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    Name:
    DpnI
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    DpnI 5 000 units
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    R0176L
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    Category:
    Restriction Enzymes
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    New England Biolabs dpn i
    DpnI
    DpnI 5 000 units
    https://www.bioz.com/result/dpn i/product/New England Biolabs
    Average 99 stars, based on 141 article reviews
    Price from $9.99 to $1999.99
    dpn i - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner"

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001082

    Polymerase linkage status of cytoplasmic versus nuclear viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with Dpn I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. ( B ) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.
    Figure Legend Snippet: Polymerase linkage status of cytoplasmic versus nuclear viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with Dpn I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. ( B ) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.

    Techniques Used: Transfection, Purification, Incubation, Proximity Ligation Assay, Produced

    Intracellular distribution and nuclease sensitivity of viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Relative nuclear distribution. DNA was extracted, after prior PK treatment, from total cells or from gradient-purified nuclei and subsequently digested with DpnI plus PsD. Serially diluted samples were loaded on the gel. Loading volumes are indicated in percent of the total sample volume, obtained from one well of a 6-well plate. One of three experiments used for quantification (see text for details) is shown. ( B ) Direct comparison of MN resistant versus total nuclear viral DNA. DNA was prepared from total cell extract treated with MN plus PK (MN), or from gradient-purified nuclei; equal aliquots of the nuclei were treated with MN plus PK before DNA extraction (MN), or with only PK followed by incubation with Dpn I plus PsD (total nuclear DNA). A six times longer exposure for the nuclear HBV samples is shown to better reveal weak signals. Quantification indicated that only ∼10% of the nuclear full-length HBV versus ≥80% of the nuclear DHBV DNA were MN resistant.
    Figure Legend Snippet: Intracellular distribution and nuclease sensitivity of viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Relative nuclear distribution. DNA was extracted, after prior PK treatment, from total cells or from gradient-purified nuclei and subsequently digested with DpnI plus PsD. Serially diluted samples were loaded on the gel. Loading volumes are indicated in percent of the total sample volume, obtained from one well of a 6-well plate. One of three experiments used for quantification (see text for details) is shown. ( B ) Direct comparison of MN resistant versus total nuclear viral DNA. DNA was prepared from total cell extract treated with MN plus PK (MN), or from gradient-purified nuclei; equal aliquots of the nuclei were treated with MN plus PK before DNA extraction (MN), or with only PK followed by incubation with Dpn I plus PsD (total nuclear DNA). A six times longer exposure for the nuclear HBV samples is shown to better reveal weak signals. Quantification indicated that only ∼10% of the nuclear full-length HBV versus ≥80% of the nuclear DHBV DNA were MN resistant.

    Techniques Used: Transfection, Purification, DNA Extraction, Incubation

    Site-specific discontinuity confirms the rcDNA nature of a substantial fraction of nuclear HBV DNA. Extensive nicking of HBV cccDNA might pretend an artifactually high ratio of rcDNA to cccDNA in the nucleus. However, nicking should occur at random whereas RC-DNA is distinctly discontinuous where the minus-strand and plus-strand DNA start. ( A ) Scheme of HBV rcDNA discontinuities. Restriction site positions are indicated with the first and last nucleotide of the recognition sequences. The Apa LI site is located immediately upstream of the plus-strand start. DNA in which the plus-strand is not sufficiently extended can not be cut. DR1 and DR2, direct repeats 1 and 2; wavy line at DR2, RNA primer at plus-strand 5′ end. ( B ) About one third of the rcDNA signal is resistant to Apa LI but not Nco I or Fsp I digestion. Cytoplasmic (treated with MN) and nuclear (treated with Dpn I) DNA preparations (both after prior PK digestion) were incubated with the indicated restriction enzymes. Consistently ( Figure S5B ), ∼35% of the rcDNA signal from the cytoplasm as well as the nucleus remained upon incubation with Apa LI (arrowheads) but not Nco I or Fsp I. Activity of Apa LI in the reactions is documented by the absence of a plasmid-derived Dpn I fragment containing internal sites for Apa LI and Fsp I but not Nco I. All samples were run on the same gel but a six-times longer exposure is shown for the nuclear samples; lane 5L on the longer exposure corresponds to lane 5 on the left panel. M, marker fragments of the indicated sizes (in kb).
    Figure Legend Snippet: Site-specific discontinuity confirms the rcDNA nature of a substantial fraction of nuclear HBV DNA. Extensive nicking of HBV cccDNA might pretend an artifactually high ratio of rcDNA to cccDNA in the nucleus. However, nicking should occur at random whereas RC-DNA is distinctly discontinuous where the minus-strand and plus-strand DNA start. ( A ) Scheme of HBV rcDNA discontinuities. Restriction site positions are indicated with the first and last nucleotide of the recognition sequences. The Apa LI site is located immediately upstream of the plus-strand start. DNA in which the plus-strand is not sufficiently extended can not be cut. DR1 and DR2, direct repeats 1 and 2; wavy line at DR2, RNA primer at plus-strand 5′ end. ( B ) About one third of the rcDNA signal is resistant to Apa LI but not Nco I or Fsp I digestion. Cytoplasmic (treated with MN) and nuclear (treated with Dpn I) DNA preparations (both after prior PK digestion) were incubated with the indicated restriction enzymes. Consistently ( Figure S5B ), ∼35% of the rcDNA signal from the cytoplasm as well as the nucleus remained upon incubation with Apa LI (arrowheads) but not Nco I or Fsp I. Activity of Apa LI in the reactions is documented by the absence of a plasmid-derived Dpn I fragment containing internal sites for Apa LI and Fsp I but not Nco I. All samples were run on the same gel but a six-times longer exposure is shown for the nuclear samples; lane 5L on the longer exposure corresponds to lane 5 on the left panel. M, marker fragments of the indicated sizes (in kb).

    Techniques Used: Incubation, Activity Assay, Plasmid Preparation, Derivative Assay, Marker

    DHBV replication in avian cells and HBV replication in human cells. ( A ) Chicken LMH cells transfected with wild-type and surface-deficient DHBV. ( B ) Human HepG2 cells transfected with wild-type and surface-deficient HBV. DNAs were extracted, after prior PK digestion, from cytoplasmic lysates and the nuclear fractions. One aliquot of the cytoplasmic lysates was treated with micrococcal nuclease (MN) before DNA extraction and analyzed without further treatment. All other samples were incubated, post extraction, with either Dpn I alone (Dpn), or Dpn I plus Plasmid safe DNAse (Dpn+PsD). Nomenclature of the various DNA species: RC, relaxed circular; DL, double strand linear; SS, single strand; CCC, covalently closed circular DNA; Pla, Dpn I restriction fragments of transfected plasmid DNA.
    Figure Legend Snippet: DHBV replication in avian cells and HBV replication in human cells. ( A ) Chicken LMH cells transfected with wild-type and surface-deficient DHBV. ( B ) Human HepG2 cells transfected with wild-type and surface-deficient HBV. DNAs were extracted, after prior PK digestion, from cytoplasmic lysates and the nuclear fractions. One aliquot of the cytoplasmic lysates was treated with micrococcal nuclease (MN) before DNA extraction and analyzed without further treatment. All other samples were incubated, post extraction, with either Dpn I alone (Dpn), or Dpn I plus Plasmid safe DNAse (Dpn+PsD). Nomenclature of the various DNA species: RC, relaxed circular; DL, double strand linear; SS, single strand; CCC, covalently closed circular DNA; Pla, Dpn I restriction fragments of transfected plasmid DNA.

    Techniques Used: Transfection, DNA Extraction, Incubation, Plasmid Preparation, Countercurrent Chromatography, Proximity Ligation Assay

    Restriction mapping of nuclear HBV rcDNA suggests genome region-selective MN accessibility. ( A ) Cytoplasmic versus nuclear MN-resistant viral DNAs. DNAs were isolated from HepG2 cells transfected with the surface-deficient HBV vector after prior MN plus PK treatment, and incubated with restriction enzymes Spe I and Nco I (ø, no restriction). Amounts equivalent to 10% of the total cytoplasmic fraction and 90% of the total nuclear fraction were loaded. ( B ) Total nuclear rcDNA versus MN resistant nuclear DNA. Viral DNA from isolated nuclei was prepared by either the PK plus Dpn I plus PsD procedure (total nuclear rcDNA), or after prior MN plus PK treatment (MN resistant nuclear DNA), and incubated with Nsi I, Eco RI or Bsp EI. Asterisks denote newly formed distinct fragments; lane ø on the right is a longer exposure of lane ø on the left. ( C ) Restriction map of HBV. The restriction sites probed in A and B are indicated. DR1 and DR2, direct repeats 1 and 2; P, covalently linked polymerase; wiggly red line, RNA primer at (+)-DNA 5′ end. Together, the patterns can consistently be explained if MN treatment removed a defined region from the rcDNA that approximately encompasses position 3000 to 500.
    Figure Legend Snippet: Restriction mapping of nuclear HBV rcDNA suggests genome region-selective MN accessibility. ( A ) Cytoplasmic versus nuclear MN-resistant viral DNAs. DNAs were isolated from HepG2 cells transfected with the surface-deficient HBV vector after prior MN plus PK treatment, and incubated with restriction enzymes Spe I and Nco I (ø, no restriction). Amounts equivalent to 10% of the total cytoplasmic fraction and 90% of the total nuclear fraction were loaded. ( B ) Total nuclear rcDNA versus MN resistant nuclear DNA. Viral DNA from isolated nuclei was prepared by either the PK plus Dpn I plus PsD procedure (total nuclear rcDNA), or after prior MN plus PK treatment (MN resistant nuclear DNA), and incubated with Nsi I, Eco RI or Bsp EI. Asterisks denote newly formed distinct fragments; lane ø on the right is a longer exposure of lane ø on the left. ( C ) Restriction map of HBV. The restriction sites probed in A and B are indicated. DR1 and DR2, direct repeats 1 and 2; P, covalently linked polymerase; wiggly red line, RNA primer at (+)-DNA 5′ end. Together, the patterns can consistently be explained if MN treatment removed a defined region from the rcDNA that approximately encompasses position 3000 to 500.

    Techniques Used: Isolation, Transfection, Plasmid Preparation, Incubation

    Core protein association of nuclear DHBV and HBV DNA. Vectors for surface-deficient DHBV and HBV genomes were transfected into Huh7 cells. IPs were performed in cytoplasmic extracts and extracts of purified nuclei containing 0.75× RIPA buffer, using antibodies against DHBV core protein (αDc) or HBV core protein (αHBc); in the mock IPs αDc was replaced by αHBc and vice versa. Immunopellets were treated with MN or not as indicated, and extracted after prior PK digestion. Purified DNAs from not MN-treated samples were digested with Dpn I. ø, extract directly treated with MN. ( A ) DHBV. M1, marker for cccDNA and rcDNA; M2, marker for single-stranded DNA; * and **, positions of cccDNA and ssDNA, respectively. ( B ) HBV. The rightmost panel shows the nuclear samples from an analogous experiment in HepG2 cells; the cytoplasmic samples are shown in Figure S7 .
    Figure Legend Snippet: Core protein association of nuclear DHBV and HBV DNA. Vectors for surface-deficient DHBV and HBV genomes were transfected into Huh7 cells. IPs were performed in cytoplasmic extracts and extracts of purified nuclei containing 0.75× RIPA buffer, using antibodies against DHBV core protein (αDc) or HBV core protein (αHBc); in the mock IPs αDc was replaced by αHBc and vice versa. Immunopellets were treated with MN or not as indicated, and extracted after prior PK digestion. Purified DNAs from not MN-treated samples were digested with Dpn I. ø, extract directly treated with MN. ( A ) DHBV. M1, marker for cccDNA and rcDNA; M2, marker for single-stranded DNA; * and **, positions of cccDNA and ssDNA, respectively. ( B ) HBV. The rightmost panel shows the nuclear samples from an analogous experiment in HepG2 cells; the cytoplasmic samples are shown in Figure S7 .

