ndei  (New England Biolabs)


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    Structured Review

    New England Biolabs ndei
    Nicking endonuclease activity of HP0268. (A) The nicking endonuclease activity at various protein concentrations (1, 2, 4 and 8 μM) after incubation at 37ºC for 30 min. OC, RC and linear are abbreviations for the nicked open-circular, relaxed circular and linear DNA, respectively. (B) The pH dependence of the DNA nicking activity. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min under various pH conditions (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). (C) Effect of metal ions on the DNA nicking activity of HP0268. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min in the presence and absence of 1 mM metal ion (Ca 2+ , Co 2+ , Ni 2+ , Fe 3+ , Mn 2+ , Mg 2+ and Cu 2+ ). Increasing concentrations (0.2, 0.4 and 1 μM) of Mn 2+ ion were used. Excess EDTA was used to remove the residual metal ions during the protein preparation. (D) The percentages of the resulting DNA conformations were plotted with regard to metal ion used. Cont. represents the substrate plasmid pET-21a(+) without HP0268, and <t>Nt.BsmAI</t> and <t>NdeI</t> represent the positive controls for the nicked and linear DNA, respectively. Commonly, 10 units of control enzyme were used in a final volume of 30 μl.
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    Images

    1) Product Images from "Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities"

    Article Title: Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv348

    Nicking endonuclease activity of HP0268. (A) The nicking endonuclease activity at various protein concentrations (1, 2, 4 and 8 μM) after incubation at 37ºC for 30 min. OC, RC and linear are abbreviations for the nicked open-circular, relaxed circular and linear DNA, respectively. (B) The pH dependence of the DNA nicking activity. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min under various pH conditions (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). (C) Effect of metal ions on the DNA nicking activity of HP0268. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min in the presence and absence of 1 mM metal ion (Ca 2+ , Co 2+ , Ni 2+ , Fe 3+ , Mn 2+ , Mg 2+ and Cu 2+ ). Increasing concentrations (0.2, 0.4 and 1 μM) of Mn 2+ ion were used. Excess EDTA was used to remove the residual metal ions during the protein preparation. (D) The percentages of the resulting DNA conformations were plotted with regard to metal ion used. Cont. represents the substrate plasmid pET-21a(+) without HP0268, and Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. Commonly, 10 units of control enzyme were used in a final volume of 30 μl.
    Figure Legend Snippet: Nicking endonuclease activity of HP0268. (A) The nicking endonuclease activity at various protein concentrations (1, 2, 4 and 8 μM) after incubation at 37ºC for 30 min. OC, RC and linear are abbreviations for the nicked open-circular, relaxed circular and linear DNA, respectively. (B) The pH dependence of the DNA nicking activity. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min under various pH conditions (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). (C) Effect of metal ions on the DNA nicking activity of HP0268. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min in the presence and absence of 1 mM metal ion (Ca 2+ , Co 2+ , Ni 2+ , Fe 3+ , Mn 2+ , Mg 2+ and Cu 2+ ). Increasing concentrations (0.2, 0.4 and 1 μM) of Mn 2+ ion were used. Excess EDTA was used to remove the residual metal ions during the protein preparation. (D) The percentages of the resulting DNA conformations were plotted with regard to metal ion used. Cont. represents the substrate plasmid pET-21a(+) without HP0268, and Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. Commonly, 10 units of control enzyme were used in a final volume of 30 μl.

    Techniques Used: Activity Assay, Incubation, Plasmid Preparation, Positron Emission Tomography

    Nuclease activity of HP0268 mutants. (A) Nicking endonuclease assay of wild-type and mutant HP0268 using gel electrophoresis. The substrate plasmid DNA was incubated with the wild-type and mutants (2 μM) at 37ºC for 30 min. Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. (B) Graph of the nicking endonuclease activities of wild-type and mutant HP0268. The DNA nicking activities of the mutants are normalized by that of the wild-type. (C) Fluorometric ribonuclease activities of wild-type and mutant HP0268. The protein concentrations were maintained at 6 μM. The fluorescence spectra are shown in a color-coded mode. In every figure, Cont. and WT indicate the reference condition of having only a buffer and the wild-type protein, respectively. The reaction buffer consisted of 20 mM Tris (pH 8.0) and 150 mM NaCl.
    Figure Legend Snippet: Nuclease activity of HP0268 mutants. (A) Nicking endonuclease assay of wild-type and mutant HP0268 using gel electrophoresis. The substrate plasmid DNA was incubated with the wild-type and mutants (2 μM) at 37ºC for 30 min. Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. (B) Graph of the nicking endonuclease activities of wild-type and mutant HP0268. The DNA nicking activities of the mutants are normalized by that of the wild-type. (C) Fluorometric ribonuclease activities of wild-type and mutant HP0268. The protein concentrations were maintained at 6 μM. The fluorescence spectra are shown in a color-coded mode. In every figure, Cont. and WT indicate the reference condition of having only a buffer and the wild-type protein, respectively. The reaction buffer consisted of 20 mM Tris (pH 8.0) and 150 mM NaCl.

    Techniques Used: Activity Assay, Mutagenesis, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation, Fluorescence

    2) Product Images from "Age-Dependent Ribosomal DNA Variations in Mice"

    Article Title: Age-Dependent Ribosomal DNA Variations in Mice

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00368-20

    Detection of relative rDNA copy number in old and young mice. (A) The position of the probe for the Southern blot analysis shown in panels B and C and recognition sites for BamHI (B) and NdeI (N) are shown. (B and C) Detection of relative rDNA copy number. Southern analysis for rDNA copy number is shown at the top. DNA was digested with BamHI and NdeI. Upper bands (4 kb) come from rDNA units without the BamHI-2 site, and lower bands (2.4 kb) are from rDNA units with the BamHI-2 site. The SWI5 gene was used as a loading control (middle). The single-copy gene SWI5 was detected on the same filter used for the experiment shown in the upper panel. Relative rDNA copy numbers were determined (bottom). The band intensities of rDNA were normalized by those of SWI5, and the values are relative to the average of rDNA values in the four young mice. The blue dots show the results from the upper-band intensities of rDNA, and the red dots are the results from the lower bands. Individual mice that were used to isolate the bone marrow cells are identified by number (ID) ( Fig. 2 ). P values are shown at the bottom of the panel. n.s., not significant.
    Figure Legend Snippet: Detection of relative rDNA copy number in old and young mice. (A) The position of the probe for the Southern blot analysis shown in panels B and C and recognition sites for BamHI (B) and NdeI (N) are shown. (B and C) Detection of relative rDNA copy number. Southern analysis for rDNA copy number is shown at the top. DNA was digested with BamHI and NdeI. Upper bands (4 kb) come from rDNA units without the BamHI-2 site, and lower bands (2.4 kb) are from rDNA units with the BamHI-2 site. The SWI5 gene was used as a loading control (middle). The single-copy gene SWI5 was detected on the same filter used for the experiment shown in the upper panel. Relative rDNA copy numbers were determined (bottom). The band intensities of rDNA were normalized by those of SWI5, and the values are relative to the average of rDNA values in the four young mice. The blue dots show the results from the upper-band intensities of rDNA, and the red dots are the results from the lower bands. Individual mice that were used to isolate the bone marrow cells are identified by number (ID) ( Fig. 2 ). P values are shown at the bottom of the panel. n.s., not significant.

