100 ng) generated from plasmids dl HIV-1 IRES (lane 2) were used as amplification controls. ( C ) HeLa cells were transfected with either the 200 ng of SV40 or ΔSV40 version of dl ΔEMCV, dl HIV-1 IRES, or the different dl VAR plasmids as previously described [23] . Cells were processed and RLuc and FLuc activities were measured. For each data point the [RLuc/(total protein)] (left panel) and the [FLuc/(total protein)] (right panel) for the SV40 positive plasmids was arbitrary set to 100%. Values are the means +/- SD from three independent experiments.
    Figure Legend Snippet: Analysis of a promoterless bicistronic construct containing the HIV-1 5′UTR sequences recovered from clinical samples. ( A ) Schematic representation of the bicistronic constructs. The SV40 promoter from dl ΔEMCV (lane 4), dl HIV-1 IRES, or dl VAR was removed to generate the equivalent promoterless (ΔSV40) vectors. ( B ) HeLa cells were transfected with DNA (200 ng) corresponding to the vectors depicted in (A) as previously described [23] . Total DNA was extracted from transfected cells and the presence of the transfected plasmids was confirmed by PCR (upper panel). The dl HIV-1 IRES plasmid (100 ng) was used as an amplification control. Total RNA was extracted from transfected cells and the presence of transcripts for the ΔSV40-dl ΔEMCV, ΔSV40-dl HIV-1 IRES, or the ΔSV40-dl VAR plasmids was evaluated by a one step RT-PCR designed to detect the bicistronic RNA (depicted in Fig. 2B ) [23] . In vitro transcribed RNA (100 ng) generated from plasmids dl HIV-1 IRES (lane 2) were used as amplification controls. ( C ) HeLa cells were transfected with either the 200 ng of SV40 or ΔSV40 version of dl ΔEMCV, dl HIV-1 IRES, or the different dl VAR plasmids as previously described [23] . Cells were processed and RLuc and FLuc activities were measured. For each data point the [RLuc/(total protein)] (left panel) and the [FLuc/(total protein)] (right panel) for the SV40 positive plasmids was arbitrary set to 100%. Values are the means +/- SD from three independent experiments.

    Techniques Used: Construct, Transfection, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Reverse Transcription Polymerase Chain Reaction, In Vitro, Generated

    Related Articles

    Polymerase Chain Reaction:

    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.

    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: .. Two units of the Klenow fragment (Gibco/BRL), 50 μM each dATP, dGTP, dCTP, and dTTP, and 5 μCi of [α-32 P]dCTP (4 Ci/mmol) were added, and the reaction mixture was incubated for a further 20 min at 37°C; 20 mM EDTA and 1% sodium dodecyl sulfate were added to stop the reaction. ..

    Agarose Gel Electrophoresis:

    Article Title: HexaPrime: A novel method for detection of coronaviruses
    Article Snippet: .. The efficiency of different SS synthesis enzymes, T7 Polymerase (Thermo Scientific, Vilnius, Lithuania), DNA Polymerase I (Thermo Scientific, Vilnius, Lithuania), and Sequenase 2.0 (Affymetrix, United Kingdom), was evaluated by means of densitometry following bands separation on a 1.5% agarose gel. .. To test whether it is possible to detect coronaviral RNA not only in cell culture but also in clinical samples, 100 μl aliquots of clinical specimens, including sputum, bronchoalveolar lavage fluid, and nose wash, which had tested negatively for all known pathogens , were spiked with 1 μl of HCoV-NL63 virus stock (final TCID50 of 400).

    Plasmid Preparation:

    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).

    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).

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    Thermo Fisher rt pcr rna
    Fbxl13 and Drc7 are evolutionarily conserved and testis-enriched genes. (A) Model of DRC localization, based on Chlamydomonas [ 13 , 14 ]. The numbers shown in the N-DRC correspond to each DRC component. FBXL13 (DRC6) and DRC7 are located next to TCTE1 (DRC5). (B) The expression of mouse Fbxl13 and Drc7 was examined by <t>RT-PCR</t> using <t>RNA</t> isolated from various organs. Both Fbxl13 and Drc7 are testis-enriched with weak expression detected in other tissues. Actb was used as a loading control. (C) The expression of Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from testis at various postnatal days. Actb was used as a loading control.
    Rt Pcr Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 525 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli bl21
    Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli <t>BL21</t> lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 151 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: 89/100, based on 25 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: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Fbxl13 and Drc7 are evolutionarily conserved and testis-enriched genes. (A) Model of DRC localization, based on Chlamydomonas [ 13 , 14 ]. The numbers shown in the N-DRC correspond to each DRC component. FBXL13 (DRC6) and DRC7 are located next to TCTE1 (DRC5). (B) The expression of mouse Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from various organs. Both Fbxl13 and Drc7 are testis-enriched with weak expression detected in other tissues. Actb was used as a loading control. (C) The expression of Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from testis at various postnatal days. Actb was used as a loading control.

    Journal: PLoS Genetics

    Article Title: Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice

    doi: 10.1371/journal.pgen.1008585

    Figure Lengend Snippet: Fbxl13 and Drc7 are evolutionarily conserved and testis-enriched genes. (A) Model of DRC localization, based on Chlamydomonas [ 13 , 14 ]. The numbers shown in the N-DRC correspond to each DRC component. FBXL13 (DRC6) and DRC7 are located next to TCTE1 (DRC5). (B) The expression of mouse Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from various organs. Both Fbxl13 and Drc7 are testis-enriched with weak expression detected in other tissues. Actb was used as a loading control. (C) The expression of Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from testis at various postnatal days. Actb was used as a loading control.

    Article Snippet: Isolation of RNA and RT-PCR RNA was isolated and purified from multiple adult tissues of C57BL/6N mice at different ages with TRIzol (15596018, Thermo Fisher Scientific).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

    Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: DNA Extraction, Lysis, Electrophoresis, Polymerase Chain Reaction, Amplification, Incubation

    Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: Fluorescence, Microscopy, DNA Extraction, Polymerase Chain Reaction

    DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: DNA Extraction, Polymerase Chain Reaction, Amplification, DNA Binding Assay, Binding Assay, Purification, Genetically Modified, Transgenic Assay, Modification

    QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: Real-time Polymerase Chain Reaction, Amplification

    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