    Techniques Used: Transfection, Purification, Marker

    2) Product Images from "Fate of methylated/unmethylated H19 imprinting control region after paternal and maternal pronuclear injection"

    Article Title: Fate of methylated/unmethylated H19 imprinting control region after paternal and maternal pronuclear injection

    Journal: Experimental Animals

    doi: 10.1538/expanim.17-0031

    Generation and in vitro methylation of H19 ICR transgene. (a) Genomic structure of the mouse Igf2/H19 locus. The Igf2 and H19 genes (open boxes) are −90 kb apart, and the expression of both genes depends on the shared 3′ enhancer (filled ovals). The H19 ICR is located within a 2.9-kbp Sac I/ Bam HI fragment. The black boxes in the enlarged map indicate the position of the CCCTC-binding factor (CTCF) binding sites. (b) A schematic map of the transgene containing H19 ICR (ICR-F) used in this study. pCpG-EGFP-SB contains 2.9 kb of the mouse H19 ICR (white box containing four black boxes), mCMV enhancer (gray box), hEF1 promoter (gray box), and EGFP (white box) sequences. CTCF1/2, CTCF3/4, and EGFP regions analyzed for methylation status are indicated in solid lines. Primers used for nested-PCR are shown by arrows. P, Pac I; D, Dpn I sites. (c) Confirmation of the methylation status in the H19 ICR transgene fragment used for microinjection. Unmethylated and methylated transgenes were analyzed by bisulfite sequencing analysis using primers shown in B. Methylated and unmethylated CpG motifs are shown as filled and open circles, respectively. Each horizontal row represents a single DNA template molecule. Gray bars indicate the location of the CTCF-binding sites.
    Figure Legend Snippet: Generation and in vitro methylation of H19 ICR transgene. (a) Genomic structure of the mouse Igf2/H19 locus. The Igf2 and H19 genes (open boxes) are −90 kb apart, and the expression of both genes depends on the shared 3′ enhancer (filled ovals). The H19 ICR is located within a 2.9-kbp Sac I/ Bam HI fragment. The black boxes in the enlarged map indicate the position of the CCCTC-binding factor (CTCF) binding sites. (b) A schematic map of the transgene containing H19 ICR (ICR-F) used in this study. pCpG-EGFP-SB contains 2.9 kb of the mouse H19 ICR (white box containing four black boxes), mCMV enhancer (gray box), hEF1 promoter (gray box), and EGFP (white box) sequences. CTCF1/2, CTCF3/4, and EGFP regions analyzed for methylation status are indicated in solid lines. Primers used for nested-PCR are shown by arrows. P, Pac I; D, Dpn I sites. (c) Confirmation of the methylation status in the H19 ICR transgene fragment used for microinjection. Unmethylated and methylated transgenes were analyzed by bisulfite sequencing analysis using primers shown in B. Methylated and unmethylated CpG motifs are shown as filled and open circles, respectively. Each horizontal row represents a single DNA template molecule. Gray bars indicate the location of the CTCF-binding sites.

    Techniques Used: In Vitro, Methylation, Expressing, Binding Assay, Nested PCR, Methylation Sequencing

    Methylation analysis of transgenic H19 ICRs at blastocyst stage. (a) Bisulfite sequencing analysis of embryos that developed to blastocyst stage after microinjection of the ICR-F into the paternal or maternal PN. The methylation status of the ICR-F was indicated as described in Fig. 2c . Genomic DNA of 5–10 blastocysts was extracted as a pool and subjected to Dpn I digestion before bisulfite treatment to eliminate the transgene fragments that were unintegrated into the genome. The result is composed of data from three pools (for the CTCF1/2 region by maternal injection using unmethylated transgene, two pools were analyzed). At least six clones were sequenced from each pool. The data from three pools were combined, because the methylation level of each pool was almost the same. (b) Ratio of the DNA methylation levels at H19 ICR (CTCF1/2 and CTCF3/4) and EGFP regions are indicated.
    Figure Legend Snippet: Methylation analysis of transgenic H19 ICRs at blastocyst stage. (a) Bisulfite sequencing analysis of embryos that developed to blastocyst stage after microinjection of the ICR-F into the paternal or maternal PN. The methylation status of the ICR-F was indicated as described in Fig. 2c . Genomic DNA of 5–10 blastocysts was extracted as a pool and subjected to Dpn I digestion before bisulfite treatment to eliminate the transgene fragments that were unintegrated into the genome. The result is composed of data from three pools (for the CTCF1/2 region by maternal injection using unmethylated transgene, two pools were analyzed). At least six clones were sequenced from each pool. The data from three pools were combined, because the methylation level of each pool was almost the same. (b) Ratio of the DNA methylation levels at H19 ICR (CTCF1/2 and CTCF3/4) and EGFP regions are indicated.

    Techniques Used: Methylation, Transgenic Assay, Methylation Sequencing, Injection, Clone Assay, DNA Methylation Assay

    3) Product Images from "Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases"

    Article Title: Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases

    Journal: Virology Journal

    doi: 10.1186/1743-422X-11-11

    Effects of HPV16 E1 mutants on HPV18 replication. (A) Schematic representation of HPV16 E1 mutants. F, FLAG-tag; ND, N-terminal domain; DBD, DNA-binding domain; OD, oligomerization domain; HD, helicase domain. (B) C33A cells were transfected with 1 ng of the HPV18 gDNA together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV18 gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the HPV18 E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.
    Figure Legend Snippet: Effects of HPV16 E1 mutants on HPV18 replication. (A) Schematic representation of HPV16 E1 mutants. F, FLAG-tag; ND, N-terminal domain; DBD, DNA-binding domain; OD, oligomerization domain; HD, helicase domain. (B) C33A cells were transfected with 1 ng of the HPV18 gDNA together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV18 gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the HPV18 E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Techniques Used: FLAG-tag, Binding Assay, Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    Replication of HPV16 or HPV18 genomes supported by homologous or heterologous E1/E2s. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication when cells were transfected 20 ng of each pF16E1 and pF16E2 (A) or 10 ng of each pF18E1 and pF18E2 (B) . Each bar represents the average of two independent experiments with the standard error of mean.
    Figure Legend Snippet: Replication of HPV16 or HPV18 genomes supported by homologous or heterologous E1/E2s. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication when cells were transfected 20 ng of each pF16E1 and pF16E2 (A) or 10 ng of each pF18E1 and pF18E2 (B) . Each bar represents the average of two independent experiments with the standard error of mean.

    Techniques Used: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    Simultaneous replication of HPV16/18 genomes in the presence of E1/E2s of both types. C33A cells were transfected with a mixture of HPV16 and HPV18 gDNAs (1 ng each) together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV16 (upper panel) and HPV18 (lower panel) gDNAs were quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of three independent experiments with the standard deviation.
    Figure Legend Snippet: Simultaneous replication of HPV16/18 genomes in the presence of E1/E2s of both types. C33A cells were transfected with a mixture of HPV16 and HPV18 gDNAs (1 ng each) together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV16 (upper panel) and HPV18 (lower panel) gDNAs were quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of three independent experiments with the standard deviation.

    Techniques Used: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Standard Deviation

    Effects of heterologous E1 or E2 on HPV16/18 replication. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to that of the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.
    Figure Legend Snippet: Effects of heterologous E1 or E2 on HPV16/18 replication. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to that of the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Techniques Used: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    4) Product Images from "Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit"

    Article Title: Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit

    Journal: Scientific Reports

    doi: 10.1038/srep41851

    Detection and quantification of replicating HBV genome. Cells were infected with Ax-CM103G-kS (kS) or Ax-CM103G-dP (dP) by the indicated MOIs, or transfected with plasmids possessing the same mutant HBV expression units. ( a ) Detection of the replicating HBV genome. Total DNA either from infected or transfected HepG2 cells were analysed using Southern blot analysis. AdV (◆), Kpn I-digested genome of adenovirus vector; rc, relaxed circular DNA genome of HBV; dsL, double-stranded linear DNA genome of HBV; ss, single stranded DNA of HBV; plasmid ( ¢ ), Hind III and Dpn I-digested plasmid fragments; mock, mock infection. Overexposure of blots and full-length blots are presented in Supplementary Figs S5 and S9 , respectively. ( b ) Replicating HBV genomes were quantified using qPCR. Total DNA from infected HepG2 or PXB cells were used to performed qPCR. n = 3. Error bars represent ± s.d.; mock, mock infection of the indicated cells; ** P
    Figure Legend Snippet: Detection and quantification of replicating HBV genome. Cells were infected with Ax-CM103G-kS (kS) or Ax-CM103G-dP (dP) by the indicated MOIs, or transfected with plasmids possessing the same mutant HBV expression units. ( a ) Detection of the replicating HBV genome. Total DNA either from infected or transfected HepG2 cells were analysed using Southern blot analysis. AdV (◆), Kpn I-digested genome of adenovirus vector; rc, relaxed circular DNA genome of HBV; dsL, double-stranded linear DNA genome of HBV; ss, single stranded DNA of HBV; plasmid ( ¢ ), Hind III and Dpn I-digested plasmid fragments; mock, mock infection. Overexposure of blots and full-length blots are presented in Supplementary Figs S5 and S9 , respectively. ( b ) Replicating HBV genomes were quantified using qPCR. Total DNA from infected HepG2 or PXB cells were used to performed qPCR. n = 3. Error bars represent ± s.d.; mock, mock infection of the indicated cells; ** P

    Techniques Used: Infection, Transfection, Mutagenesis, Expressing, Southern Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

    5) Product Images from "DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO"

    Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO

    Journal: Molecular Systems Biology

    doi: 10.1038/msb.2010.54

    The DAM identification (DamID) procedure in C. elegans . ( A ) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with Dpn I, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with Dpn II (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. ( B ) Schematic of plasmid constructs used for preparation of transgenic strains. ( C , D ) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. ( E ) DAF-16∷DAM methylation profile for ist-1 , one of several evolutionarily conserved FoxO targets identified. ( F ) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.
    Figure Legend Snippet: The DAM identification (DamID) procedure in C. elegans . ( A ) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with Dpn I, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with Dpn II (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. ( B ) Schematic of plasmid constructs used for preparation of transgenic strains. ( C , D ) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. ( E ) DAF-16∷DAM methylation profile for ist-1 , one of several evolutionarily conserved FoxO targets identified. ( F ) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.