    Techniques Used: Mouse Assay, Southern Blot

    3) Product Images from "Efficient method for site-directed mutagenesis in large plasmids without subcloning"

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177788

    Validation of the URMAC method by insertion (I), substitution (S), or deletion (D) of some restriction sites in pUC18 plasmid. (A) Illustration of the Modification Target (NdeI restriction site) relative to the flanking restriction sites and location of the Starter Primers SP1 and SP2. After the first PCR, the Starter DNA migrated as expected, 532 bp on a 1% agarose gel (photo, arrow at right). A 100 bp DNA size ladder is shown in left lane for comparison. (B) Diagram of the strategy for I, S, or D using the Closed Starter DNA circularized from the PCR product in (A) as template and the Opener/Mutagenic Primers . The top photo shows the PCR product, Intermediate DNA , which contained the mutations. The bottom photo shows the Modified DNA after enrichment PCR step using SP1 and SP2. (C) Validation of URMAC mutagenesis for the three different types of mutations by restriction analysis. Fig 2C shows bands of expected DNA fragment size after digestion with respective restriction enzymes. In the control Starter PCR lane, only DNA treated with NdeI enzyme, cut the DNA into two fragments of 382 150 bp. Untreated DNA or DNA treated with MluI remained at the full size of 532 bp. In the Insertion lane, both NdeI and MluI cut the DNA at the expected sizes of 382 150 for NdeI and 383 149 for MluI. In the Substitution lane, only MluI cut the DNA producing the expected 383 149 bp bands. In the Deletion lane, none of the enzymes cut the DNA, leaving the bands at their original Modified DNA size.
    Figure Legend Snippet: Validation of the URMAC method by insertion (I), substitution (S), or deletion (D) of some restriction sites in pUC18 plasmid. (A) Illustration of the Modification Target (NdeI restriction site) relative to the flanking restriction sites and location of the Starter Primers SP1 and SP2. After the first PCR, the Starter DNA migrated as expected, 532 bp on a 1% agarose gel (photo, arrow at right). A 100 bp DNA size ladder is shown in left lane for comparison. (B) Diagram of the strategy for I, S, or D using the Closed Starter DNA circularized from the PCR product in (A) as template and the Opener/Mutagenic Primers . The top photo shows the PCR product, Intermediate DNA , which contained the mutations. The bottom photo shows the Modified DNA after enrichment PCR step using SP1 and SP2. (C) Validation of URMAC mutagenesis for the three different types of mutations by restriction analysis. Fig 2C shows bands of expected DNA fragment size after digestion with respective restriction enzymes. In the control Starter PCR lane, only DNA treated with NdeI enzyme, cut the DNA into two fragments of 382 150 bp. Untreated DNA or DNA treated with MluI remained at the full size of 532 bp. In the Insertion lane, both NdeI and MluI cut the DNA at the expected sizes of 382 150 for NdeI and 383 149 for MluI. In the Substitution lane, only MluI cut the DNA producing the expected 383 149 bp bands. In the Deletion lane, none of the enzymes cut the DNA, leaving the bands at their original Modified DNA size.

    Techniques Used: Plasmid Preparation, Modification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mutagenesis

    4) Product Images from "A programmable omnipotent Argonaute nuclease from mesophilic bacteria Kurthia massiliensis"

    Article Title: A programmable omnipotent Argonaute nuclease from mesophilic bacteria Kurthia massiliensis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1278

    Double stranded plasmid DNA and highly structured RNA cleavage by KmAgo. ( A ) Schematic overview of the pUC19 target plasmid. Black polylines indicate target sites while percentages indicate the GC-content of the 80 bp segments in which these target sites are located. ( B ) Pre-assembled KmAgo-gDNA complexes targeting various pUC19 segments were incubated with pUC19. Cleavage products were incubated with NdeI or ScaI and were further analysed by agarose gel electrophoresis. M, molecular weight marker; Lin, linearized plasmid. ( C ) Schematic overview of the HIV-1 ΔDIS 5′UTR. Arrows with different colors indicate the target region and the corresponding gDNAs are numbered from 1 to 11 with the corresponding colours. ( D ) Substrates and products generated by the assay described in (C) were resolved by denaturing PAGE (8%) revealing cleavability of the highly structured RNA by KmAgo–gDNA complex.
    Figure Legend Snippet: Double stranded plasmid DNA and highly structured RNA cleavage by KmAgo. ( A ) Schematic overview of the pUC19 target plasmid. Black polylines indicate target sites while percentages indicate the GC-content of the 80 bp segments in which these target sites are located. ( B ) Pre-assembled KmAgo-gDNA complexes targeting various pUC19 segments were incubated with pUC19. Cleavage products were incubated with NdeI or ScaI and were further analysed by agarose gel electrophoresis. M, molecular weight marker; Lin, linearized plasmid. ( C ) Schematic overview of the HIV-1 ΔDIS 5′UTR. Arrows with different colors indicate the target region and the corresponding gDNAs are numbered from 1 to 11 with the corresponding colours. ( D ) Substrates and products generated by the assay described in (C) were resolved by denaturing PAGE (8%) revealing cleavability of the highly structured RNA by KmAgo–gDNA complex.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Molecular Weight, Marker, Generated, Polyacrylamide Gel Electrophoresis

    5) Product Images from "Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells"

    Article Title: Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells

    Journal: bioRxiv

    doi: 10.1101/2020.07.10.196840

    Detection of relative rDNA copy number in old and young mice (A) Position of the probe for Southern blot analysis in ( BC ) and recognition sites for BamHI and NdeI are shown. ( BC ) Detection of relative rDNA copy number. (Top panel) Southern analysis for rDNA copy number. DNA was digested with BamHI and NdeI. Upper bands (4 kb) come from rDNA units without BamHI-2 site and lower bands (2.4 kb) from rDNA units with BamHI-2 site. (Middle panel) Detection of SWI5 gene as a loading control. A single copy gene SWI5 was detected on the same filter used in the upper panel. (Bottom panel) Relative amount of rDNA copy number. The band intensities of rDNA were normalized by those of SWI5 and the values are relative to the average of rDNA values in the four young mice. The blue dots show the results from the upper band intensities of rDNA and the red dots are the results from the lower bands. ID# is the identification number of individual mice that were used to isolate the bone marrow cells ( Figure 1 ). p values are shown at the bottom of the panel. n.s. is “not significant”.
    Figure Legend Snippet: Detection of relative rDNA copy number in old and young mice (A) Position of the probe for Southern blot analysis in ( BC ) and recognition sites for BamHI and NdeI are shown. ( BC ) Detection of relative rDNA copy number. (Top panel) Southern analysis for rDNA copy number. DNA was digested with BamHI and NdeI. Upper bands (4 kb) come from rDNA units without BamHI-2 site and lower bands (2.4 kb) from rDNA units with BamHI-2 site. (Middle panel) Detection of SWI5 gene as a loading control. A single copy gene SWI5 was detected on the same filter used in the upper panel. (Bottom panel) Relative amount of rDNA copy number. The band intensities of rDNA were normalized by those of SWI5 and the values are relative to the average of rDNA values in the four young mice. The blue dots show the results from the upper band intensities of rDNA and the red dots are the results from the lower bands. ID# is the identification number of individual mice that were used to isolate the bone marrow cells ( Figure 1 ). p values are shown at the bottom of the panel. n.s. is “not significant”.

    Techniques Used: Mouse Assay, Southern Blot

    6) Product Images from "Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi‐C"

