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    NdeI
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    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    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 "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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    Related Articles

    Ligation:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: .. General materials T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) as a 10x stock, 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)) as a 5x stock, restriction endonucleases EcoRV, NruI, BstNI, Hpy188I, NdeI, BamHI, as well as λ DNA, 1 M DTT, 6x Purple Loading Dye with SDS, Proteinase K and 10 mM ATP were obtained from New England Biolabs (NEB, Ipswich, MA). .. Tris-HCl (1 M pH 7.5 @ 25°C) was obtained from Amresco (Solon, OH).

    Random Primed:

    Article Title: Enhanced transgene expression in rice following selection controlled by weak promoters
    Article Snippet: .. About 3 μg genomic DNA per sample was digested by Nde I (NEB, Beverly, MA) and hybridized with a HPT probe which was prepared using a random primed digoxigenin-DNA labeling kit (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. .. Primers for probe amplification are listed in Additional file : Table S1.

    Labeling:

    Article Title: Enhanced transgene expression in rice following selection controlled by weak promoters
    Article Snippet: .. About 3 μg genomic DNA per sample was digested by Nde I (NEB, Beverly, MA) and hybridized with a HPT probe which was prepared using a random primed digoxigenin-DNA labeling kit (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. .. Primers for probe amplification are listed in Additional file : Table S1.

    Generated:

    Article Title: DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI
    Article Snippet: .. Linear DNA substrates were generated by incubating 4 nM plasmid DNA with 1 U/μl of AlwNI or 2 U/μl of NdeI in NEBuffer 2 (New England Biolabs, MA, USA). .. The DNA was purified by phenol/chloroform and chloroform extraction followed by ethanol precipitation.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation
    Article Snippet: .. Some cultures were pretreated with 10 µM of H89 (N -[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride, Sigma, #B1427) for 10 min. To differentiate products of Snap25 endogenous mRNA transcripts from exons 5a and 5b, we digested 1.0 µl of 32 P-labelled-PCR products with AvaII and NdeI restriction enzymes in buffer 4 (New England Biolabs) in a 10 -µl reaction at 37°C for 1 h and run in 6% denaturing polyacrylamide gel electrophoresis (PAGE) gel. .. For minigene splicing reporters, 24 cycles of PCR were carried out and products resolved in 3% agarose gels stained with ethidium bromide.

    DNA Labeling:

    Article Title: Enhanced transgene expression in rice following selection controlled by weak promoters
    Article Snippet: .. About 3 μg genomic DNA per sample was digested by Nde I (NEB, Beverly, MA) and hybridized with a HPT probe which was prepared using a random primed digoxigenin-DNA labeling kit (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. .. Primers for probe amplification are listed in Additional file : Table S1.

    Plasmid Preparation:

    Article Title: DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI
    Article Snippet: .. Linear DNA substrates were generated by incubating 4 nM plasmid DNA with 1 U/μl of AlwNI or 2 U/μl of NdeI in NEBuffer 2 (New England Biolabs, MA, USA). .. The DNA was purified by phenol/chloroform and chloroform extraction followed by ethanol precipitation.

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    New England Biolabs ndei restriction enzyme
    The <t>NdeI</t> Restriction Profiles of DPYD Gene IVS14+1G > A Polymorphism on 3% Agarose Gel Electrophoresis (1X TBE). Lane M shows DNA ladder (100bp); Lane 1 shows undigested <t>PCR</t> product (198 bp); Lane 2 shows wild type genotype (181 bp); Lane 3 shows Heterozygous genotype (181 bp and 154 bp); Lane 4 shows negative control.
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    The NdeI Restriction Profiles of DPYD Gene IVS14+1G > A Polymorphism on 3% Agarose Gel Electrophoresis (1X TBE). Lane M shows DNA ladder (100bp); Lane 1 shows undigested PCR product (198 bp); Lane 2 shows wild type genotype (181 bp); Lane 3 shows Heterozygous genotype (181 bp and 154 bp); Lane 4 shows negative control.

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    Article Title: Low Incidence of the DPD IVS14+1G > A Polymorphism in Jordanian Breast and Colorectal Cancer patients

    doi: 10.22034/APJCP.2017.18.6.1651

    Figure Lengend Snippet: The NdeI Restriction Profiles of DPYD Gene IVS14+1G > A Polymorphism on 3% Agarose Gel Electrophoresis (1X TBE). Lane M shows DNA ladder (100bp); Lane 1 shows undigested PCR product (198 bp); Lane 2 shows wild type genotype (181 bp); Lane 3 shows Heterozygous genotype (181 bp and 154 bp); Lane 4 shows negative control.