    Techniques Used: Binding Assay, Methylation, Amplification, Labeling, Plasmid Preparation, Construct, Transgenic Assay, Expressing

    6) Product Images from "A tool kit for rapid cloning and expression of recombinant antibodies"

    Article Title: A tool kit for rapid cloning and expression of recombinant antibodies

    Journal: Scientific Reports

    doi: 10.1038/srep05885

    Schematic representation of PIPE cloning strategy for swapping antibody variable regions. pVITRO1-IgE/κ vector is PCR linearized by V H and V K flanking primer pairs in two independent PCR reactions, resulting in two vector fragments subsequently treated with Dpn I. Simultaneously, the CSPG4 specific V H and V K are PCR amplified for generation of vector fragment terminal end-homology. The Dpn I-treated vector fragments are mixed with unpurified V H and V K , the single-stranded DNA fragments anneal directionally across the complementary sequences and nicks and gaps are repaired in vivo after transformation, generating pVITRO1-CSPG4-IgE/κ expression vector.
    Figure Legend Snippet: Schematic representation of PIPE cloning strategy for swapping antibody variable regions. pVITRO1-IgE/κ vector is PCR linearized by V H and V K flanking primer pairs in two independent PCR reactions, resulting in two vector fragments subsequently treated with Dpn I. Simultaneously, the CSPG4 specific V H and V K are PCR amplified for generation of vector fragment terminal end-homology. The Dpn I-treated vector fragments are mixed with unpurified V H and V K , the single-stranded DNA fragments anneal directionally across the complementary sequences and nicks and gaps are repaired in vivo after transformation, generating pVITRO1-CSPG4-IgE/κ expression vector.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, In Vivo, Transformation Assay, Expressing

    7) Product Images from "Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli"

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    Journal: Frontiers in Public Health

    doi: 10.3389/fpubh.2016.00131

    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).
    Figure Legend Snippet: Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Techniques Used: Mutagenesis, Methylation, Quantitative RT-PCR, Expressing, Marker, Negative Control

    Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.
    Figure Legend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .
    Figure Legend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Techniques Used: Mutagenesis, Amplification, Negative Control, Polymerase Chain Reaction, Methylation

    8) Product Images from "Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition"

    Article Title: Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03688-w

    Helraiser circle replication and donor site repair in HEK293T cells. a Schematic of the Helraiser heteroduplex LacZ donor plasmid (pHelR(mm)-Cam-LacZ) and resulting Helraiser circle. The red cross indicates the mismatch position within the transposon sequence, and the red circle marks the position of the mismatch used in the analysis of the Helraiser circles. b Experimental design of transposon circle replication assay using heteroduplex pHelR(mm)-Cam-LacZ donor plasmid. As shown, Dpn I digestion of LMW DNA reaction products can be used to distinguish between transposition of the (+) strand and the (−) strand. c Proportion of the transposon circles containing precise LTS-to-RTS junctions before and after Dpn I digestion of electroporated LMW DNA. The data are presented as n = 3 biological replicates. d Results of the transposon circle replication assay with pHelR(mm)-Cam-LacZ plasmid. The data are presented as a mean ± s.e.m., n = 3 biological replicates. e Schematic representation of possible outcomes of the transposon circle replication assay with heteroduplex pHelR(mm)-Cam-LacZ donors. Purple line: (+) strand of transposon donor; green line: (−) strand of transposon donor; solid line: methylated DNA; dashed line: unmethylated DNA; thin black line: plasmid backbone
    Figure Legend Snippet: Helraiser circle replication and donor site repair in HEK293T cells. a Schematic of the Helraiser heteroduplex LacZ donor plasmid (pHelR(mm)-Cam-LacZ) and resulting Helraiser circle. The red cross indicates the mismatch position within the transposon sequence, and the red circle marks the position of the mismatch used in the analysis of the Helraiser circles. b Experimental design of transposon circle replication assay using heteroduplex pHelR(mm)-Cam-LacZ donor plasmid. As shown, Dpn I digestion of LMW DNA reaction products can be used to distinguish between transposition of the (+) strand and the (−) strand. c Proportion of the transposon circles containing precise LTS-to-RTS junctions before and after Dpn I digestion of electroporated LMW DNA. The data are presented as n = 3 biological replicates. d Results of the transposon circle replication assay with pHelR(mm)-Cam-LacZ plasmid. The data are presented as a mean ± s.e.m., n = 3 biological replicates. e Schematic representation of possible outcomes of the transposon circle replication assay with heteroduplex pHelR(mm)-Cam-LacZ donors. Purple line: (+) strand of transposon donor; green line: (−) strand of transposon donor; solid line: methylated DNA; dashed line: unmethylated DNA; thin black line: plasmid backbone

    Techniques Used: Plasmid Preparation, Chick Chorioallantoic Membrane Assay, Sequencing, Methylation

    9) Product Images from "Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase"

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000439

    A3A inhibits AAV2 and MVM DNA replication. (A) Inhibition of wild-type AAV replication. U2OS cells were transfected with plasmids for APOBEC3 proteins and then infected with AAV and adenovirus. HA-tagged APOBEC3 (red) and AAV Rep proteins (green) were detected by immunofluorescence using specific antibodies. (B) Inhibition of wild-type MVM replication. Southern blot detection of low molecular weight DNA extracted from A9 cells cotransfected with an infectious MVM clone together with APOBEC3A expression plasmids. The DNA was digested with Dpn -I, separated by gel electrophoresis and hybridized with a radiolabeled MVM probe. Left line is a marker (M). Replicative intermediates of ssDNA (SS), monomer (M), and dimer (D) are indicated to the right. Panel below shows immunoblots for APOBEC3 and the NS1 protein of MVM.
    Figure Legend Snippet: A3A inhibits AAV2 and MVM DNA replication. (A) Inhibition of wild-type AAV replication. U2OS cells were transfected with plasmids for APOBEC3 proteins and then infected with AAV and adenovirus. HA-tagged APOBEC3 (red) and AAV Rep proteins (green) were detected by immunofluorescence using specific antibodies. (B) Inhibition of wild-type MVM replication. Southern blot detection of low molecular weight DNA extracted from A9 cells cotransfected with an infectious MVM clone together with APOBEC3A expression plasmids. The DNA was digested with Dpn -I, separated by gel electrophoresis and hybridized with a radiolabeled MVM probe. Left line is a marker (M). Replicative intermediates of ssDNA (SS), monomer (M), and dimer (D) are indicated to the right. Panel below shows immunoblots for APOBEC3 and the NS1 protein of MVM.

    Techniques Used: Inhibition, Transfection, Infection, Hemagglutination Assay, Immunofluorescence, Southern Blot, Molecular Weight, Expressing, Nucleic Acid Electrophoresis, Marker, Western Blot

    A3A mutants inhibit AAV DNA replication. (A) Titration of wild-type and mutant A3A expression vectors in rAAVLuc production assays. Production of rAAVLuc was assessed by transduction of target cells and quantitation of luciferase activity. Presented is the average of four independent experiments normalized to vector alone control (mock). The panels below show immunoblots to detect HA-tagged wild-type and mutant A3A proteins in transfected 293T cell lysates. (B) Southern blot detection of low molecular weight DNA extracted from 293T cells transfected for rAAVLuc production in the presence of mock (1 µg) A3G (1 µg), A3A (1, 0.1, 0.01 and 0.001 µg) and mutant A3A expression vectors (1 µg). The DNA was digested with Dpn -I, separated by gel electrophoresis, and hybridized with a radiolabeled luciferase probe.
    Figure Legend Snippet: A3A mutants inhibit AAV DNA replication. (A) Titration of wild-type and mutant A3A expression vectors in rAAVLuc production assays. Production of rAAVLuc was assessed by transduction of target cells and quantitation of luciferase activity. Presented is the average of four independent experiments normalized to vector alone control (mock). The panels below show immunoblots to detect HA-tagged wild-type and mutant A3A proteins in transfected 293T cell lysates. (B) Southern blot detection of low molecular weight DNA extracted from 293T cells transfected for rAAVLuc production in the presence of mock (1 µg) A3G (1 µg), A3A (1, 0.1, 0.01 and 0.001 µg) and mutant A3A expression vectors (1 µg). The DNA was digested with Dpn -I, separated by gel electrophoresis, and hybridized with a radiolabeled luciferase probe.

    Techniques Used: Titration, Mutagenesis, Expressing, Transduction, Quantitation Assay, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Hemagglutination Assay, Transfection, Southern Blot, Molecular Weight, Nucleic Acid Electrophoresis

    10) Product Images from "N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena"

    Article Title: N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx883

    6mA is preferentially associated with RNA Polymerase II-transcribed genes. ( A ) Comparison of 6mA methylation levels on bulk genomic DNA (MAC, blue), Polymerase II transcribed gene regions (Pol II, green) and rDNA (Pol I, red). ( B ) No 6mA methylation is localized on RNA Polymerase III transcribed genes. Pol III genes are listed in Supplementary Table S2 . ( C ) qPCR validation of nine selected GATC sites located on genes transcribed by different RNA polymerases (Pol I, II and III). Genomic DNA of SB210 was digested at 37°C with Dpn I for 30 min or overnight or with Dpn II overnight. qPCR was performed with primers flanking GATC sites or with internal control primers ( Supplementary Table S3 ). Y -axis represents methylation level that is reflected by normalized Ct value difference between digested and undigested samples. H1-H3: highly methylated sites. I1: intermediately methylated sites. N1-N5: unmethylated sites. ( D ) Methylation levels of 6mA distributed in the 1 kb region downstream the TSS of genes with different expression levels. Genes are ranked from high to low by their expression levels and divided into 10 quantiles (Q1-Q10). Each 6mA is shown as a green dot. Median of methylation level in each group of genes is marked with a red line. Inter-quartile ranges (IQR) are plotted as brown boxes. Confidence intervals are marked with blue lines. Ten groups of genes with different expression levels are divided into four homogeneous subsets according to ANOVA analysis (significance of each subset compared with others are 0.040, 0.016, 1.000 and 1.000, respectively, under condition of α = 0.01; see Supplementary Table S4 ). ( E ) Correlation matrix of different attributes of genes: (i) 6mA amount in 1 kb region downstream TSS, (ii) relative distance of 6mA to nucleosome dyad in this region, (iii) methylation level of 6mA in this region and (iv) gene expression levels (log 10 ). Correlation coefficients and correlation color dots were shown in the higher triangle of the correlation matrix ( P
    Figure Legend Snippet: 6mA is preferentially associated with RNA Polymerase II-transcribed genes. ( A ) Comparison of 6mA methylation levels on bulk genomic DNA (MAC, blue), Polymerase II transcribed gene regions (Pol II, green) and rDNA (Pol I, red). ( B ) No 6mA methylation is localized on RNA Polymerase III transcribed genes. Pol III genes are listed in Supplementary Table S2 . ( C ) qPCR validation of nine selected GATC sites located on genes transcribed by different RNA polymerases (Pol I, II and III). Genomic DNA of SB210 was digested at 37°C with Dpn I for 30 min or overnight or with Dpn II overnight. qPCR was performed with primers flanking GATC sites or with internal control primers ( Supplementary Table S3 ). Y -axis represents methylation level that is reflected by normalized Ct value difference between digested and undigested samples. H1-H3: highly methylated sites. I1: intermediately methylated sites. N1-N5: unmethylated sites. ( D ) Methylation levels of 6mA distributed in the 1 kb region downstream the TSS of genes with different expression levels. Genes are ranked from high to low by their expression levels and divided into 10 quantiles (Q1-Q10). Each 6mA is shown as a green dot. Median of methylation level in each group of genes is marked with a red line. Inter-quartile ranges (IQR) are plotted as brown boxes. Confidence intervals are marked with blue lines. Ten groups of genes with different expression levels are divided into four homogeneous subsets according to ANOVA analysis (significance of each subset compared with others are 0.040, 0.016, 1.000 and 1.000, respectively, under condition of α = 0.01; see Supplementary Table S4 ). ( E ) Correlation matrix of different attributes of genes: (i) 6mA amount in 1 kb region downstream TSS, (ii) relative distance of 6mA to nucleosome dyad in this region, (iii) methylation level of 6mA in this region and (iv) gene expression levels (log 10 ). Correlation coefficients and correlation color dots were shown in the higher triangle of the correlation matrix ( P

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Expressing

    11) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).
    Figure Legend Snippet: Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation, Synthesized, Staining, Activity Assay, Methylation

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    Article Snippet: PCR was performed using PfuTurbo DNA polymerase (Stratagene) with an elongation time corresponding to 2 min for each kb. .. The PCR products were treated with DpnI (20 U, New England BioLabs) at 37°C for 1 h. Plasmid mutants were ligated using T4 DNA Ligase (New England BioLabs) at 37°C for 2 h. The mutant DNA was transformed into E. coli XL1-competent cells. .. DNA was extracted from selected colonies by mini-prep extraction (Promega).

    Article Title: A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein
    Article Snippet: The PCR reaction system (25 μL) consisted of the following components: 2.5 μL of 10× pfx buffer (Invitrogen), 0.5 μL of 40 mmol/L dNTP mix (10 mmol/L each), 1.0 μL of forward/reverse primers (10 pmol/μL), 1.0 μL of pET28-fadR ec as template (5 ng/μL), 0.5 μL of Platinum pfx (2.5 U/μL, Invitrogen), and 17.5 μL of distilled sterilized H2 O. .. To remove the residual template plasmid pET28-fadR ec, the gel purified PCR products were digested for 1 h with DpnI (20 U/μL, NEB) at 37°C. .. Subsequently, they were transformed into chemically-competent cells of DH5α and the inserts of purified plasmids were verified by direct DNA sequencing.