    Article Title: Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi‐C

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20188293

    Diagram of the workflow (related to Fig 1 ) Annotation (SK1 background) corresponds to CDS, ARS, telomere regions, retrotransposable elements, mating type loci, tRNA, Sn/Sno RNA, rDNA, ncRNA, intron motives, and TATA boxes. All those features but CDS and transposons were labeled as “forbidden”, preventing any nucleotide substitution in these regions. DpnII, HindIII, SacI, EcoRI, NdeI, SacII, SalI, XbaI, and XhoI. Putative restriction sites are DNA sequences differing with only one base pair from a RS recognized by a RE. The sequence modifications were allowed only in non‐forbidden positions. In CDS, silent mutations were introduced. When two sites overlapped, the minimum changes needed were selected. When possible, we favored A ↔ G and C ↔ T substitutions. A validation step to test whether or not the deletion of one site creates a new site was performed after each modification, and if so, a new modification was sought for. Modifications to generate new sites were also only introduced at non‐forbidden positions. Only silent mutations were introduced within coding regions. 583 × 150 kb windows with 10‐kb overlaps were generated over the entire genome, excluding telomeres and 75 kb from each side of centromeres. Here, 400, 1,500, 2,000 and 6,000 bp. For each 150‐kb window and each interval, the following steps were performed: for each enzyme, for each starting point: putative sites within the first bin of the window (0− 0+spacing). find the putative sites at position n +1 at a distance interval ± 10% from position n until the end of window. For each window, a score is calculated as follows: for each interval, a score is calculated for each enzyme based on the median absolute deviation (MAD). the best enzyme exhibiting the lowest score was chosen for each interval. Each spacing must have a different enzyme, so multiple combinations of enzymes were computed for each window. The window score is calculated as the sum of the four chosen interval scores. A final step of manual curation was performed to introduced PCRTags (Richardson et al , 2017 ).
    Figure Legend Snippet: Diagram of the workflow (related to Fig 1 ) Annotation (SK1 background) corresponds to CDS, ARS, telomere regions, retrotransposable elements, mating type loci, tRNA, Sn/Sno RNA, rDNA, ncRNA, intron motives, and TATA boxes. All those features but CDS and transposons were labeled as “forbidden”, preventing any nucleotide substitution in these regions. DpnII, HindIII, SacI, EcoRI, NdeI, SacII, SalI, XbaI, and XhoI. Putative restriction sites are DNA sequences differing with only one base pair from a RS recognized by a RE. The sequence modifications were allowed only in non‐forbidden positions. In CDS, silent mutations were introduced. When two sites overlapped, the minimum changes needed were selected. When possible, we favored A ↔ G and C ↔ T substitutions. A validation step to test whether or not the deletion of one site creates a new site was performed after each modification, and if so, a new modification was sought for. Modifications to generate new sites were also only introduced at non‐forbidden positions. Only silent mutations were introduced within coding regions. 583 × 150 kb windows with 10‐kb overlaps were generated over the entire genome, excluding telomeres and 75 kb from each side of centromeres. Here, 400, 1,500, 2,000 and 6,000 bp. For each 150‐kb window and each interval, the following steps were performed: for each enzyme, for each starting point: putative sites within the first bin of the window (0− 0+spacing). find the putative sites at position n +1 at a distance interval ± 10% from position n until the end of window. For each window, a score is calculated as follows: for each interval, a score is calculated for each enzyme based on the median absolute deviation (MAD). the best enzyme exhibiting the lowest score was chosen for each interval. Each spacing must have a different enzyme, so multiple combinations of enzymes were computed for each window. The window score is calculated as the sum of the four chosen interval scores. A final step of manual curation was performed to introduced PCRTags (Richardson et al , 2017 ).

    Techniques Used: Labeling, Sequencing, Modification, Generated

    7) Product Images from "The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner"

    Article Title: The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr768

    Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.
    Figure Legend Snippet: Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.

    Techniques Used: Methylation, Plasmid Preparation, Sequencing, Activity Assay

    CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.
    Figure Legend Snippet: CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.

    Techniques Used: Methylation, Plasmid Preparation, Marker, Molecular Weight

    8) Product Images from "Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli"

    Article Title: Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.11261

    Polymerase Chain Reaction Amplification and Restriction Enzyme Analyses of Different Plasmids by NdeI and XhoI Restriction Enzymes and Comparison of Undigested and Digested Patterns of Plasmids With orf2.1 and orf2.2 on Agarose Gel Electrophoresis PCR amplification and restriction enzyme analyses of plasmids pBluescript II SK-ORF2.1, pET30a-ORF2.1, pET30a-ORF2.2, and pET30a+ without ORF2.1 by NdeI and XhoI restriction enzymes. Lane 1, the 1 kb DNA marker; Lane 2, the undigested plasmid pET30a+; Lane 3, the digested plasmid pET30a+; Lane 4, the undigested pBluescript II SK-ORF2.1; Lane 5, the digested pBluescript II SK-ORF2.1; Lane 6, the undigested plasmid pET30a-ORF2.1; Lane 7, the digested plasmid pET30a- ORF2.1; Lane 8, the amplified orf2.1 gene by PCR (with T7 promoter and T7 terminator primers); Lane 9, the undigested plasmid pET30a-ORF2.2; Lane 10, the digested plasmid pET30a-ORF2.2; Lane 11, the amplified orf2.2 gene by PCR (with T7 promoter and T7 terminator primers); Lane 13, the 1kb DNA marker; Lane 14, the amplified orf2.1 gene by PCR; and Lane 15, the amplified orf2.2 gene by PCR.
    Figure Legend Snippet: Polymerase Chain Reaction Amplification and Restriction Enzyme Analyses of Different Plasmids by NdeI and XhoI Restriction Enzymes and Comparison of Undigested and Digested Patterns of Plasmids With orf2.1 and orf2.2 on Agarose Gel Electrophoresis PCR amplification and restriction enzyme analyses of plasmids pBluescript II SK-ORF2.1, pET30a-ORF2.1, pET30a-ORF2.2, and pET30a+ without ORF2.1 by NdeI and XhoI restriction enzymes. Lane 1, the 1 kb DNA marker; Lane 2, the undigested plasmid pET30a+; Lane 3, the digested plasmid pET30a+; Lane 4, the undigested pBluescript II SK-ORF2.1; Lane 5, the digested pBluescript II SK-ORF2.1; Lane 6, the undigested plasmid pET30a-ORF2.1; Lane 7, the digested plasmid pET30a- ORF2.1; Lane 8, the amplified orf2.1 gene by PCR (with T7 promoter and T7 terminator primers); Lane 9, the undigested plasmid pET30a-ORF2.2; Lane 10, the digested plasmid pET30a-ORF2.2; Lane 11, the amplified orf2.2 gene by PCR (with T7 promoter and T7 terminator primers); Lane 13, the 1kb DNA marker; Lane 14, the amplified orf2.1 gene by PCR; and Lane 15, the amplified orf2.2 gene by PCR.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    9) Product Images from "DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI"

    Article Title: DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp790

    DNA site requirements for cleavage by LlaGI. ( A and B ) Plasmid substrates with no sites, one-site or two indirectly-repeated sites were incubated with either saturating BamHI (B) or LlaGI (L) for 1 h. Substrates and products were separated by agarose gel electrophoresis as indicated. ( C and D ) Plasmid substrates with two directly-repeated sites (pHT-12) or two indirectly-repeated sites (pHH-12) were cleaved with either AlwNI (A) or NdeI (N) to produce the linear DNA indicated. Sequences of the LlaGI sites are in Figure 3 . The parental plasmids and linear DNA were then incubated with saturating LlaGI for 1 h. Substrates and products were separated by agarose gel electrophoresis as indicated. See main text for full details. Under these assay conditions, an additional slowly-migrating band was observed which we assign to a LlaGI-DNA bandshift. Gels labelled as in Figure 3 .
    Figure Legend Snippet: DNA site requirements for cleavage by LlaGI. ( A and B ) Plasmid substrates with no sites, one-site or two indirectly-repeated sites were incubated with either saturating BamHI (B) or LlaGI (L) for 1 h. Substrates and products were separated by agarose gel electrophoresis as indicated. ( C and D ) Plasmid substrates with two directly-repeated sites (pHT-12) or two indirectly-repeated sites (pHH-12) were cleaved with either AlwNI (A) or NdeI (N) to produce the linear DNA indicated. Sequences of the LlaGI sites are in Figure 3 . The parental plasmids and linear DNA were then incubated with saturating LlaGI for 1 h. Substrates and products were separated by agarose gel electrophoresis as indicated. See main text for full details. Under these assay conditions, an additional slowly-migrating band was observed which we assign to a LlaGI-DNA bandshift. Gels labelled as in Figure 3 .