    Article Snippet: The PCR product (198bp) was digested for overnight at 37°C using the NdeI restriction enzyme (New England Biolab, Inc.).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control

    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)

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

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

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

    Figure Lengend 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)

    Article Snippet: Restriction enzymes NdeI, KpnI-HF, and XhoI with supplied buffers and 100× BSA solution (New England Biolabs).

    Techniques: Polymerase Cycling Assembly, Selection, Sequencing

    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 .

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkp790

    Figure Lengend 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 .

    Article Snippet: Linear DNA substrates were generated by incubating 4 nM plasmid DNA with 1 U/μl of AlwNI or 2 U/μl of NdeI in NEBuffer 2 (New England Biolabs, MA, USA).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    (A). Comparison of in vitro chaperone activity of α-crystallin and purified sHsp18 . Enzymes (2 U) were heat inactivated ( Sma I at 37°C for 90 min and Nde I at 45°C for 90 min) in the presence or absence of molecular chaperones (0.2 μg) and assayed for the cleavage of 1 μg of plasmid DNA. Lanes represent, λ Hind III marker (lane 1), digested plasmid (lane 2), uncut plasmid (lane 3), plasmid digested with- heat inactivated Sma I (lane 4), heat inactivated Nde I (lane 5), heat inactivated Sma I in the presence of sHsp18 (lane 6), heat inactivated Sma I in the presence of α-crystallin (lane 7), heat inactivated Nde I in the presence of sHsp18 (lane 8) and heat inactivated Nde I in the presence of α-crystallin (lane 9). (B). sHsp18 can act as a molecular chaperone in wide range of physiological temperatures. 0.2 μg sHsp18 or BSA was incubated with 2 U of Sma I at different temperatures for 90 min, and the cleavage of plasmid DNA was assayed at 25°C for 3 hrs. Lanes represent, λ Hind III marker (lane 1), uncut plasmid (lane 2), plasmid digested with Sma I as control (lane 3), plasmid incubated with heat inactivated Sma I without and with sHsp18 at 30°C (lanes 4–5); at 35°C (lanes 6–7), at 40°C (lanes 8–9), at 45°C (lanes 11–12), plasmid incubated with Sma I with BSA at 35°C, 40°C and 45°C (lanes 12–14). (C). Preheating of molecular chaperones does not affect chaperone activity. Lanes represent, λ Hind III marker (lane 1), undigested plasmid (lane 2), plasmid digested with- Sma I (lane 3), Sma I with preheated sHsp18 at 100°C for 5 min (lane 4) and with preheated α-crystallin at 100°C for 5 min (lane 5).

    Journal: BMC Microbiology

    Article Title: Functional characterization of a small heat shock protein from Mycobacterium leprae

    doi: 10.1186/1471-2180-8-208

    Figure Lengend Snippet: (A). Comparison of in vitro chaperone activity of α-crystallin and purified sHsp18 . Enzymes (2 U) were heat inactivated ( Sma I at 37°C for 90 min and Nde I at 45°C for 90 min) in the presence or absence of molecular chaperones (0.2 μg) and assayed for the cleavage of 1 μg of plasmid DNA. Lanes represent, λ Hind III marker (lane 1), digested plasmid (lane 2), uncut plasmid (lane 3), plasmid digested with- heat inactivated Sma I (lane 4), heat inactivated Nde I (lane 5), heat inactivated Sma I in the presence of sHsp18 (lane 6), heat inactivated Sma I in the presence of α-crystallin (lane 7), heat inactivated Nde I in the presence of sHsp18 (lane 8) and heat inactivated Nde I in the presence of α-crystallin (lane 9). (B). sHsp18 can act as a molecular chaperone in wide range of physiological temperatures. 0.2 μg sHsp18 or BSA was incubated with 2 U of Sma I at different temperatures for 90 min, and the cleavage of plasmid DNA was assayed at 25°C for 3 hrs. Lanes represent, λ Hind III marker (lane 1), uncut plasmid (lane 2), plasmid digested with Sma I as control (lane 3), plasmid incubated with heat inactivated Sma I without and with sHsp18 at 30°C (lanes 4–5); at 35°C (lanes 6–7), at 40°C (lanes 8–9), at 45°C (lanes 11–12), plasmid incubated with Sma I with BSA at 35°C, 40°C and 45°C (lanes 12–14). (C). Preheating of molecular chaperones does not affect chaperone activity. Lanes represent, λ Hind III marker (lane 1), undigested plasmid (lane 2), plasmid digested with- Sma I (lane 3), Sma I with preheated sHsp18 at 100°C for 5 min (lane 4) and with preheated α-crystallin at 100°C for 5 min (lane 5).

    Article Snippet: In this assay, restriction enzymes Sma I and Nde I (New England Biolabs, Beverly, MA) were used, according to the manufacturer's recommendations.

    Techniques: In Vitro, Activity Assay, Purification, Plasmid Preparation, Marker, Activated Clotting Time Assay, Incubation