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: Briefly, Phusion DNA polymerase (NEB) was used to linearly amplify and mutate the pESO-H7 template with primers H7sdmF 5’-GCCTCTCGATTCACCTTCACTAGTTAATAAATGCACTGGGTCCGC-3’ and H7sdmR 5’-CTGGCGGACCCAGTGCATTTATTAACTAGTGAAGGTGAATCGAGAGGCCAG-3’. .. The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time. .. After successful transformation into XL1 Blue Supercompetent cells (Agilent) following the manufacturer's protocol, plasmid DNA was rescued from individual colonies using the Zyppy kit (Zymo Research), and introduction of the desired mutations comprising pESO-H7sdm ( ) was verified with bi-directional sequencing using the primers BTSeqF 5’-CTGCTCCGAACAATAAAGATTCTAC-3’ and BTSeqR 5’-GTATGTGTAAAGTTGGTAACGGAAC-3’.

    Article Title: Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase
    Article Snippet: Briefly, 25ng pET21b containing the full-length SAUGI with the stop codon was used as the template DNA in a 50 μl reaction mixture containing one of the primer pairs. .. After PCR, the reaction products were incubated with 1.5 μl (9 U) of DpnI (New England Biolabs) for 1 h to digest the methylated parent plasmids. .. The resulting plasmids were transformed into DH5 alpha E. coli cells and successful mutagenesis was confirmed by DNA sequencing.

    Neutralization:

    Article Title: Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41
    Article Snippet: Briefly, using a plasmid encoding a neutralization-resistant HIV-1 Env protein as a template, we designed primers containing the desired mutation. .. We amplified the plasmid template with each forward and reverse primer using Pfu Turbo (Stratagene), digested the reaction with DpnI (New England Biolabs), and transformed into DH5α cells by electroporation.

    TA Cloning:

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems
    Article Snippet: The library was then purified with QIAquick PCR purification kit and subjected to a DpnI digest according to manufacturer's instructions (NEB). .. The re-amplified library was then precipitated with PEG–MgCl2 , blunted with T4 DNA polymerase, assembled and purified by PCR.

    Construct:

    Article Title: Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2
    Article Snippet: The conditions for large-scale PCR to construct the randomized libraries was described previously ( , ). .. After PCR, the reaction mixture was treated with Dpn I (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid, then subjected to purification using a QIA-Quick column (Qiagen).

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: The sequence of the 5′-UTR of these constructs is reported in the . .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Real-time Polymerase Chain Reaction:

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing
    Article Snippet: Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). .. Adaptor-ligated DNA segments were PCR amplified for 15 cycles, purified by AMPure XP beads and suspended in 20 μl of resuspension buffer to yield the sequencing library.

    Incubation:

    Article Title: Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase
    Article Snippet: Briefly, 25ng pET21b containing the full-length SAUGI with the stop codon was used as the template DNA in a 50 μl reaction mixture containing one of the primer pairs. .. After PCR, the reaction products were incubated with 1.5 μl (9 U) of DpnI (New England Biolabs) for 1 h to digest the methylated parent plasmids. .. The resulting plasmids were transformed into DH5 alpha E. coli cells and successful mutagenesis was confirmed by DNA sequencing.

    Expressing:

    Article Title: Proteomic Analysis Reveals CACN-1 Is a Component of the Spliceosome in Caenorhabditis elegans
    Article Snippet: The injection solution contained 20 ng/µL transgene, 20 ng/µL sur-5 ::gfp , and 60 ng/µL N2 genomic DNA digested with Dpn I (New England Biolabs, Ipswich, MA). .. The green fluorescent protein marker allowed tracking and maintenance of the transgenic line, UN1305 xbEx1305[hsp16.2p ::flag ::ha ::cacn-1 , sur-5 ::GFP] .

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: Paragraph title: Expression systems and plasmid construction ... Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene). .. The substitutions in ZβZBP1 were generated by mutagenesis PCR with hZBP1 N141D Y145A F (5′-ACA GCA AAA GAT GTG GAC CGA GAC TTG GCC AGG ATG AAG AGC AGG-3′) and hZBP1 N141D Y145A R (5′-GGG CGA GGA CTT GGT CGA GCT CCC TCT TG-3′) in one step.

    Article Title: Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production
    Article Snippet: The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 µl of PCR products were digested with 1 µl DpnI (New England Biolabs Beijing, China) at 37°C for 4 hrs to remove the template plasmid. .. The mutants were confirmed by DNA sequencing of the TCS1 plasmids.

    Article Title: The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation
    Article Snippet: Paragraph title: Cloning and expression of Hes1 mutants ... All Hes1 mutants were amplified by PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pEASY-blut-Flag-Hes1 as a template (20 ng), following by DpnI (NEB) digestion.

    Modification:

    Article Title: Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface
    Article Snippet: Low-molecular-weight DNA was extracted using a modified version of the Hirt extract procedure ( ). .. The extracts were digested with the restriction enzyme DpnI (New England BioLabs) to digest input DNA.

    Transformation Assay:

    Article Title: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations
    Article Snippet: The samples were then digested by Dpn I (NEB R0176L) according to the manufacturer's manual. .. Since single colony picking after bacterial transformation of mutagenesis PCR product is a rate-limiting step, we rigorously optimized this step.

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB). .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Article Title: Quadruplex DNA in long terminal repeats in maize LTR retrotransposons inhibits the expression of a reporter gene in yeast
    Article Snippet: Mutations in G4 forming sequences in cloned LTRs were introduced using single mutagenic primers for each LTR and Q5 polymerase (recommended conditions, Additional file ). .. The products were treated with DpnI (NEB) and 1 μl was used for XL-1 blue electrocompetent cell (Agilent) transformation. .. We used the S. cerevisiae strain CM100 (MATα, can1–100 oc, his3, leu2, trp1, ura3–52) for the lacZ expression assay.

    Article Title: O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
    Article Snippet: The p3XFLAG-OGT-siRNA-resistant vector was generated by directed mutagenesis using Phusion® Hot start (NEB), p3XFLAG-OGT as temple and, 5′-agcagggaaaactgcaggaagctctgatgcattataaagaagcgatcaggatttcccctacctttgctgatgcctactc-3′ and 5′-gagtaggcatcagcaaaggtaggggaaatcctgatcgcttctttataatgcatcagagcttcctgcagttttccctgct-3′ as primers. .. Prior to transformation in DH5α, template was digested for 2 h at 37 °C by 1 U of DpnI (NEB). .. Positive clones were screened by sequencing.

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Site-specific mutations were generated by PCR mutagenesis using KOD Hot start polymerase (Novagen) and primers containing the desired mutation following the Quick Change protocol (Stratagene). .. Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene). .. For two proximate amino acid substitutions in ZαZBP1 two mutagenesis PCRs were performed consecutively.

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: The wild type PF13_0159 locus was subcloned into the StrataClone vector (Agilent Technologies) and PCR was performed using the overlapping primers, D110A SENSE and ANTISENSE (Table S1 in ), containing the desired mutation. .. To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α. .. Prior to assays, purified PfNMNAT-6×HIS protein was dialyzed into reaction buffer containing 100 mM HEPES and 10 mM MgCl2 at pH 7.5 overnight at 4° using 3500 Da MWCO dialysis tubing from Fisherbrand.

    Article Title: Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41
    Article Snippet: The primers used and PCR conditions are described in . .. We amplified the plasmid template with each forward and reverse primer using Pfu Turbo (Stratagene), digested the reaction with DpnI (New England Biolabs), and transformed into DH5α cells by electroporation. .. Colonies were screened for presence of HIV-1 Env by restriction digestion with MluI and NotI (New England Biolabs), and the entire HIV-1 Env variant was sequenced using BigDye (Applied Biosystems).

    Article Title: The Pivotal Roles of TIA Proteins in 5? Splice-Site Selection of Alu Exons and Across Evolution
    Article Snippet: PCR was performed using PfuTurbo DNA polymerase (Stratagene) with an elongation time corresponding to 2 min for each kb. .. The PCR products were treated with DpnI (20 U, New England BioLabs) at 37°C for 1 h. Plasmid mutants were ligated using T4 DNA Ligase (New England BioLabs) at 37°C for 2 h. The mutant DNA was transformed into E. coli XL1-competent cells. .. DNA was extracted from selected colonies by mini-prep extraction (Promega).

    Article Title: Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production
    Article Snippet: The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 µl of PCR products were digested with 1 µl DpnI (New England Biolabs Beijing, China) at 37°C for 4 hrs to remove the template plasmid. .. The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 µl of PCR products were digested with 1 µl DpnI (New England Biolabs Beijing, China) at 37°C for 4 hrs to remove the template plasmid.

    Article Title: A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein
    Article Snippet: To remove the residual template plasmid pET28-fadR ec, the gel purified PCR products were digested for 1 h with DpnI (20 U/μL, NEB) at 37°C. .. Subsequently, they were transformed into chemically-competent cells of DH5α and the inserts of purified plasmids were verified by direct DNA sequencing.

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: To allow homologous recombination (and to preemptively silence any yeast receiving reclosed vector during the transformation), site-directed mutagenesis was used to create a unique restriction enzyme site (SpeI) followed by a double stop codon in the middle of CDRH1. .. The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time.

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. .. The nicked synthesized plasmid DNAs with the desired mutations were transformed into E. coli TOP10 chemically competent cells (Invitrogen).

    Derivative Assay:

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: All the ribozyme and riboswitch constructs presented in the paper were realized inserting an engineered twister ribozyme derived from the env-9 motif (ref. ) in the 5′-UTR of the eGFP gene. .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Countercurrent Chromatography:

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene). .. For two proximate amino acid substitutions in ZαZBP1 two mutagenesis PCRs were performed consecutively.

    Electroporation:

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB). .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Article Title: Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41
    Article Snippet: The primers used and PCR conditions are described in . .. We amplified the plasmid template with each forward and reverse primer using Pfu Turbo (Stratagene), digested the reaction with DpnI (New England Biolabs), and transformed into DH5α cells by electroporation. .. Colonies were screened for presence of HIV-1 Env by restriction digestion with MluI and NotI (New England Biolabs), and the entire HIV-1 Env variant was sequenced using BigDye (Applied Biosystems).

    Flow Cytometry:

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: Protein was purified over Ni Sepharose 6 Fast Flow resin (GE Healthcare) and eluted with 500 mM imidazole phosphate buffer. .. To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α.

    Gas Chromatography:

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: The reading frame of EGFP in pEGFP-N1 was replaced by monomeric red fluorescent protein (mRFP) that was amplified by mRFP1F AgeI (5′-TTG T AA CCG GT G GCC ACC ATG GCC TCC TCC GAG-3′) and mRFP1R NotI (5′-TTA ATC GCG GCC GC T ATT AGG CGC CGG TGG AGT GGC GGC-3′) with indicated enzymes, yielding pmRFP-N1. .. Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene).

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time. .. The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time.

    Ligation:

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing
    Article Snippet: Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). .. Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S).

    Synthesized:

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: The following conditions were used for plasmid replication: 15–17 cycles of denaturation at 95°C for 30 sec, primer annealing for 1 min at 55°C, followed by extension at 68°C for 1 min per 1 kb amplified. .. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. .. The nicked synthesized plasmid DNAs with the desired mutations were transformed into E. coli TOP10 chemically competent cells (Invitrogen).

    Introduce:

    Article Title: Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41
    Article Snippet: To introduce amino acid changes into the neutralization-resistant backgrounds, we used site-directed mutagenesis according to the instructions for Quik-Change site-directed mutagenesis kit (Stratagene). .. We amplified the plasmid template with each forward and reverse primer using Pfu Turbo (Stratagene), digested the reaction with DpnI (New England Biolabs), and transformed into DH5α cells by electroporation.

    Article Title: The Pivotal Roles of TIA Proteins in 5? Splice-Site Selection of Alu Exons and Across Evolution
    Article Snippet: Site-directed mutagenesis was carried out to introduce mutations into the ADAR2 and U1 minigenes by PCR using oligonucleotide primers containing the desired mutations. .. The PCR products were treated with DpnI (20 U, New England BioLabs) at 37°C for 1 h. Plasmid mutants were ligated using T4 DNA Ligase (New England BioLabs) at 37°C for 2 h. The mutant DNA was transformed into E. coli XL1-competent cells.