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    10) Product Images from "Supercoiling and looping promote DNA base accessibility and coordination among distant sites"

    Article Title: Supercoiling and looping promote DNA base accessibility and coordination among distant sites

    Journal: Nature Communications

    doi: 10.1038/s41467-021-25936-2

    Bal-31 and S1 nuclease cleave at specific DNA sites containing exposed bases. a Map of the 336 bp minicircle sequence showing the positions of the restriction enzymes used, the location of the three major Bal-31/S1 nuclease cleavage sites, and the att R integrase site. The numbers on the inside of the circle indicate the designated start/end of the minicircle sequence (see “Methods”) and also the positions of each restriction site along the listed sequence. Dashed lines indicate the distances between each restriction cleavage site. Intrinsic curvature in att R is centered around the MseI site (Supplementary Fig. 1 ). b Minicircle DNA was cleaved with Bal-31, deproteinized, then subsequently cleaved with each of the restriction enzymes, and the products were separated by agarose gel electrophoresis. The 336 bp Lk = 29; Δ Lk = −3; σ = −0.095 topoisomer is shown. Mr 1 : 100 bp DNA ladder; Mr 2 : low molecular weight DNA ladder; S: supercoiled (336 bp Lk = 29; Δ Lk = −3), N: nicked 336 bp minicircle; L: 336 bp minicircle linearized by EcoRV; -, B, N, M, E, X: minicircle incubated with Bal-31 for 1 min followed by incubation with a second restriction enzyme as indicated (-: no second enzyme; B: BbvCI; N: NdeI; M: MseI; E: EcoRV; X: XmnI). This assay was performed at least two times for each topoisomer with very similar results. c Mapping of S1 nuclease cleavage sites (336 bp; Lk = 29; Δ Lk = −3 topoisomer is shown). The experiment was performed following the same protocol as for Bal-31. This assay was performed once for each topoisomer. d Relative Bal-31 cleavage at each of the three sites as a function of Lk . e Relative S1 nuclease cleavage at each of the three sites as a function of Lk . N.D. not determined. f Model showing localization of sites 1 and 2 to the superhelical apices.
    Figure Legend Snippet: Bal-31 and S1 nuclease cleave at specific DNA sites containing exposed bases. a Map of the 336 bp minicircle sequence showing the positions of the restriction enzymes used, the location of the three major Bal-31/S1 nuclease cleavage sites, and the att R integrase site. The numbers on the inside of the circle indicate the designated start/end of the minicircle sequence (see “Methods”) and also the positions of each restriction site along the listed sequence. Dashed lines indicate the distances between each restriction cleavage site. Intrinsic curvature in att R is centered around the MseI site (Supplementary Fig. 1 ). b Minicircle DNA was cleaved with Bal-31, deproteinized, then subsequently cleaved with each of the restriction enzymes, and the products were separated by agarose gel electrophoresis. The 336 bp Lk = 29; Δ Lk = −3; σ = −0.095 topoisomer is shown. Mr 1 : 100 bp DNA ladder; Mr 2 : low molecular weight DNA ladder; S: supercoiled (336 bp Lk = 29; Δ Lk = −3), N: nicked 336 bp minicircle; L: 336 bp minicircle linearized by EcoRV; -, B, N, M, E, X: minicircle incubated with Bal-31 for 1 min followed by incubation with a second restriction enzyme as indicated (-: no second enzyme; B: BbvCI; N: NdeI; M: MseI; E: EcoRV; X: XmnI). This assay was performed at least two times for each topoisomer with very similar results. c Mapping of S1 nuclease cleavage sites (336 bp; Lk = 29; Δ Lk = −3 topoisomer is shown). The experiment was performed following the same protocol as for Bal-31. This assay was performed once for each topoisomer. d Relative Bal-31 cleavage at each of the three sites as a function of Lk . e Relative S1 nuclease cleavage at each of the three sites as a function of Lk . N.D. not determined. f Model showing localization of sites 1 and 2 to the superhelical apices.

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Incubation

    11) Product Images from "Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells"

    Article Title: Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

    Journal: Genes & Development

    doi: 10.1101/gad.1369805

    Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.
    Figure Legend Snippet: Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.

    Techniques Used: Plasmid Preparation, Binding Assay, In Vivo, Isolation, Agarose Gel Electrophoresis, Southern Blot, Marker, Transfection, Activity Assay, Luciferase

    12) Product Images from "A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies"

    Article Title: A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0244464

    Assessment for restriction digest efficiencies. (A) Agarose gel electrophoresis of restriction digests. 80 ng DNA was applied to each lane. Lane M: DNA marker; lane 1 5: mNeonGreen template DNA (prior to digestion); lane 2: DNA digested by NcoI for 1 h; lane 3: DNA digested by NcoI-HF for 1 h; lane 4: DNA digested by NcoI-HF for 15 min; lane 6: DNA digested by NcoI-HF for 24 h; lane 7: DNA digested by BtgI for 24 h. (B) Digital CFPS using DNA digest solution. The NcoI-HF (left) or NdeI (right)-digested (for 15 min) mNeonGreen DNA solution was mixed with CFPS components and introduced into FemDA. Fluorescent proteins were synthesized in the droplet containing undigested DNA. Scale bar: 10 μm. (C) Simultaneous measurement of restriction digest efficiency of multiple enzymes with multiplexed digital CFPS. BamHI-HF-digested mTurquoise2 (cyan) DNA, NdeI-digested mNeonGreen (green) DNA, and EcoRI-HF-digested mScarlet (red) DNA were mixed and used for a single CFPS reaction on FemDA. The digestion time for each was 15 min. Scale bar: 10 μm. (D) Sum of Gaussian distributions of equal peak-to-peak intervals of fluorescence intensities of droplets. The NdeI-digested (for 1 h) DNA solution was used for this digital CFPS. The blue square represents the observed probability of droplets containing different numbers of template DNA molecules. The red cross represents the corresponding probability fitted by the Poisson distribution. The observed probability and the fitted probability were consistent with each other, proving the Poisson distribution of single DNA molecules. (E) Restriction digest efficiencies (digested versus the initial quantity of template DNA, expressed in %) of NcoI-HF at different time points. (E) Measurement of enzyme kinetics and Michaelis-Menten curve fitting for NdeI. (G) Restriction digest efficiencies (left y-axis marked in black) of NdeI at different time points and the number of false-positive colonies (right y-axis marked in red) caused by the undigested DNA in a model transformation experiment. Each reaction or transformation was performed in triplicate. Error bars: 1 standard deviation.
    Figure Legend Snippet: Assessment for restriction digest efficiencies. (A) Agarose gel electrophoresis of restriction digests. 80 ng DNA was applied to each lane. Lane M: DNA marker; lane 1 5: mNeonGreen template DNA (prior to digestion); lane 2: DNA digested by NcoI for 1 h; lane 3: DNA digested by NcoI-HF for 1 h; lane 4: DNA digested by NcoI-HF for 15 min; lane 6: DNA digested by NcoI-HF for 24 h; lane 7: DNA digested by BtgI for 24 h. (B) Digital CFPS using DNA digest solution. The NcoI-HF (left) or NdeI (right)-digested (for 15 min) mNeonGreen DNA solution was mixed with CFPS components and introduced into FemDA. Fluorescent proteins were synthesized in the droplet containing undigested DNA. Scale bar: 10 μm. (C) Simultaneous measurement of restriction digest efficiency of multiple enzymes with multiplexed digital CFPS. BamHI-HF-digested mTurquoise2 (cyan) DNA, NdeI-digested mNeonGreen (green) DNA, and EcoRI-HF-digested mScarlet (red) DNA were mixed and used for a single CFPS reaction on FemDA. The digestion time for each was 15 min. Scale bar: 10 μm. (D) Sum of Gaussian distributions of equal peak-to-peak intervals of fluorescence intensities of droplets. The NdeI-digested (for 1 h) DNA solution was used for this digital CFPS. The blue square represents the observed probability of droplets containing different numbers of template DNA molecules. The red cross represents the corresponding probability fitted by the Poisson distribution. The observed probability and the fitted probability were consistent with each other, proving the Poisson distribution of single DNA molecules. (E) Restriction digest efficiencies (digested versus the initial quantity of template DNA, expressed in %) of NcoI-HF at different time points. (E) Measurement of enzyme kinetics and Michaelis-Menten curve fitting for NdeI. (G) Restriction digest efficiencies (left y-axis marked in black) of NdeI at different time points and the number of false-positive colonies (right y-axis marked in red) caused by the undigested DNA in a model transformation experiment. Each reaction or transformation was performed in triplicate. Error bars: 1 standard deviation.

