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.
    Ndei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 "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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    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 "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

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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

    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:

    Telomere length distribution by simulation and single-telomere Southern blot. (A) The protein-counting mechanism is based on the measurement of telomere length by Rap1/Rif1/Rif2 binding to telomeric repeats and an inhibition of telomerase elongation depending on the number/concentration of these complexes. (B) Trajectories of 10 independent telomeres over 100 divisions. Depending on the length, a telomere (blue trajectory) can shorten by length a over several consecutive divisions (solid arrowhead) and then be elongated by a random length b (shaded arrowhead). (C) Telomere length distribution at equilibrium. Equation 1 was iterated 500 times starting with 100,000 telomeres drawn from an uniform distribution between 200 and 400 bp, and the resulting telomere length distribution was plotted (bin size, 3 bp). (D) Representative single-telomere Southern blot. Six independent TLC1 wild-type spores derived from TLC1/tlc1 Δ heterozygous diploids were grown for 24 hr on YPD plates and then transferred to liquid culture for another 24 hr at exponential growth (for a total of ∼30 population doublings). The Southern blot was performed on Nde I- and Bst EII-digested genomic DNA using simultaneously oT355 (green) and oT360 (red) fluorescent probes, designed to detect I-L and VI-R telomeres, respectively. (E) Single-telomere Southern blot with oT355 and oT360 fluorescent probes performed on telomerase-positive (wild type, WT) or negative ( tlc1 Δ) cells. TLC1/tlc1 Δ diploid cells were sporulated, and the four spores from a tetrad were grown for 24 hr on a plate and then in liquid YPD medium for the total indicated population doublings (PD). (F) Comparison of experimental and simulated data of telomere length distribution. Plot of 117 experimental measurements of telomere length from single-telomere Southern blot as in D (black line, bin size, 20 bp) and 1000 simulated values L i ¯ ∈ [ | 1 : 1000 | ] (colored lines, bin size, 20 bp) for 20, 30, …, 70 divisions. The latter were obtained as follows: after drawing randomly 100 initial lengths L i 0 from the theoretical distribution in C, we applied the dynamics of Equation 1 to 1000 telomeres with initial length L i 0 to get, after 50 divisions, a mean length L i ¯ . This was done for each plot, corresponding to 20, 30,…, or 70 divisions (blue to orange lines).

    Journal: Genetics

    Article Title: The Length of the Shortest Telomere as the Major Determinant of the Onset of Replicative Senescence

    doi: 10.1534/genetics.113.152322

    Figure Lengend Snippet: Telomere length distribution by simulation and single-telomere Southern blot. (A) The protein-counting mechanism is based on the measurement of telomere length by Rap1/Rif1/Rif2 binding to telomeric repeats and an inhibition of telomerase elongation depending on the number/concentration of these complexes. (B) Trajectories of 10 independent telomeres over 100 divisions. Depending on the length, a telomere (blue trajectory) can shorten by length a over several consecutive divisions (solid arrowhead) and then be elongated by a random length b (shaded arrowhead). (C) Telomere length distribution at equilibrium. Equation 1 was iterated 500 times starting with 100,000 telomeres drawn from an uniform distribution between 200 and 400 bp, and the resulting telomere length distribution was plotted (bin size, 3 bp). (D) Representative single-telomere Southern blot. Six independent TLC1 wild-type spores derived from TLC1/tlc1 Δ heterozygous diploids were grown for 24 hr on YPD plates and then transferred to liquid culture for another 24 hr at exponential growth (for a total of ∼30 population doublings). The Southern blot was performed on Nde I- and Bst EII-digested genomic DNA using simultaneously oT355 (green) and oT360 (red) fluorescent probes, designed to detect I-L and VI-R telomeres, respectively. (E) Single-telomere Southern blot with oT355 and oT360 fluorescent probes performed on telomerase-positive (wild type, WT) or negative ( tlc1 Δ) cells. TLC1/tlc1 Δ diploid cells were sporulated, and the four spores from a tetrad were grown for 24 hr on a plate and then in liquid YPD medium for the total indicated population doublings (PD). (F) Comparison of experimental and simulated data of telomere length distribution. Plot of 117 experimental measurements of telomere length from single-telomere Southern blot as in D (black line, bin size, 20 bp) and 1000 simulated values L i ¯ ∈ [ | 1 : 1000 | ] (colored lines, bin size, 20 bp) for 20, 30, …, 70 divisions. The latter were obtained as follows: after drawing randomly 100 initial lengths L i 0 from the theoretical distribution in C, we applied the dynamics of Equation 1 to 1000 telomeres with initial length L i 0 to get, after 50 divisions, a mean length L i ¯ . This was done for each plot, corresponding to 20, 30,…, or 70 divisions (blue to orange lines).

    Article Snippet: Nde I, Bst EII, and Bst NI enzymes were purchased from New England Biolabs.

    Techniques: Southern Blot, Binding Assay, Inhibition, Concentration Assay, Derivative Assay