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: To introduce SNPs into the MEK -encoding sequence, paired synthetic primers that encoded the desired mutations were synthesized (See Table for primer sequences; Sigma-Aldrich) and utilized for mutagenic primer-directed replication of both plasmid strands with high-fidelity PfuUltra DNA polymerase (Agilent). .. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA.

    Generated:

    Article Title: O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
    Article Snippet: The p3XFLAG-OGT-siRNA-resistant vector was generated by directed mutagenesis using Phusion® Hot start (NEB), p3XFLAG-OGT as temple and, 5′-agcagggaaaactgcaggaagctctgatgcattataaagaagcgatcaggatttcccctacctttgctgatgcctactc-3′ and 5′-gagtaggcatcagcaaaggtaggggaaatcctgatcgcttctttataatgcatcagagcttcctgcagttttccctgct-3′ as primers. .. Prior to transformation in DH5α, template was digested for 2 h at 37 °C by 1 U of DpnI (NEB).

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Site-specific mutations were generated by PCR mutagenesis using KOD Hot start polymerase (Novagen) and primers containing the desired mutation following the Quick Change protocol (Stratagene). .. Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene).

    Article Title: The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation
    Article Snippet: A dominant-negative Hes1 (dnHes1) was generated by mutating E43, K44, and R47 in the basic region to A. Hes1(ΔHLH) and Hes1(ΔWPRW) deletion mutants were generated by deleting HLH domain (amino acids 48-92) and the last 6 amino acids of Hes1 respectively. .. All Hes1 mutants were amplified by PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pEASY-blut-Flag-Hes1 as a template (20 ng), following by DpnI (NEB) digestion.

    DNA Sequencing:

    Article Title: Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production
    Article Snippet: The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 µl of PCR products were digested with 1 µl DpnI (New England Biolabs Beijing, China) at 37°C for 4 hrs to remove the template plasmid. .. Aliquots of (2 µl) digestive products were transformed into E. coli Rosetta (BL21, DE3) competent cells (Stratagene, CA, USA).

    Electroporation Bacterial Transformation:

    Article Title: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations
    Article Snippet: The samples were then digested by Dpn I (NEB R0176L) according to the manufacturer's manual. .. After digestion, samples were transformed into competent E. coli .

    Injection:

    Article Title: Proteomic Analysis Reveals CACN-1 Is a Component of the Spliceosome in Caenorhabditis elegans
    Article Snippet: Young adult N2 hermaphrodites were injected using a microINJECTOR apparatus (Tritech Research, Los Angeles, CA). .. The injection solution contained 20 ng/µL transgene, 20 ng/µL sur-5 ::gfp , and 60 ng/µL N2 genomic DNA digested with Dpn I (New England Biolabs, Ipswich, MA). .. The green fluorescent protein marker allowed tracking and maintenance of the transgenic line, UN1305 xbEx1305[hsp16.2p ::flag ::ha ::cacn-1 , sur-5 ::GFP] .

    Recombinant:

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene). .. The substitutions in ZβZBP1 were generated by mutagenesis PCR with hZBP1 N141D Y145A F (5′-ACA GCA AAA GAT GTG GAC CGA GAC TTG GCC AGG ATG AAG AGC AGG-3′) and hZBP1 N141D Y145A R (5′-GGG CGA GGA CTT GGT CGA GCT CCC TCT TG-3′) in one step.

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: Paragraph title: Purification of PfNMNAT recombinant protein ... To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α.

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: The negative control for surface display and soluble scFv assays was an anti-hen egg lysozyme antibody, scFv D1.3 [ ]. scFv H7 was previously subcloned into the pCT-ESO backbone to create the plasmid pESO-H7 [ ]. pESO-H7 formed the basis for constructing a recombinant, histidine-saturation library focused on scFv H7 CDRH1. .. The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time.

    Cellular Antioxidant Activity Assay:

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene). .. For two proximate amino acid substitutions in ZαZBP1 two mutagenesis PCRs were performed consecutively.

    Methylation:

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: As control, the pUC19 plasmid was used to transform C2925 cells and One Shot TOP10 cells, which have a normal methylation system. .. Bacterial genomic DNA was isolated from 3 ml LBamp cultures from individual colonies using the DNeasy Tissue kit (Qiagen, 69504). gDNA (1 µg) was digested with 10 units of Dpn I (NEB, R0176S) for 1 h at 37°C.

    Article Title: Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase
    Article Snippet: Briefly, 25ng pET21b containing the full-length SAUGI with the stop codon was used as the template DNA in a 50 μl reaction mixture containing one of the primer pairs. .. After PCR, the reaction products were incubated with 1.5 μl (9 U) of DpnI (New England Biolabs) for 1 h to digest the methylated parent plasmids. .. The resulting plasmids were transformed into DH5 alpha E. coli cells and successful mutagenesis was confirmed by DNA sequencing.

    Mutagenesis:

    Article Title: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations
    Article Snippet: Paragraph title: Construction of mutant alleles using high-throughput site-directed mutagenesis PCR ... The samples were then digested by Dpn I (NEB R0176L) according to the manufacturer's manual.

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: This mutation results in a frame-shift starting from the amino acid 353 and probably in the destruction of the C-terminal helix which forms the tetramerization domain of the Lac repressor . .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Article Title: Quadruplex DNA in long terminal repeats in maize LTR retrotransposons inhibits the expression of a reporter gene in yeast
    Article Snippet: Paragraph title: Cloning and mutagenesis ... The products were treated with DpnI (NEB) and 1 μl was used for XL-1 blue electrocompetent cell (Agilent) transformation.

    Article Title: O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
    Article Snippet: The p3XFLAG-OGT-siRNA-resistant vector was generated by directed mutagenesis using Phusion® Hot start (NEB), p3XFLAG-OGT as temple and, 5′-agcagggaaaactgcaggaagctctgatgcattataaagaagcgatcaggatttcccctacctttgctgatgcctactc-3′ and 5′-gagtaggcatcagcaaaggtaggggaaatcctgatcgcttctttataatgcatcagagcttcctgcagttttccctgct-3′ as primers. .. Prior to transformation in DH5α, template was digested for 2 h at 37 °C by 1 U of DpnI (NEB).

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Site-specific mutations were generated by PCR mutagenesis using KOD Hot start polymerase (Novagen) and primers containing the desired mutation following the Quick Change protocol (Stratagene). .. Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene).

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: The wild type PF13_0159 locus was subcloned into the StrataClone vector (Agilent Technologies) and PCR was performed using the overlapping primers, D110A SENSE and ANTISENSE (Table S1 in ), containing the desired mutation. .. To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α.

    Article Title: Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41
    Article Snippet: Paragraph title: Cloning and Generation of Wild-Type and Mutant HIV-1 Viral Envelope Proteins ... We amplified the plasmid template with each forward and reverse primer using Pfu Turbo (Stratagene), digested the reaction with DpnI (New England Biolabs), and transformed into DH5α cells by electroporation.

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems
    Article Snippet: epPCR libraries of AGT were prepared with the GeneMorph II Random Mutagenesis Kit. .. The library was then purified with QIAquick PCR purification kit and subjected to a DpnI digest according to manufacturer's instructions (NEB).

    Article Title: The Pivotal Roles of TIA Proteins in 5? Splice-Site Selection of Alu Exons and Across Evolution
    Article Snippet: PCR was performed using PfuTurbo DNA polymerase (Stratagene) with an elongation time corresponding to 2 min for each kb. .. The PCR products were treated with DpnI (20 U, New England BioLabs) at 37°C for 1 h. Plasmid mutants were ligated using T4 DNA Ligase (New England BioLabs) at 37°C for 2 h. The mutant DNA was transformed into E. coli XL1-competent cells. .. DNA was extracted from selected colonies by mini-prep extraction (Promega).

    Article Title: Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production
    Article Snippet: Paragraph title: Site-directed mutagenesis of tea caffeine synthase (TCS1) ... The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 µl of PCR products were digested with 1 µl DpnI (New England Biolabs Beijing, China) at 37°C for 4 hrs to remove the template plasmid.

    Article Title: A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein
    Article Snippet: Paragraph title: Site-directed mutagenesis ... To remove the residual template plasmid pET28-fadR ec, the gel purified PCR products were digested for 1 h with DpnI (20 U/μL, NEB) at 37°C.

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: Paragraph title: Histidine-saturation mutagenesis of scFv H7 CDRH1 and library construction ... The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time.

    Article Title: Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase
    Article Snippet: Paragraph title: Site directed mutagenesis of SAUGI ... After PCR, the reaction products were incubated with 1.5 μl (9 U) of DpnI (New England Biolabs) for 1 h to digest the methylated parent plasmids.

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: The following conditions were used for plasmid replication: 15–17 cycles of denaturation at 95°C for 30 sec, primer annealing for 1 min at 55°C, followed by extension at 68°C for 1 min per 1 kb amplified. .. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. .. The nicked synthesized plasmid DNAs with the desired mutations were transformed into E. coli TOP10 chemically competent cells (Invitrogen).

    Isolation:

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: As control, the pUC19 plasmid was used to transform C2925 cells and One Shot TOP10 cells, which have a normal methylation system. .. Bacterial genomic DNA was isolated from 3 ml LBamp cultures from individual colonies using the DNeasy Tissue kit (Qiagen, 69504). gDNA (1 µg) was digested with 10 units of Dpn I (NEB, R0176S) for 1 h at 37°C. .. The products were run in a 1% agarose gel.

    Transfection:

    Article Title: The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation
    Article Snippet: All Hes1 mutants were amplified by PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pEASY-blut-Flag-Hes1 as a template (20 ng), following by DpnI (NEB) digestion. .. All Hes1 mutants were amplified by PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pEASY-blut-Flag-Hes1 as a template (20 ng), following by DpnI (NEB) digestion.

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: Paragraph title: MEK allele plasmid construction and transfection for in vitro studies ... The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA.

    Labeling:

    Article Title: Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface
    Article Snippet: The extracts were digested with the restriction enzyme DpnI (New England BioLabs) to digest input DNA. .. The membranes were hybridized overnight in 0.75 nylon wash buffer (40.6 g Na2 HPO4 , 18.65 g EDTA, and 500 g SDS in 3.58 liters of double-distilled H2 O, pH 7.2) at 65°C with a radiolabeled Rep probe (fw primer, 5′-AACTGGACCAATGAGAACTTTCC-3′; rv primer, 5′-A AAAAGTCTTTGACTTCCTGCTT-3′) or an ampicillin probe (fw primer, 5′-AATCAGTGAGGCACCTATCTCAGC-3′; rv primer, 5′-AACTCGGTCGCCGCATACACTATT-3′) to control for DpnI digestion.

    Purification:

    Article Title: Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2
    Article Snippet: The conditions for large-scale PCR to construct the randomized libraries was described previously ( , ). .. After PCR, the reaction mixture was treated with Dpn I (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid, then subjected to purification using a QIA-Quick column (Qiagen). .. The products were then verified by agarose gel electrophoresis.

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing
    Article Snippet: Penicillium chrysogenum DNA was extracted by DNeasy Plant Mini Kit (Qiagen, Cat. no. 69104). .. Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). .. Digested DNA segments were further sheared by Bioruptor (Diagenode) to ∼300 bp.

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB). .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Article Title: Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface
    Article Snippet: The low-molecular-weight DNA was then purified by phenol extraction, followed by sodium acetate and isopropanol precipitation. .. The extracts were digested with the restriction enzyme DpnI (New England BioLabs) to digest input DNA.

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: Paragraph title: Purification of PfNMNAT recombinant protein ... To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α.

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems
    Article Snippet: This was followed by a final extension step for 10 min at 72°C. .. The library was then purified with QIAquick PCR purification kit and subjected to a DpnI digest according to manufacturer's instructions (NEB). .. A portion of the library (50 ng or 7 × 1010 templates in a volume of 100 μl) was then re-amplified with Taq DNA polymerase under the same conditions described earlier to prepare suitable substrates for the assembly process except that amplification featured 25 cycles instead of 30.