    Techniques Used: Agarose Gel Electrophoresis, Marker, Synthesized, Fluorescence, Transformation Assay, Standard Deviation

    DNA digest analysis using capillary electrophoresis (Agilent Bioanalyzer) and slab gel electrophoresis. The measured digest efficiency (in % unit, from Table 1 ) was labeled beside the corresponding RE site on the template DNA map. On capillary electrophoresis (middle), lane 1~15: mNeonGreen DNA restriction digest with (in the order from left to right) XbaI, NdeI, NheI-HF, BmtI-HF, BamHI-HF, XcmI, PflMI, BstEII-HF, HpaI, BbsI-HF, BsgI, AfeI, BstXI, StuI, and BsrGI-HF, respectively. On agarose slab gel electrophoresis (bottom), lane M 1 : Hi-Lo DNA marker; lane 16: template DNA (prior to digestion, 1008 bp); lane 2~17: digested by (in the order from left to right) XbaI, NdeI, NheI-HF, BmtI-HF, BamHI-HF, XcmI, PflMI, BstEII-HF, HpaI, BbsI-HF, BsgI, AfeI (newly purchased), AfeI (old stock), BstXI, StuI, BsrGI-HF; lane M 2 : 100 bp DNA ladder; label “Y”: undigested DNA was detectable on the slab gel; label “N”: undigested DNA was not detectable on the slab gel. 100 ng (calculated based on the stock concentration of template DNA solution measured using NanoDrop) DNA was applied to each lane of the slab gel.
    Figure Legend Snippet: DNA digest analysis using capillary electrophoresis (Agilent Bioanalyzer) and slab gel electrophoresis. The measured digest efficiency (in % unit, from Table 1 ) was labeled beside the corresponding RE site on the template DNA map. On capillary electrophoresis (middle), lane 1~15: mNeonGreen DNA restriction digest with (in the order from left to right) XbaI, NdeI, NheI-HF, BmtI-HF, BamHI-HF, XcmI, PflMI, BstEII-HF, HpaI, BbsI-HF, BsgI, AfeI, BstXI, StuI, and BsrGI-HF, respectively. On agarose slab gel electrophoresis (bottom), lane M 1 : Hi-Lo DNA marker; lane 16: template DNA (prior to digestion, 1008 bp); lane 2~17: digested by (in the order from left to right) XbaI, NdeI, NheI-HF, BmtI-HF, BamHI-HF, XcmI, PflMI, BstEII-HF, HpaI, BbsI-HF, BsgI, AfeI (newly purchased), AfeI (old stock), BstXI, StuI, BsrGI-HF; lane M 2 : 100 bp DNA ladder; label “Y”: undigested DNA was detectable on the slab gel; label “N”: undigested DNA was not detectable on the slab gel. 100 ng (calculated based on the stock concentration of template DNA solution measured using NanoDrop) DNA was applied to each lane of the slab gel.

    Techniques Used: Electrophoresis, Nucleic Acid Electrophoresis, Labeling, Marker, Concentration Assay

    13) Product Images from "Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes"

    Article Title: Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-62703-968-0_14

    Example of assembly PCR primers and introduction of variation via degenerate codons. Each colored selection represents a single assembly primer; the sum of all primers is designed to produce the entire coding sequence shown. An NdeI restriction site (CATATG) has been added to the N-terminal end of the sequence, and an XhoI restriction site (CTCGAG) has been added to the C-terminal end. The magnified inset towards the C-terminal end of the sequence gives an example of introducing variation using the degenerate codon “RAA.” The “R” base designates the introduction of either a guanine (G) or adenine (A) base at that position, resulting in a translated protein sequence with either glutamic acid (E) or lysine (K)
    Figure Legend Snippet: Example of assembly PCR primers and introduction of variation via degenerate codons. Each colored selection represents a single assembly primer; the sum of all primers is designed to produce the entire coding sequence shown. An NdeI restriction site (CATATG) has been added to the N-terminal end of the sequence, and an XhoI restriction site (CTCGAG) has been added to the C-terminal end. The magnified inset towards the C-terminal end of the sequence gives an example of introducing variation using the degenerate codon “RAA.” The “R” base designates the introduction of either a guanine (G) or adenine (A) base at that position, resulting in a translated protein sequence with either glutamic acid (E) or lysine (K)

    Techniques Used: Polymerase Cycling Assembly, Selection, Sequencing

    14) Product Images from "Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair"

    Article Title: Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2016.03.003

    Interference between BER and end joining of Tg5. A. Either the Tg5 substrate or a corresponding unmodified substrate was incubated in extracts containing XLF for the times indicated, then cut with NdeI and PstI and analyzed as in . B. Quantitative
    Figure Legend Snippet: Interference between BER and end joining of Tg5. A. Either the Tg5 substrate or a corresponding unmodified substrate was incubated in extracts containing XLF for the times indicated, then cut with NdeI and PstI and analyzed as in . B. Quantitative

    Techniques Used: Incubation

    Ligation of Tg-containing substrates by purified Ku, X4L4 and XLF. A. The indicated substrates were incubated with 10 nM Ku, 40 nM X4L4 and 50 or 100 nM XLF as indicated for 4 hr, then deproteinized and cut with NdeI and PstI and analyzed on a sequencing
    Figure Legend Snippet: Ligation of Tg-containing substrates by purified Ku, X4L4 and XLF. A. The indicated substrates were incubated with 10 nM Ku, 40 nM X4L4 and 50 or 100 nM XLF as indicated for 4 hr, then deproteinized and cut with NdeI and PstI and analyzed on a sequencing

    Techniques Used: Ligation, Purification, Incubation, Sequencing

    Presence of Tg in end joining products. Tg-containing or unmodified substrates were incubated for 6 hr in Bustel extracts supplemented with XLF and ddTTP as indicated. Samples were deproteinized and cut with NdeI and PstI, then denatured and annealed
    Figure Legend Snippet: Presence of Tg in end joining products. Tg-containing or unmodified substrates were incubated for 6 hr in Bustel extracts supplemented with XLF and ddTTP as indicated. Samples were deproteinized and cut with NdeI and PstI, then denatured and annealed

    Techniques Used: Incubation

    Time course for Tg3 and Tg1 end joining and effect of dideoxynucleotides. A. and B. Tg3 was incubated in extracts containing ddTTP, ddCTP and/or XLF for the times indicated, then cut with NdeI and PstI and analyzed as in . One sample in (A.) was
    Figure Legend Snippet: Time course for Tg3 and Tg1 end joining and effect of dideoxynucleotides. A. and B. Tg3 was incubated in extracts containing ddTTP, ddCTP and/or XLF for the times indicated, then cut with NdeI and PstI and analyzed as in . One sample in (A.) was

    Techniques Used: Incubation

    15) Product Images from "Unique Properties of the Alpha-Helical DNA-Binding Protein KfrA Encoded by the IncU Incompatibility Group Plasmid RA3 and Its Host-Dependent Role in Plasmid Maintenance"

    Article Title: Unique Properties of the Alpha-Helical DNA-Binding Protein KfrA Encoded by the IncU Incompatibility Group Plasmid RA3 and Its Host-Dependent Role in Plasmid Maintenance

