ndei (New England Biolabs)


Structured Review

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
https://www.bioz.com/result/ndei/product/New England Biolabs
Average 97 stars, based on 1 article reviews
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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

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

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

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

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

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

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

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

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

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

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

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

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