    Article Title: A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein
    Article Snippet: The PCR reaction system (25 μL) consisted of the following components: 2.5 μL of 10× pfx buffer (Invitrogen), 0.5 μL of 40 mmol/L dNTP mix (10 mmol/L each), 1.0 μL of forward/reverse primers (10 pmol/μL), 1.0 μL of pET28-fadR ec as template (5 ng/μL), 0.5 μL of Platinum pfx (2.5 U/μL, Invitrogen), and 17.5 μL of distilled sterilized H2 O. .. To remove the residual template plasmid pET28-fadR ec, the gel purified PCR products were digested for 1 h with DpnI (20 U/μL, NEB) at 37°C. .. Subsequently, they were transformed into chemically-competent cells of DH5α and the inserts of purified plasmids were verified by direct DNA sequencing.

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: The following conditions were used for plasmid replication: 15–17 cycles of denaturation at 95°C for 30 sec, primer annealing for 1 min at 55°C, followed by extension at 68°C for 1 min per 1 kb amplified. .. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. .. The nicked synthesized plasmid DNAs with the desired mutations were transformed into E. coli TOP10 chemically competent cells (Invitrogen).

    Sequencing:

    Article Title: Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2
    Article Snippet: After PCR, the reaction mixture was treated with Dpn I (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid, then subjected to purification using a QIA-Quick column (Qiagen). .. After PCR, the reaction mixture was treated with Dpn I (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid, then subjected to purification using a QIA-Quick column (Qiagen).

    Article Title: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations
    Article Snippet: To generate mutant alleles, sequence-verified single-colony wild-type clones and their corresponding mutagenic primers were aliquoted into individual wells of 96-well PCR plates. .. The samples were then digested by Dpn I (NEB R0176L) according to the manufacturer's manual.

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing
    Article Snippet: Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). .. After purification by AMPure XP beads (Beckman Coulter, Cat. no. A63880), DNA segments were end-repaired, followed by 3′-adenylation and adaptor ligation according to standard Illumina TruSeq DNA sample preparation procedures.

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: The sequence of the 5′-UTR of these constructs is reported in the . .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB).

    Article Title: Quadruplex DNA in long terminal repeats in maize LTR retrotransposons inhibits the expression of a reporter gene in yeast
    Article Snippet: The products were treated with DpnI (NEB) and 1 μl was used for XL-1 blue electrocompetent cell (Agilent) transformation. .. The products were treated with DpnI (NEB) and 1 μl was used for XL-1 blue electrocompetent cell (Agilent) transformation.

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems
    Article Snippet: The library was then purified with QIAquick PCR purification kit and subjected to a DpnI digest according to manufacturer's instructions (NEB). .. The re-amplified library was then precipitated with PEG–MgCl2 , blunted with T4 DNA polymerase, assembled and purified by PCR.

    Article Title: The Pivotal Roles of TIA Proteins in 5? Splice-Site Selection of Alu Exons and Across Evolution
    Article Snippet: Mutations creating deletions in wild-type minigenes were performed by PCR using 5′ phosphorylated primers flanking the sequence to be deleted (see Supplementary Table 1 in for list of primers). .. The PCR products were treated with DpnI (20 U, New England BioLabs) at 37°C for 1 h. Plasmid mutants were ligated using T4 DNA Ligase (New England BioLabs) at 37°C for 2 h. The mutant DNA was transformed into E. coli XL1-competent cells.

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: To introduce SNPs into the MEK -encoding sequence, paired synthetic primers that encoded the desired mutations were synthesized (See Table for primer sequences; Sigma-Aldrich) and utilized for mutagenic primer-directed replication of both plasmid strands with high-fidelity PfuUltra DNA polymerase (Agilent). .. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA.

    Concentration Assay:

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing
    Article Snippet: Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). .. Adaptor-ligated DNA segments were PCR amplified for 15 cycles, purified by AMPure XP beads and suspended in 20 μl of resuspension buffer to yield the sequencing library.

    Sample Prep:

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing
    Article Snippet: Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). .. Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains
    Article Snippet: Following PCR with 18 cycles the products were digested with DpnI (New England Biolabs) and transformed into ultracompetent XL-10 Escherichia coli (Stratagene). .. For two proximate amino acid substitutions in ZαZBP1 two mutagenesis PCRs were performed consecutively.

    Silver Staining:

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: Purity of the 26 kDa fusion protein was verified by silver stain SDS/PAGE. .. To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α.

    Chromatin Immunoprecipitation:

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing
    Article Snippet: Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). .. Adaptor-ligated DNA segments were PCR amplified for 15 cycles, purified by AMPure XP beads and suspended in 20 μl of resuspension buffer to yield the sequencing library.

    SDS Page:

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: Purity of the 26 kDa fusion protein was verified by silver stain SDS/PAGE. .. To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α.

    Plasmid Preparation:

    Article Title: Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2
    Article Snippet: The conditions for large-scale PCR to construct the randomized libraries was described previously ( , ). .. After PCR, the reaction mixture was treated with Dpn I (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid, then subjected to purification using a QIA-Quick column (Qiagen). .. The products were then verified by agarose gel electrophoresis.

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: As control, the pUC19 plasmid was used to transform C2925 cells and One Shot TOP10 cells, which have a normal methylation system. .. Bacterial genomic DNA was isolated from 3 ml LBamp cultures from individual colonies using the DNeasy Tissue kit (Qiagen, 69504). gDNA (1 µg) was digested with 10 units of Dpn I (NEB, R0176S) for 1 h at 37°C.

    Article Title: Twister ribozymes as highly versatile expression platforms for artificial riboswitches
    Article Snippet: The twister ribozyme and the twister-based theophylline and TPP aptazymes developed in E. coli were introduced into the pET16b_eGFP plasmid by whole-plasmid PCR with Phusion Hot Start 2 Polymerase (NEB) using primers with the designed ribozyme sequences contained into the 5′-overhang ends ( ). .. Subsequently to the PCR, the template plasmid was digested using the enzyme DpnI (NEB). .. The PCR products were purified by band purification after gel electrophoresis (0.8% GTQ agarose—Roth).

    Article Title: O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
    Article Snippet: The p3XFLAG-OGT-siRNA-resistant vector was generated by directed mutagenesis using Phusion® Hot start (NEB), p3XFLAG-OGT as temple and, 5′-agcagggaaaactgcaggaagctctgatgcattataaagaagcgatcaggatttcccctacctttgctgatgcctactc-3′ and 5′-gagtaggcatcagcaaaggtaggggaaatcctgatcgcttctttataatgcatcagagcttcctgcagttttccctgct-3′ as primers. .. Prior to transformation in DH5α, template was digested for 2 h at 37 °C by 1 U of DpnI (NEB).

    Article Title: Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum
    Article Snippet: The wild type PF13_0159 locus was subcloned into the StrataClone vector (Agilent Technologies) and PCR was performed using the overlapping primers, D110A SENSE and ANTISENSE (Table S1 in ), containing the desired mutation. .. To recover the mutated plasmid, the native template was digested with DpnI (New England Biolabs) and then transformed into E. coli strain DH5α. .. Prior to assays, purified PfNMNAT-6×HIS protein was dialyzed into reaction buffer containing 100 mM HEPES and 10 mM MgCl2 at pH 7.5 overnight at 4° using 3500 Da MWCO dialysis tubing from Fisherbrand.

    Article Title: Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41
    Article Snippet: The primers used and PCR conditions are described in . .. We amplified the plasmid template with each forward and reverse primer using Pfu Turbo (Stratagene), digested the reaction with DpnI (New England Biolabs), and transformed into DH5α cells by electroporation. .. Colonies were screened for presence of HIV-1 Env by restriction digestion with MluI and NotI (New England Biolabs), and the entire HIV-1 Env variant was sequenced using BigDye (Applied Biosystems).

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems
    Article Snippet: The library was then purified with QIAquick PCR purification kit and subjected to a DpnI digest according to manufacturer's instructions (NEB). .. The re-amplified library was then precipitated with PEG–MgCl2 , blunted with T4 DNA polymerase, assembled and purified by PCR.

    Article Title: The Pivotal Roles of TIA Proteins in 5? Splice-Site Selection of Alu Exons and Across Evolution
    Article Snippet: PCR was performed using PfuTurbo DNA polymerase (Stratagene) with an elongation time corresponding to 2 min for each kb. .. The PCR products were treated with DpnI (20 U, New England BioLabs) at 37°C for 1 h. Plasmid mutants were ligated using T4 DNA Ligase (New England BioLabs) at 37°C for 2 h. The mutant DNA was transformed into E. coli XL1-competent cells. .. DNA was extracted from selected colonies by mini-prep extraction (Promega).

    Article Title: A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein
    Article Snippet: The PCR reaction system (25 μL) consisted of the following components: 2.5 μL of 10× pfx buffer (Invitrogen), 0.5 μL of 40 mmol/L dNTP mix (10 mmol/L each), 1.0 μL of forward/reverse primers (10 pmol/μL), 1.0 μL of pET28-fadR ec as template (5 ng/μL), 0.5 μL of Platinum pfx (2.5 U/μL, Invitrogen), and 17.5 μL of distilled sterilized H2 O. .. To remove the residual template plasmid pET28-fadR ec, the gel purified PCR products were digested for 1 h with DpnI (20 U/μL, NEB) at 37°C. .. Subsequently, they were transformed into chemically-competent cells of DH5α and the inserts of purified plasmids were verified by direct DNA sequencing.

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: To allow homologous recombination (and to preemptively silence any yeast receiving reclosed vector during the transformation), site-directed mutagenesis was used to create a unique restriction enzyme site (SpeI) followed by a double stop codon in the middle of CDRH1. .. The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time.

    Article Title: The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation
    Article Snippet: The full-length murine Hes1 cDNA with an N-terminal FLAG tag was cloned into the pEasy-blunt cloning vector (Transgene). .. All Hes1 mutants were amplified by PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pEASY-blut-Flag-Hes1 as a template (20 ng), following by DpnI (NEB) digestion.

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: Paragraph title: MEK allele plasmid construction and transfection for in vitro studies ... The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA.

    Dominant Negative Mutation:

    Article Title: The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation
    Article Snippet: A dominant-negative Hes1 (dnHes1) was generated by mutating E43, K44, and R47 in the basic region to A. Hes1(ΔHLH) and Hes1(ΔWPRW) deletion mutants were generated by deleting HLH domain (amino acids 48-92) and the last 6 amino acids of Hes1 respectively. .. All Hes1 mutants were amplified by PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pEASY-blut-Flag-Hes1 as a template (20 ng), following by DpnI (NEB) digestion.

    Negative Control:

    Article Title: Proteomic Analysis Reveals CACN-1 Is a Component of the Spliceosome in Caenorhabditis elegans
    Article Snippet: The injection solution contained 20 ng/µL transgene, 20 ng/µL sur-5 ::gfp , and 60 ng/µL N2 genomic DNA digested with Dpn I (New England Biolabs, Ipswich, MA). .. The green fluorescent protein marker allowed tracking and maintenance of the transgenic line, UN1305 xbEx1305[hsp16.2p ::flag ::ha ::cacn-1 , sur-5 ::GFP] .

    Article Title: O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
    Article Snippet: Control siRNA (siRNA univ. negative control) and siRNA against OGT (GGAGGCUAUUCGAAUCAGU[dT][dT] forward, ACUGAUUCGAAUAGCCUCC[dT][dT] reverse) were purchased from Sigma-Aldrich. siRNA against OGA (siGENOME Human MGEA5 (10724) siRNA SMARTpool) was from Dharmacon (GE Healthcare Europe GmbH, Velizy-Villacoublay, France). .. Prior to transformation in DH5α, template was digested for 2 h at 37 °C by 1 U of DpnI (NEB).