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01771-20

    Analysis of HTH motif in KfrA RA3. (A) Ability of KfrA deletion variants to repress the kfrA promoter. E. coli C600K carrying pAKB4.70 ( kfrAp-xylE ) was transformed with the expression vector pGBT30 or its derivatives encoding WT KfrA and the truncated derivatives. Double transformants were grown without or with 0.5 mM IPTG to induce tacp and protein overproduction. XylE activities were assayed in extracts from the exponentially growing cultures and are shown relative to the XylE activity in C600K(pAKB4.70)(pGBT30) cells. Since WT KfrA exhibits strong repression activity (even in the absence of inducer due to the leakiness of tacp ), the bracketed part of the diagram is enlarged and presented using a different scale. (B) Schematic presentation of KfrA. The color code is as in Fig. 2A . The protein region with the putative HTH motif is enlarged, and the predicted secondary structure (H, helix; C, coil) is shown. (C) The C tip of KfrA RA3 is responsible for nonspecific DNA binding activity. His 6 -tagged KfrA and two deletion variants were analyzed by EMSA. Different concentrations (0 to 0.2 μM) of His 6 -tagged proteins were incubated for 30 min at 37°C with 10 nM pESB2.68 DNA (pUC18 carrying kfrAp ) cleaved into two EcoRI-NdeI fragments: a shorter one with the kfrAp sequence (approximately 0.3 kb) and a longer one used as a control (2.4 kb). Nucleoprotein complexes were separated by electrophoresis on 1.7% agarose gels in 1× TBE buffer and visualized by ethidium bromide staining. The graph presents the quantitative analysis of results carried out with ImageJ 1.52N software ( 71 ). The percentage of unbound DNA was plotted against the protein concentration applied; continuous lines correspond to the kfrAp fragments (probe), and dashed lines correspond to the control fragments (vector) in these three reactions. (D) Alanine scanning of KfrA HTH motif. The putative HTH motif residues were replaced by alanines. Mutagenized kfrA alleles were cloned into the expression vector pGBT30 and tested in a two-plasmid regulatory system for the ability to repress kfrAp-xylE transcriptional fusion (pAKB4.70). Double transformants of E. coli C600K were grown without or with 0.5 mM IPTG to induce tacp and protein overproduction. XylE activities were assayed in extracts from exponentially growing cultures and shown on diagram relative to the activity of C600K(pAKB4.70)(pGBT30) using a logarithmic scale. KfrA* represents KfrA derivatives with amino acid substitutions. Amino acids shaded in dark gray indicate those in which Ala substitutions led to significant impairment, Ala substitutions for those shaded in light gray led to partial impairment, and strains with Ala substitutions for unshaded amino acids retained WT repression ability. The last two sections on the right correspond to the XylE activities in the control strains C600K(pAKB4.70)(pGBT30) and C600K(pAKB4.70)(pESB5.58 tacp-kfrA ) grown with and without IPTG. (E) DNA binding activity of KfrA single-substitution variants in vitro . Selected His 6 -tagged KfrA variants were used in the EMSA with NdeI-EcoRI-cut pESB2.68 (pUC18 carrying kfrAp ) as described in the legend to panel C. The graph illustrates the quantitative analysis of the results obtained with the use of ImageJ 1.52N software ( 71 ). The percentage of unbound DNA (probe) was plotted against the protein concentration applied.
    Figure Legend Snippet: Analysis of HTH motif in KfrA RA3. (A) Ability of KfrA deletion variants to repress the kfrA promoter. E. coli C600K carrying pAKB4.70 ( kfrAp-xylE ) was transformed with the expression vector pGBT30 or its derivatives encoding WT KfrA and the truncated derivatives. Double transformants were grown without or with 0.5 mM IPTG to induce tacp and protein overproduction. XylE activities were assayed in extracts from the exponentially growing cultures and are shown relative to the XylE activity in C600K(pAKB4.70)(pGBT30) cells. Since WT KfrA exhibits strong repression activity (even in the absence of inducer due to the leakiness of tacp ), the bracketed part of the diagram is enlarged and presented using a different scale. (B) Schematic presentation of KfrA. The color code is as in Fig. 2A . The protein region with the putative HTH motif is enlarged, and the predicted secondary structure (H, helix; C, coil) is shown. (C) The C tip of KfrA RA3 is responsible for nonspecific DNA binding activity. His 6 -tagged KfrA and two deletion variants were analyzed by EMSA. Different concentrations (0 to 0.2 μM) of His 6 -tagged proteins were incubated for 30 min at 37°C with 10 nM pESB2.68 DNA (pUC18 carrying kfrAp ) cleaved into two EcoRI-NdeI fragments: a shorter one with the kfrAp sequence (approximately 0.3 kb) and a longer one used as a control (2.4 kb). Nucleoprotein complexes were separated by electrophoresis on 1.7% agarose gels in 1× TBE buffer and visualized by ethidium bromide staining. The graph presents the quantitative analysis of results carried out with ImageJ 1.52N software ( 71 ). The percentage of unbound DNA was plotted against the protein concentration applied; continuous lines correspond to the kfrAp fragments (probe), and dashed lines correspond to the control fragments (vector) in these three reactions. (D) Alanine scanning of KfrA HTH motif. The putative HTH motif residues were replaced by alanines. Mutagenized kfrA alleles were cloned into the expression vector pGBT30 and tested in a two-plasmid regulatory system for the ability to repress kfrAp-xylE transcriptional fusion (pAKB4.70). Double transformants of E. coli C600K were grown without or with 0.5 mM IPTG to induce tacp and protein overproduction. XylE activities were assayed in extracts from exponentially growing cultures and shown on diagram relative to the activity of C600K(pAKB4.70)(pGBT30) using a logarithmic scale. KfrA* represents KfrA derivatives with amino acid substitutions. Amino acids shaded in dark gray indicate those in which Ala substitutions led to significant impairment, Ala substitutions for those shaded in light gray led to partial impairment, and strains with Ala substitutions for unshaded amino acids retained WT repression ability. The last two sections on the right correspond to the XylE activities in the control strains C600K(pAKB4.70)(pGBT30) and C600K(pAKB4.70)(pESB5.58 tacp-kfrA ) grown with and without IPTG. (E) DNA binding activity of KfrA single-substitution variants in vitro . Selected His 6 -tagged KfrA variants were used in the EMSA with NdeI-EcoRI-cut pESB2.68 (pUC18 carrying kfrAp ) as described in the legend to panel C. The graph illustrates the quantitative analysis of the results obtained with the use of ImageJ 1.52N software ( 71 ). The percentage of unbound DNA (probe) was plotted against the protein concentration applied.

    Techniques Used: Transformation Assay, Expressing, Plasmid Preparation, Activity Assay, Binding Assay, Incubation, Sequencing, Electrophoresis, Staining, Software, Protein Concentration, Clone Assay, In Vitro

    Analysis of KfrA binding site (O K ). (A) DNA sequence of minimal kfrAp . The −35 and −10 motifs of the promoter are marked in yellow. Five 9-nt repeats (DR) surrounding the −10 motif (three direct repeats in one direction and two in the opposite orientation) are numbered and indicated with lines. (B) Ability of KfrA to bind to a DR(s) in vitro . Oligonucleotides corresponding to various combinations of DRs (in number and orientation) were cloned into pUC18; purified plasmid DNA was digested with NdeI and EcoRI and used in EMSAs with different concentrations (0 to 0.1 μM) of purified His 6 -KfrA. DNA-protein complexes were separated by electrophoresis on 1.7% agarose gels in 1× TBE buffer and visualized by ethidium bromide staining. Schemes of DR combinations cloned in pUC18 are shown below the gels. The graph illustrates the quantitative analysis of results carried out with ImageJ 1.52N software ( 71 ). The percentage of unbound DNA (probe) was plotted against the concentrations of KfrA applied. (C) Analysis of kfrAp variants with different combinations of DRs in vivo . Double-stranded oligonucleotides encompassing the −35 and −10 motifs of kfrAp and DRs in various numbers and orientations were cloned into the promoter probe vector pPT01. Sequences of the oligonucleotide inserts are shown, with promoter motifs highlighted in yellow and DRs in green. Lowercase letters indicate introduced substitutions in comparison with the WT sequence. Repeats listed in the DR column were retained intact. pPT01 derivatives were introduced into E. coli C600K and analyzed in the two-plasmid regulatory system with empty expression vector pGBT30( tacp ) or pESB5.58( tacp-kfrA ) in trans . Cultures of double transformants were grown without or with 0.5 mM IPTG to induce protein overproduction and XylE activity was measured in extracts of exponential-phase cultures. The KfrA repression index and the standard deviation (SD) were calculated for each promoter region as a ratio of XylE activity in the presence of pGBT30 to the XylE activity in the presence of pESB5.58 grown under the same conditions. The assays were repeated at least 3 times.
    Figure Legend Snippet: Analysis of KfrA binding site (O K ). (A) DNA sequence of minimal kfrAp . The −35 and −10 motifs of the promoter are marked in yellow. Five 9-nt repeats (DR) surrounding the −10 motif (three direct repeats in one direction and two in the opposite orientation) are numbered and indicated with lines. (B) Ability of KfrA to bind to a DR(s) in vitro . Oligonucleotides corresponding to various combinations of DRs (in number and orientation) were cloned into pUC18; purified plasmid DNA was digested with NdeI and EcoRI and used in EMSAs with different concentrations (0 to 0.1 μM) of purified His 6 -KfrA. DNA-protein complexes were separated by electrophoresis on 1.7% agarose gels in 1× TBE buffer and visualized by ethidium bromide staining. Schemes of DR combinations cloned in pUC18 are shown below the gels. The graph illustrates the quantitative analysis of results carried out with ImageJ 1.52N software ( 71 ). The percentage of unbound DNA (probe) was plotted against the concentrations of KfrA applied. (C) Analysis of kfrAp variants with different combinations of DRs in vivo . Double-stranded oligonucleotides encompassing the −35 and −10 motifs of kfrAp and DRs in various numbers and orientations were cloned into the promoter probe vector pPT01. Sequences of the oligonucleotide inserts are shown, with promoter motifs highlighted in yellow and DRs in green. Lowercase letters indicate introduced substitutions in comparison with the WT sequence. Repeats listed in the DR column were retained intact. pPT01 derivatives were introduced into E. coli C600K and analyzed in the two-plasmid regulatory system with empty expression vector pGBT30( tacp ) or pESB5.58( tacp-kfrA ) in trans . Cultures of double transformants were grown without or with 0.5 mM IPTG to induce protein overproduction and XylE activity was measured in extracts of exponential-phase cultures. The KfrA repression index and the standard deviation (SD) were calculated for each promoter region as a ratio of XylE activity in the presence of pGBT30 to the XylE activity in the presence of pESB5.58 grown under the same conditions. The assays were repeated at least 3 times.