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: The negative control for surface display and soluble scFv assays was an anti-hen egg lysozyme antibody, scFv D1.3 [ ]. scFv H7 was previously subcloned into the pCT-ESO backbone to create the plasmid pESO-H7 [ ]. pESO-H7 formed the basis for constructing a recombinant, histidine-saturation library focused on scFv H7 CDRH1. .. The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time.

    Selection:

    Article Title: Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2
    Article Snippet: After PCR, the reaction mixture was treated with Dpn I (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid, then subjected to purification using a QIA-Quick column (Qiagen). .. After PCR, the reaction mixture was treated with Dpn I (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid, then subjected to purification using a QIA-Quick column (Qiagen).

    Agarose Gel Electrophoresis:

    Article Title: Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production
    Article Snippet: PCR was performed using phusion high-fidelity DNA polymerase (Finnzymes, Espoo, Finland) with the reaction settings at 98°C for 50 s, 1 cycle, followed by 25 cycles at 98°C for 10 s, 60°C for 15 s, and 72°C for 6 min; the final extension was 72°C for 10 min. .. The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 µl of PCR products were digested with 1 µl DpnI (New England Biolabs Beijing, China) at 37°C for 4 hrs to remove the template plasmid. .. Aliquots of (2 µl) digestive products were transformed into E. coli Rosetta (BL21, DE3) competent cells (Stratagene, CA, USA).

    In Vitro:

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: Paragraph title: MEK allele plasmid construction and transfection for in vitro studies ... The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA.

    Size-exclusion Chromatography:

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. .. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA.

    Transgenic Assay:

    Article Title: Proteomic Analysis Reveals CACN-1 Is a Component of the Spliceosome in Caenorhabditis elegans
    Article Snippet: Paragraph title: Nematode strains and generation of transgenic animals ... The injection solution contained 20 ng/µL transgene, 20 ng/µL sur-5 ::gfp , and 60 ng/µL N2 genomic DNA digested with Dpn I (New England Biolabs, Ipswich, MA).

    Functional Assay:

    Article Title: Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae
    Article Snippet: The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. .. Eight to ten transformed colonies for every desired mutation were screened for plasmid DNA using the Qiagen Miniprep Kit and the manufacturer’s instructions (Qiagen).

    Activation Assay:

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems
    Article Snippet: The library was then purified with QIAquick PCR purification kit and subjected to a DpnI digest according to manufacturer's instructions (NEB). .. The library was then purified with QIAquick PCR purification kit and subjected to a DpnI digest according to manufacturer's instructions (NEB).

    FLAG-tag:

    Article Title: The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation
    Article Snippet: The full-length murine Hes1 cDNA with an N-terminal FLAG tag was cloned into the pEasy-blunt cloning vector (Transgene). .. All Hes1 mutants were amplified by PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pEASY-blut-Flag-Hes1 as a template (20 ng), following by DpnI (NEB) digestion.

    High Throughput Screening Assay:

    Article Title: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations
    Article Snippet: Paragraph title: Construction of mutant alleles using high-throughput site-directed mutagenesis PCR ... The samples were then digested by Dpn I (NEB R0176L) according to the manufacturer's manual.

    Lysis:

    Article Title: Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface
    Article Snippet: Briefly, cells were lysed in Hirt lysis buffer (0.6% SDS, 10 mM Tris, pH 7.5, 10 mM EDTA) and treated with proteinase K (Thermo Fisher) to digest proteins. .. The extracts were digested with the restriction enzyme DpnI (New England BioLabs) to digest input DNA.

    Homologous Recombination:

    Article Title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation
    Article Snippet: To allow homologous recombination (and to preemptively silence any yeast receiving reclosed vector during the transformation), site-directed mutagenesis was used to create a unique restriction enzyme site (SpeI) followed by a double stop codon in the middle of CDRH1. .. The resulting PCR product was digested with DpnI (NEB), ethanol precipitated in the presence of sodium acetate and pellet paint (Covagen), rehydrated in 1x TE, and digested with DpnI a second time.

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  • 99
    New England Biolabs dpn i
    Polymerase linkage status of cytoplasmic versus nuclear viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with <t>Dpn</t> I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. ( B ) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.
    Dpn I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dpn i/product/New England Biolabs
    Average 99 stars, based on 141 article reviews
    Price from $9.99 to $1999.99
    dpn i - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs dpn i enzyme
    Chimera construction or insertion with a short DNA fragment . To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single <t>PCR</t> amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. Dpn I digestion and transformation are the same as in Figure 1.
    Dpn I Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dpn i enzyme/product/New England Biolabs
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    dpn i enzyme - by Bioz Stars, 2019-12
    99/100 stars
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    Polymerase linkage status of cytoplasmic versus nuclear viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with Dpn I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. ( B ) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.

    Journal: PLoS Pathogens

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    doi: 10.1371/journal.ppat.1001082

    Figure Lengend Snippet: Polymerase linkage status of cytoplasmic versus nuclear viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with Dpn I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. ( B ) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.

    Article Snippet: Dpn I (NEB) and Plasmid-Safe DNase (Epicentre Biotechnologies) were applied as suggested by the manufacturers.

    Techniques: Transfection, Purification, Incubation, Proximity Ligation Assay, Produced

    Intracellular distribution and nuclease sensitivity of viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Relative nuclear distribution. DNA was extracted, after prior PK treatment, from total cells or from gradient-purified nuclei and subsequently digested with DpnI plus PsD. Serially diluted samples were loaded on the gel. Loading volumes are indicated in percent of the total sample volume, obtained from one well of a 6-well plate. One of three experiments used for quantification (see text for details) is shown. ( B ) Direct comparison of MN resistant versus total nuclear viral DNA. DNA was prepared from total cell extract treated with MN plus PK (MN), or from gradient-purified nuclei; equal aliquots of the nuclei were treated with MN plus PK before DNA extraction (MN), or with only PK followed by incubation with Dpn I plus PsD (total nuclear DNA). A six times longer exposure for the nuclear HBV samples is shown to better reveal weak signals. Quantification indicated that only ∼10% of the nuclear full-length HBV versus ≥80% of the nuclear DHBV DNA were MN resistant.

    Journal: PLoS Pathogens

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    doi: 10.1371/journal.ppat.1001082

    Figure Lengend Snippet: Intracellular distribution and nuclease sensitivity of viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Relative nuclear distribution. DNA was extracted, after prior PK treatment, from total cells or from gradient-purified nuclei and subsequently digested with DpnI plus PsD. Serially diluted samples were loaded on the gel. Loading volumes are indicated in percent of the total sample volume, obtained from one well of a 6-well plate. One of three experiments used for quantification (see text for details) is shown. ( B ) Direct comparison of MN resistant versus total nuclear viral DNA. DNA was prepared from total cell extract treated with MN plus PK (MN), or from gradient-purified nuclei; equal aliquots of the nuclei were treated with MN plus PK before DNA extraction (MN), or with only PK followed by incubation with Dpn I plus PsD (total nuclear DNA). A six times longer exposure for the nuclear HBV samples is shown to better reveal weak signals. Quantification indicated that only ∼10% of the nuclear full-length HBV versus ≥80% of the nuclear DHBV DNA were MN resistant.

    Article Snippet: Dpn I (NEB) and Plasmid-Safe DNase (Epicentre Biotechnologies) were applied as suggested by the manufacturers.

    Techniques: Transfection, Purification, DNA Extraction, Incubation

    Site-specific discontinuity confirms the rcDNA nature of a substantial fraction of nuclear HBV DNA. Extensive nicking of HBV cccDNA might pretend an artifactually high ratio of rcDNA to cccDNA in the nucleus. However, nicking should occur at random whereas RC-DNA is distinctly discontinuous where the minus-strand and plus-strand DNA start. ( A ) Scheme of HBV rcDNA discontinuities. Restriction site positions are indicated with the first and last nucleotide of the recognition sequences. The Apa LI site is located immediately upstream of the plus-strand start. DNA in which the plus-strand is not sufficiently extended can not be cut. DR1 and DR2, direct repeats 1 and 2; wavy line at DR2, RNA primer at plus-strand 5′ end. ( B ) About one third of the rcDNA signal is resistant to Apa LI but not Nco I or Fsp I digestion. Cytoplasmic (treated with MN) and nuclear (treated with Dpn I) DNA preparations (both after prior PK digestion) were incubated with the indicated restriction enzymes. Consistently ( Figure S5B ), ∼35% of the rcDNA signal from the cytoplasm as well as the nucleus remained upon incubation with Apa LI (arrowheads) but not Nco I or Fsp I. Activity of Apa LI in the reactions is documented by the absence of a plasmid-derived Dpn I fragment containing internal sites for Apa LI and Fsp I but not Nco I. All samples were run on the same gel but a six-times longer exposure is shown for the nuclear samples; lane 5L on the longer exposure corresponds to lane 5 on the left panel. M, marker fragments of the indicated sizes (in kb).

    Journal: PLoS Pathogens

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    doi: 10.1371/journal.ppat.1001082

    Figure Lengend Snippet: Site-specific discontinuity confirms the rcDNA nature of a substantial fraction of nuclear HBV DNA. Extensive nicking of HBV cccDNA might pretend an artifactually high ratio of rcDNA to cccDNA in the nucleus. However, nicking should occur at random whereas RC-DNA is distinctly discontinuous where the minus-strand and plus-strand DNA start. ( A ) Scheme of HBV rcDNA discontinuities. Restriction site positions are indicated with the first and last nucleotide of the recognition sequences. The Apa LI site is located immediately upstream of the plus-strand start. DNA in which the plus-strand is not sufficiently extended can not be cut. DR1 and DR2, direct repeats 1 and 2; wavy line at DR2, RNA primer at plus-strand 5′ end. ( B ) About one third of the rcDNA signal is resistant to Apa LI but not Nco I or Fsp I digestion. Cytoplasmic (treated with MN) and nuclear (treated with Dpn I) DNA preparations (both after prior PK digestion) were incubated with the indicated restriction enzymes. Consistently ( Figure S5B ), ∼35% of the rcDNA signal from the cytoplasm as well as the nucleus remained upon incubation with Apa LI (arrowheads) but not Nco I or Fsp I. Activity of Apa LI in the reactions is documented by the absence of a plasmid-derived Dpn I fragment containing internal sites for Apa LI and Fsp I but not Nco I. All samples were run on the same gel but a six-times longer exposure is shown for the nuclear samples; lane 5L on the longer exposure corresponds to lane 5 on the left panel. M, marker fragments of the indicated sizes (in kb).

    Article Snippet: Dpn I (NEB) and Plasmid-Safe DNase (Epicentre Biotechnologies) were applied as suggested by the manufacturers.

    Techniques: Incubation, Activity Assay, Plasmid Preparation, Derivative Assay, Marker

    DHBV replication in avian cells and HBV replication in human cells. ( A ) Chicken LMH cells transfected with wild-type and surface-deficient DHBV. ( B ) Human HepG2 cells transfected with wild-type and surface-deficient HBV. DNAs were extracted, after prior PK digestion, from cytoplasmic lysates and the nuclear fractions. One aliquot of the cytoplasmic lysates was treated with micrococcal nuclease (MN) before DNA extraction and analyzed without further treatment. All other samples were incubated, post extraction, with either Dpn I alone (Dpn), or Dpn I plus Plasmid safe DNAse (Dpn+PsD). Nomenclature of the various DNA species: RC, relaxed circular; DL, double strand linear; SS, single strand; CCC, covalently closed circular DNA; Pla, Dpn I restriction fragments of transfected plasmid DNA.

    Journal: PLoS Pathogens

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    doi: 10.1371/journal.ppat.1001082

    Figure Lengend Snippet: DHBV replication in avian cells and HBV replication in human cells. ( A ) Chicken LMH cells transfected with wild-type and surface-deficient DHBV. ( B ) Human HepG2 cells transfected with wild-type and surface-deficient HBV. DNAs were extracted, after prior PK digestion, from cytoplasmic lysates and the nuclear fractions. One aliquot of the cytoplasmic lysates was treated with micrococcal nuclease (MN) before DNA extraction and analyzed without further treatment. All other samples were incubated, post extraction, with either Dpn I alone (Dpn), or Dpn I plus Plasmid safe DNAse (Dpn+PsD). Nomenclature of the various DNA species: RC, relaxed circular; DL, double strand linear; SS, single strand; CCC, covalently closed circular DNA; Pla, Dpn I restriction fragments of transfected plasmid DNA.

    Article Snippet: Dpn I (NEB) and Plasmid-Safe DNase (Epicentre Biotechnologies) were applied as suggested by the manufacturers.

    Techniques: Transfection, DNA Extraction, Incubation, Plasmid Preparation, Countercurrent Chromatography, Proximity Ligation Assay

    Restriction mapping of nuclear HBV rcDNA suggests genome region-selective MN accessibility. ( A ) Cytoplasmic versus nuclear MN-resistant viral DNAs. DNAs were isolated from HepG2 cells transfected with the surface-deficient HBV vector after prior MN plus PK treatment, and incubated with restriction enzymes Spe I and Nco I (ø, no restriction). Amounts equivalent to 10% of the total cytoplasmic fraction and 90% of the total nuclear fraction were loaded. ( B ) Total nuclear rcDNA versus MN resistant nuclear DNA. Viral DNA from isolated nuclei was prepared by either the PK plus Dpn I plus PsD procedure (total nuclear rcDNA), or after prior MN plus PK treatment (MN resistant nuclear DNA), and incubated with Nsi I, Eco RI or Bsp EI. Asterisks denote newly formed distinct fragments; lane ø on the right is a longer exposure of lane ø on the left. ( C ) Restriction map of HBV. The restriction sites probed in A and B are indicated. DR1 and DR2, direct repeats 1 and 2; P, covalently linked polymerase; wiggly red line, RNA primer at (+)-DNA 5′ end. Together, the patterns can consistently be explained if MN treatment removed a defined region from the rcDNA that approximately encompasses position 3000 to 500.

    Journal: PLoS Pathogens

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    doi: 10.1371/journal.ppat.1001082

    Figure Lengend Snippet: Restriction mapping of nuclear HBV rcDNA suggests genome region-selective MN accessibility. ( A ) Cytoplasmic versus nuclear MN-resistant viral DNAs. DNAs were isolated from HepG2 cells transfected with the surface-deficient HBV vector after prior MN plus PK treatment, and incubated with restriction enzymes Spe I and Nco I (ø, no restriction). Amounts equivalent to 10% of the total cytoplasmic fraction and 90% of the total nuclear fraction were loaded. ( B ) Total nuclear rcDNA versus MN resistant nuclear DNA. Viral DNA from isolated nuclei was prepared by either the PK plus Dpn I plus PsD procedure (total nuclear rcDNA), or after prior MN plus PK treatment (MN resistant nuclear DNA), and incubated with Nsi I, Eco RI or Bsp EI. Asterisks denote newly formed distinct fragments; lane ø on the right is a longer exposure of lane ø on the left. ( C ) Restriction map of HBV. The restriction sites probed in A and B are indicated. DR1 and DR2, direct repeats 1 and 2; P, covalently linked polymerase; wiggly red line, RNA primer at (+)-DNA 5′ end. Together, the patterns can consistently be explained if MN treatment removed a defined region from the rcDNA that approximately encompasses position 3000 to 500.

    Article Snippet: Dpn I (NEB) and Plasmid-Safe DNase (Epicentre Biotechnologies) were applied as suggested by the manufacturers.

    Techniques: Isolation, Transfection, Plasmid Preparation, Incubation

    Core protein association of nuclear DHBV and HBV DNA. Vectors for surface-deficient DHBV and HBV genomes were transfected into Huh7 cells. IPs were performed in cytoplasmic extracts and extracts of purified nuclei containing 0.75× RIPA buffer, using antibodies against DHBV core protein (αDc) or HBV core protein (αHBc); in the mock IPs αDc was replaced by αHBc and vice versa. Immunopellets were treated with MN or not as indicated, and extracted after prior PK digestion. Purified DNAs from not MN-treated samples were digested with Dpn I. ø, extract directly treated with MN. ( A ) DHBV. M1, marker for cccDNA and rcDNA; M2, marker for single-stranded DNA; * and **, positions of cccDNA and ssDNA, respectively. ( B ) HBV. The rightmost panel shows the nuclear samples from an analogous experiment in HepG2 cells; the cytoplasmic samples are shown in Figure S7 .

    Journal: PLoS Pathogens

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    doi: 10.1371/journal.ppat.1001082

    Figure Lengend Snippet: Core protein association of nuclear DHBV and HBV DNA. Vectors for surface-deficient DHBV and HBV genomes were transfected into Huh7 cells. IPs were performed in cytoplasmic extracts and extracts of purified nuclei containing 0.75× RIPA buffer, using antibodies against DHBV core protein (αDc) or HBV core protein (αHBc); in the mock IPs αDc was replaced by αHBc and vice versa. Immunopellets were treated with MN or not as indicated, and extracted after prior PK digestion. Purified DNAs from not MN-treated samples were digested with Dpn I. ø, extract directly treated with MN. ( A ) DHBV. M1, marker for cccDNA and rcDNA; M2, marker for single-stranded DNA; * and **, positions of cccDNA and ssDNA, respectively. ( B ) HBV. The rightmost panel shows the nuclear samples from an analogous experiment in HepG2 cells; the cytoplasmic samples are shown in Figure S7 .

    Article Snippet: Dpn I (NEB) and Plasmid-Safe DNase (Epicentre Biotechnologies) were applied as suggested by the manufacturers.

    Techniques: Transfection, Purification, Marker

    Effects of HPV16 E1 mutants on HPV18 replication. (A) Schematic representation of HPV16 E1 mutants. F, FLAG-tag; ND, N-terminal domain; DBD, DNA-binding domain; OD, oligomerization domain; HD, helicase domain. (B) C33A cells were transfected with 1 ng of the HPV18 gDNA together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV18 gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the HPV18 E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Journal: Virology Journal

    Article Title: Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases

    doi: 10.1186/1743-422X-11-11

    Figure Lengend Snippet: Effects of HPV16 E1 mutants on HPV18 replication. (A) Schematic representation of HPV16 E1 mutants. F, FLAG-tag; ND, N-terminal domain; DBD, DNA-binding domain; OD, oligomerization domain; HD, helicase domain. (B) C33A cells were transfected with 1 ng of the HPV18 gDNA together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV18 gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the HPV18 E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Article Snippet: To digest the transfected DNA, 2 μl of the DNA sample was incubated with 10 U of Dpn I in 20 μl of 1 × reaction buffer (New England Biolabs, Ipswich, MA, USA) for 2 h at 37°C, followed by heat inactivation of Dpn I at 80°C for 20 min.

    Techniques: FLAG-tag, Binding Assay, Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    Replication of HPV16 or HPV18 genomes supported by homologous or heterologous E1/E2s. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication when cells were transfected 20 ng of each pF16E1 and pF16E2 (A) or 10 ng of each pF18E1 and pF18E2 (B) . Each bar represents the average of two independent experiments with the standard error of mean.

    Journal: Virology Journal

    Article Title: Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases

    doi: 10.1186/1743-422X-11-11

    Figure Lengend Snippet: Replication of HPV16 or HPV18 genomes supported by homologous or heterologous E1/E2s. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication when cells were transfected 20 ng of each pF16E1 and pF16E2 (A) or 10 ng of each pF18E1 and pF18E2 (B) . Each bar represents the average of two independent experiments with the standard error of mean.

    Article Snippet: To digest the transfected DNA, 2 μl of the DNA sample was incubated with 10 U of Dpn I in 20 μl of 1 × reaction buffer (New England Biolabs, Ipswich, MA, USA) for 2 h at 37°C, followed by heat inactivation of Dpn I at 80°C for 20 min.

    Techniques: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    Simultaneous replication of HPV16/18 genomes in the presence of E1/E2s of both types. C33A cells were transfected with a mixture of HPV16 and HPV18 gDNAs (1 ng each) together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV16 (upper panel) and HPV18 (lower panel) gDNAs were quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of three independent experiments with the standard deviation.

    Journal: Virology Journal

    Article Title: Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases

    doi: 10.1186/1743-422X-11-11

    Figure Lengend Snippet: Simultaneous replication of HPV16/18 genomes in the presence of E1/E2s of both types. C33A cells were transfected with a mixture of HPV16 and HPV18 gDNAs (1 ng each) together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV16 (upper panel) and HPV18 (lower panel) gDNAs were quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of three independent experiments with the standard deviation.

    Article Snippet: To digest the transfected DNA, 2 μl of the DNA sample was incubated with 10 U of Dpn I in 20 μl of 1 × reaction buffer (New England Biolabs, Ipswich, MA, USA) for 2 h at 37°C, followed by heat inactivation of Dpn I at 80°C for 20 min.

    Techniques: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Standard Deviation

    Effects of heterologous E1 or E2 on HPV16/18 replication. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to that of the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Journal: Virology Journal

    Article Title: Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases

    doi: 10.1186/1743-422X-11-11

    Figure Lengend Snippet: Effects of heterologous E1 or E2 on HPV16/18 replication. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to that of the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Article Snippet: To digest the transfected DNA, 2 μl of the DNA sample was incubated with 10 U of Dpn I in 20 μl of 1 × reaction buffer (New England Biolabs, Ipswich, MA, USA) for 2 h at 37°C, followed by heat inactivation of Dpn I at 80°C for 20 min.

    Techniques: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Methylation, Quantitative RT-PCR, Expressing, Marker, Negative Control

    Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Amplification, Negative Control, Polymerase Chain Reaction, Methylation

    Chimera construction or insertion with a short DNA fragment . To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single PCR amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. Dpn I digestion and transformation are the same as in Figure 1.

    Journal: BMC Biotechnology

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    doi: 10.1186/1472-6750-11-92

    Figure Lengend Snippet: Chimera construction or insertion with a short DNA fragment . To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single PCR amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. Dpn I digestion and transformation are the same as in Figure 1.

    Article Snippet: After confirmation of PCR products, 1 μl of Dpn I enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately.

    Techniques: FLAG-tag, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Gas Chromatography, Transformation Assay

    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Journal: BMC Biotechnology

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    doi: 10.1186/1472-6750-11-92

    Figure Lengend Snippet: Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Article Snippet: After confirmation of PCR products, 1 μl of Dpn I enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transformation Assay, Negative Control, Construct

    The procedures for FastCloning: Step 1. PCR amplification of vector and insert. Note that the primer pair for insert amplification has 16-base tails overlapping with the PCR-amplified vector ends. Step 2. Dpn I digestion. The parent DNA templates (if in a plasmid) for PCR amplification needs to be methylated in order to be compatible to Dpn I digestion. Although the detailed mechanism is not known, it is likely that the 3' exonuclease activity of the high fidelity DNA polymerase directly creates sticky ends for the overlapped regions of the vector and insert during Dpn I digestion, allowing them to form a circular construct with nicks. Step 3. transformation into competent E. coli . cells. The nicks will be repaired after transformation into the bacteria.

    Journal: BMC Biotechnology

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    doi: 10.1186/1472-6750-11-92

    Figure Lengend Snippet: The procedures for FastCloning: Step 1. PCR amplification of vector and insert. Note that the primer pair for insert amplification has 16-base tails overlapping with the PCR-amplified vector ends. Step 2. Dpn I digestion. The parent DNA templates (if in a plasmid) for PCR amplification needs to be methylated in order to be compatible to Dpn I digestion. Although the detailed mechanism is not known, it is likely that the 3' exonuclease activity of the high fidelity DNA polymerase directly creates sticky ends for the overlapped regions of the vector and insert during Dpn I digestion, allowing them to form a circular construct with nicks. Step 3. transformation into competent E. coli . cells. The nicks will be repaired after transformation into the bacteria.

    Article Snippet: After confirmation of PCR products, 1 μl of Dpn I enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately.

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Methylation, Activity Assay, Construct, Transformation Assay