    Techniques Used: Binding Assay, Sequencing, In Vitro, Clone Assay, Purification, Plasmid Preparation, Electrophoresis, Staining, Software, In Vivo, Expressing, Activity Assay, Standard Deviation

    16) Product Images from "Defining characteristics of Tn5 Transposase non-specific DNA binding"

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl179

    Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.
    Figure Legend Snippet: Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Techniques Used: Plasmid Preparation, In Vitro, Incubation, Labeling, Agarose Gel Electrophoresis

    17) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    18) Product Images from "Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells"

    Article Title: Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

    Journal: Genes & Development

    doi: 10.1101/gad.1369805

    Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.
    Figure Legend Snippet: Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.

    Techniques Used: Plasmid Preparation, Binding Assay, In Vivo, Isolation, Agarose Gel Electrophoresis, Southern Blot, Marker, Transfection, Activity Assay, Luciferase

    19) Product Images from "An exogenous chloroplast genome for complex sequence manipulation in algae"

    Article Title: An exogenous chloroplast genome for complex sequence manipulation in algae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1008

    Characterization of the cloned C. reinhardtii chloroplast genome in vivo . ( A ) A nested set representing the presence of increasing numbers of markers in primary transformants of pCr03 into a psbD knockout strain as determined by PCR ( Table 2 ; primers used as follows: M1, 11 606 and 11 607; M2, 5512 and 5513; M3, 11 456 and 11 457; and M4, 14 067 and 14 068.). The broken circle shows the subset of transformants with M1, M2, M3 and M4 that gave rise to the same genotype upon rescreening. ( B–E ) Southern blot analysis of EcoRI (B, C and E) or NdeI (D) digests (see ‘Materials and Methods’ section). Probes were specific for sequences adjacent to integration sites for M1 (B), M2 (C), M3 (D) and M4 (E). All samples are arranged as follows: Lane L, 1 kb DNA ladder (Invitrogen; Carlsbad, CA); lane 1, wild-type; lane 2, purified pCr03; and lane 3, a representative algae clone containing all unique markers. A single band in lane 3 indicates homoplasmic integration of the marker, while two bands indicate heteroplasmy with the wild-type locus.
    Figure Legend Snippet: Characterization of the cloned C. reinhardtii chloroplast genome in vivo . ( A ) A nested set representing the presence of increasing numbers of markers in primary transformants of pCr03 into a psbD knockout strain as determined by PCR ( Table 2 ; primers used as follows: M1, 11 606 and 11 607; M2, 5512 and 5513; M3, 11 456 and 11 457; and M4, 14 067 and 14 068.). The broken circle shows the subset of transformants with M1, M2, M3 and M4 that gave rise to the same genotype upon rescreening. ( B–E ) Southern blot analysis of EcoRI (B, C and E) or NdeI (D) digests (see ‘Materials and Methods’ section). Probes were specific for sequences adjacent to integration sites for M1 (B), M2 (C), M3 (D) and M4 (E). All samples are arranged as follows: Lane L, 1 kb DNA ladder (Invitrogen; Carlsbad, CA); lane 1, wild-type; lane 2, purified pCr03; and lane 3, a representative algae clone containing all unique markers. A single band in lane 3 indicates homoplasmic integration of the marker, while two bands indicate heteroplasmy with the wild-type locus.

    Techniques Used: Clone Assay, In Vivo, Knock-Out, Polymerase Chain Reaction, Southern Blot, Purification, Marker

    20) Product Images from "The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics"

    Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.03439004

    Concept of the asymmetric directional T-vector. ( A ) Construction of pGEX-4T3-PRESAT and direct cloning of PCR product in pGEX-4T3-PRESAT. ( B ) The schematic representation of the ORF selection method using potential restriction enzyme site. The figure illustrates the case in which NcoI is chosen as the second restriction enzyme for selection. The rear PCR primer is designed with 5′-GG at the 5′ end, so that only the ligated plasmid with insert in the reverse orientation will have the NcoI site at the TA-cloning position. For NdeI selection, the rear primer with 5′-ATG is used instead of the 5′-GG primer.
    Figure Legend Snippet: Concept of the asymmetric directional T-vector. ( A ) Construction of pGEX-4T3-PRESAT and direct cloning of PCR product in pGEX-4T3-PRESAT. ( B ) The schematic representation of the ORF selection method using potential restriction enzyme site. The figure illustrates the case in which NcoI is chosen as the second restriction enzyme for selection. The rear PCR primer is designed with 5′-GG at the 5′ end, so that only the ligated plasmid with insert in the reverse orientation will have the NcoI site at the TA-cloning position. For NdeI selection, the rear primer with 5′-ATG is used instead of the 5′-GG primer.

    Techniques Used: Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Selection, TA Cloning

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    New England Biolabs ndei
    Nicking endonuclease activity of HP0268. (A) The nicking endonuclease activity at various protein concentrations (1, 2, 4 and 8 μM) after incubation at 37ºC for 30 min. OC, RC and linear are abbreviations for the nicked open-circular, relaxed circular and linear DNA, respectively. (B) The pH dependence of the DNA nicking activity. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min under various pH conditions (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). (C) Effect of metal ions on the DNA nicking activity of HP0268. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min in the presence and absence of 1 mM metal ion (Ca 2+ , Co 2+ , Ni 2+ , Fe 3+ , Mn 2+ , Mg 2+ and Cu 2+ ). Increasing concentrations (0.2, 0.4 and 1 μM) of Mn 2+ ion were used. Excess EDTA was used to remove the residual metal ions during the protein preparation. (D) The percentages of the resulting DNA conformations were plotted with regard to metal ion used. Cont. represents the substrate plasmid pET-21a(+) without HP0268, and <t>Nt.BsmAI</t> and <t>NdeI</t> represent the positive controls for the nicked and linear DNA, respectively. Commonly, 10 units of control enzyme were used in a final volume of 30 μl.
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    Nicking endonuclease activity of HP0268. (A) The nicking endonuclease activity at various protein concentrations (1, 2, 4 and 8 μM) after incubation at 37ºC for 30 min. OC, RC and linear are abbreviations for the nicked open-circular, relaxed circular and linear DNA, respectively. (B) The pH dependence of the DNA nicking activity. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min under various pH conditions (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). (C) Effect of metal ions on the DNA nicking activity of HP0268. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min in the presence and absence of 1 mM metal ion (Ca 2+ , Co 2+ , Ni 2+ , Fe 3+ , Mn 2+ , Mg 2+ and Cu 2+ ). Increasing concentrations (0.2, 0.4 and 1 μM) of Mn 2+ ion were used. Excess EDTA was used to remove the residual metal ions during the protein preparation. (D) The percentages of the resulting DNA conformations were plotted with regard to metal ion used. Cont. represents the substrate plasmid pET-21a(+) without HP0268, and Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. Commonly, 10 units of control enzyme were used in a final volume of 30 μl.

    Journal: Nucleic Acids Research

    Article Title: Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities

    doi: 10.1093/nar/gkv348

    Figure Lengend Snippet: Nicking endonuclease activity of HP0268. (A) The nicking endonuclease activity at various protein concentrations (1, 2, 4 and 8 μM) after incubation at 37ºC for 30 min. OC, RC and linear are abbreviations for the nicked open-circular, relaxed circular and linear DNA, respectively. (B) The pH dependence of the DNA nicking activity. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min under various pH conditions (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). (C) Effect of metal ions on the DNA nicking activity of HP0268. The substrate plasmid DNA was incubated with 4 μM HP0268 at 37ºC for 30 min in the presence and absence of 1 mM metal ion (Ca 2+ , Co 2+ , Ni 2+ , Fe 3+ , Mn 2+ , Mg 2+ and Cu 2+ ). Increasing concentrations (0.2, 0.4 and 1 μM) of Mn 2+ ion were used. Excess EDTA was used to remove the residual metal ions during the protein preparation. (D) The percentages of the resulting DNA conformations were plotted with regard to metal ion used. Cont. represents the substrate plasmid pET-21a(+) without HP0268, and Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. Commonly, 10 units of control enzyme were used in a final volume of 30 μl.

    Article Snippet: The commercial enzymes Nt.BsmAI and NdeI (NEB, Inc.) were used as references to validate the DNA nicking and double-cutting activity of HP0268, respectively.

    Techniques: Activity Assay, Incubation, Plasmid Preparation, Positron Emission Tomography

    Nuclease activity of HP0268 mutants. (A) Nicking endonuclease assay of wild-type and mutant HP0268 using gel electrophoresis. The substrate plasmid DNA was incubated with the wild-type and mutants (2 μM) at 37ºC for 30 min. Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. (B) Graph of the nicking endonuclease activities of wild-type and mutant HP0268. The DNA nicking activities of the mutants are normalized by that of the wild-type. (C) Fluorometric ribonuclease activities of wild-type and mutant HP0268. The protein concentrations were maintained at 6 μM. The fluorescence spectra are shown in a color-coded mode. In every figure, Cont. and WT indicate the reference condition of having only a buffer and the wild-type protein, respectively. The reaction buffer consisted of 20 mM Tris (pH 8.0) and 150 mM NaCl.

    Journal: Nucleic Acids Research

    Article Title: Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities

    doi: 10.1093/nar/gkv348

    Figure Lengend Snippet: Nuclease activity of HP0268 mutants. (A) Nicking endonuclease assay of wild-type and mutant HP0268 using gel electrophoresis. The substrate plasmid DNA was incubated with the wild-type and mutants (2 μM) at 37ºC for 30 min. Nt.BsmAI and NdeI represent the positive controls for the nicked and linear DNA, respectively. (B) Graph of the nicking endonuclease activities of wild-type and mutant HP0268. The DNA nicking activities of the mutants are normalized by that of the wild-type. (C) Fluorometric ribonuclease activities of wild-type and mutant HP0268. The protein concentrations were maintained at 6 μM. The fluorescence spectra are shown in a color-coded mode. In every figure, Cont. and WT indicate the reference condition of having only a buffer and the wild-type protein, respectively. The reaction buffer consisted of 20 mM Tris (pH 8.0) and 150 mM NaCl.

    Article Snippet: The commercial enzymes Nt.BsmAI and NdeI (NEB, Inc.) were used as references to validate the DNA nicking and double-cutting activity of HP0268, respectively.

    Techniques: Activity Assay, Mutagenesis, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation, Fluorescence

    Detection of relative rDNA copy number in old and young mice. (A) The position of the probe for the Southern blot analysis shown in panels B and C and recognition sites for BamHI (B) and NdeI (N) are shown. (B and C) Detection of relative rDNA copy number. Southern analysis for rDNA copy number is shown at the top. DNA was digested with BamHI and NdeI. Upper bands (4 kb) come from rDNA units without the BamHI-2 site, and lower bands (2.4 kb) are from rDNA units with the BamHI-2 site. The SWI5 gene was used as a loading control (middle). The single-copy gene SWI5 was detected on the same filter used for the experiment shown in the upper panel. Relative rDNA copy numbers were determined (bottom). The band intensities of rDNA were normalized by those of SWI5, and the values are relative to the average of rDNA values in the four young mice. The blue dots show the results from the upper-band intensities of rDNA, and the red dots are the results from the lower bands. Individual mice that were used to isolate the bone marrow cells are identified by number (ID) ( Fig. 2 ). P values are shown at the bottom of the panel. n.s., not significant.

    Journal: Molecular and Cellular Biology

    Article Title: Age-Dependent Ribosomal DNA Variations in Mice

    doi: 10.1128/MCB.00368-20

    Figure Lengend Snippet: Detection of relative rDNA copy number in old and young mice. (A) The position of the probe for the Southern blot analysis shown in panels B and C and recognition sites for BamHI (B) and NdeI (N) are shown. (B and C) Detection of relative rDNA copy number. Southern analysis for rDNA copy number is shown at the top. DNA was digested with BamHI and NdeI. Upper bands (4 kb) come from rDNA units without the BamHI-2 site, and lower bands (2.4 kb) are from rDNA units with the BamHI-2 site. The SWI5 gene was used as a loading control (middle). The single-copy gene SWI5 was detected on the same filter used for the experiment shown in the upper panel. Relative rDNA copy numbers were determined (bottom). The band intensities of rDNA were normalized by those of SWI5, and the values are relative to the average of rDNA values in the four young mice. The blue dots show the results from the upper-band intensities of rDNA, and the red dots are the results from the lower bands. Individual mice that were used to isolate the bone marrow cells are identified by number (ID) ( Fig. 2 ). P values are shown at the bottom of the panel. n.s., not significant.

    Article Snippet: For Southern blot analysis, 150 ng of mouse DNA was digested with 10 units of BamHI-HF ( and ), NdeI ( and ), and SacII ( ) (all restriction enzymes, NEB) overnight at 37°C.

    Techniques: Mouse Assay, Southern Blot

    TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

    Journal: Virology Journal

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein

    doi: 10.1186/1743-422X-9-279

    Figure Lengend Snippet: TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

    Article Snippet: Both plasmid pET28a (Novagen) and CP were digested with Nde I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in pET28a (pET28a-WT-His-TMV-CP12 , pET28a-TR-His-TMV-CP19 , pET28a-TR-His-TMV-CP62 , and pET28a-TR-His-TMV-CP68 ).

    Techniques: Agarose Gel Electrophoresis, Clone Assay, Amplification, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

    Restriction maps of the mutS-rpoS chromosomal region. Approximate locations of restriction sites for five restriction enzymes: Eco RV (E), Nde I (N), Acc I (A), Csp 45 (C), and Nsp I (Ns). The pattern of restriction sites is conserved among strains of each pathogenic group with the exception of the second Nsp I site in mutS [(Ns)*], which is present in EPEC 2 strains but absent in EHEC 2 strains. A distinct Nsp I map was obtained for 921-B4 (not shown). The novel DNA segment found in EPEC and EHEC strains is located at the 3′ end of rpoS and is highlighted with the gray bar.

    Journal: Journal of Bacteriology

    Article Title: Gene Conservation and Loss in the mutS-rpoS Genomic Region of Pathogenic Escherichia coli

    doi:

    Figure Lengend Snippet: Restriction maps of the mutS-rpoS chromosomal region. Approximate locations of restriction sites for five restriction enzymes: Eco RV (E), Nde I (N), Acc I (A), Csp 45 (C), and Nsp I (Ns). The pattern of restriction sites is conserved among strains of each pathogenic group with the exception of the second Nsp I site in mutS [(Ns)*], which is present in EPEC 2 strains but absent in EHEC 2 strains. A distinct Nsp I map was obtained for 921-B4 (not shown). The novel DNA segment found in EPEC and EHEC strains is located at the 3′ end of rpoS and is highlighted with the gray bar.

    Article Snippet: To estimate the length of the mutS-rpoS genomic region and to map the length variation, the long PCR amplicons were digested with five restriction enzymes, Eco RV and Nde I (New England Biolabs, Beverly, Mass.), Csp 45 (Promega, Madison, Wis.), and Acc I and Nsp I (GIBCO-BRL).

    Techniques: