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  • 99
    New England Biolabs ndei
    <t>DNA</t> 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 <t>NdeI</t> (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 .
    Ndei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher fastdigest ndei
    <t>DNA</t> 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 <t>NdeI</t> (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 .
    Fastdigest Ndei, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega ndei
    Schematics. (A) c92 gene cloned into pSeSD1 overexpression vector showing the location of the FLAG and His tags on the C-terminal end of the protein (underlined in the amino acid sequence); the <t>NdeI</t> (CATATG) and <t>SalI</t> (GTCGAC) sites are underlined in the nucleotide sequence. (B) The c92 c53a mutant construct, showing the change of a serine residue (TCA) to a premature stop codon (TAA); the transmembrane domain is underlined in the amino acid sequence.
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    99
    Millipore ndei
    Cloning, expression, and purification of rPCNA. (A) Specific PCR of <t>PCNA.</t> Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; <t>NdeI-</t> and BamHI-digested pTZ57R/T-PCNA. (C)
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    98
    Thermo Fisher ndei
    Design and cloning of SDF1-ELP. (A) Cloning of SDF-ELP was done using a peT25B+ expression vector. SDF1 was fused to ELP using the <t>XbaI</t> and <t>NdeI</t> restriction sites. The plasmid with SDF1-ELP was mutated to put a 6X Histidine tag in frame with the protein
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    Image Search Results


    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

    Isolation of a KARRE from the upstream 3′ splice site of the exon 5a of Snap25 gene based on forskolin reduction of 5a/5b ratios in PC12 cells during neuronal differentiation. ( A ) Forskolin-induced neuronal differentiation of PC12 cells. Shown are representative images of PC12 cells without or with forskolin addition for 18 h. ( B ) Diagram of the alternative splicing of the exons 5a and 5b of the Snap25 gene. Arrows: sites of restriction enzymes NdeI (5a) and AvaII (5b) used to digest the PCR products. Arrowheads: PCR primers. ( C ) RT-PCR products from untreated (−) and treated PC12 cells. ETOH: ethanol, vehicle for forskolin and Dex (dexamethasone); H89: a PKA-specific inhibitor; NC: PCR negative control; M: molecular size marker. ( D) Diagram of the deletion/replacement mutants of Snap25 splicing reporters and their exon inclusion levels in the presence of PKAm or active PKA. Asterisks: indicating P value levels in one tail, paired Student’s t -test (*** P

    Journal: Nucleic Acids Research

    Article Title: Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation

    doi: 10.1093/nar/gks504

    Figure Lengend Snippet: Isolation of a KARRE from the upstream 3′ splice site of the exon 5a of Snap25 gene based on forskolin reduction of 5a/5b ratios in PC12 cells during neuronal differentiation. ( A ) Forskolin-induced neuronal differentiation of PC12 cells. Shown are representative images of PC12 cells without or with forskolin addition for 18 h. ( B ) Diagram of the alternative splicing of the exons 5a and 5b of the Snap25 gene. Arrows: sites of restriction enzymes NdeI (5a) and AvaII (5b) used to digest the PCR products. Arrowheads: PCR primers. ( C ) RT-PCR products from untreated (−) and treated PC12 cells. ETOH: ethanol, vehicle for forskolin and Dex (dexamethasone); H89: a PKA-specific inhibitor; NC: PCR negative control; M: molecular size marker. ( D) Diagram of the deletion/replacement mutants of Snap25 splicing reporters and their exon inclusion levels in the presence of PKAm or active PKA. Asterisks: indicating P value levels in one tail, paired Student’s t -test (*** P

    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.

    Techniques: Isolation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Marker

    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

    Schematics. (A) c92 gene cloned into pSeSD1 overexpression vector showing the location of the FLAG and His tags on the C-terminal end of the protein (underlined in the amino acid sequence); the NdeI (CATATG) and SalI (GTCGAC) sites are underlined in the nucleotide sequence. (B) The c92 c53a mutant construct, showing the change of a serine residue (TCA) to a premature stop codon (TAA); the transmembrane domain is underlined in the amino acid sequence.

    Journal: Journal of Virology

    Article Title: Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures ▿ Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures ▿ †

    doi: 10.1128/JVI.00379-11

    Figure Lengend Snippet: Schematics. (A) c92 gene cloned into pSeSD1 overexpression vector showing the location of the FLAG and His tags on the C-terminal end of the protein (underlined in the amino acid sequence); the NdeI (CATATG) and SalI (GTCGAC) sites are underlined in the nucleotide sequence. (B) The c92 c53a mutant construct, showing the change of a serine residue (TCA) to a premature stop codon (TAA); the transmembrane domain is underlined in the amino acid sequence.

    Article Snippet: The purified PCR products were then digested with NdeI (Promega) and SalI (Promega) at 37°C for 1 h. The restriction digests were purified as described above with a Qiagen QIAquick PCR purification kit and eluted in 30 μl ddH2 O.

    Techniques: Clone Assay, Over Expression, Plasmid Preparation, Sequencing, Mutagenesis, Construct

    Confirmation of KH1 deletion in L. infantum . ( a ) Comparison of the copy number of KH1 gene in the genomic DNA of the mutant lines. The copy number was determined by quantitative real-time PCR relative to the housekeeping genes GAPDH , using the comparative C T method (2 −ΔΔCT Method). ( b ) Schematic representation of KH1 locus before and after the cassettes integration. The cleavage sites of Nde I and Pvu II restriction enzymes and the length of the generated fragments were represented. ( c , d ) Southern blot analysis performed with 10 µg of genomic DNA of L. infantum wild-type (WT), the mutants Δ Likh1 + , Δ Likh1 and Δ Likh1 [pSP72αZEOα/ KH1 ] were digested with Nde I and Pvu II restriction enzymes. The membranes were hybridized with 32P -labelled probes, that reconize the KH1 5′UTR ( c ) or Kh1 gene ( d ). The normalization was performed by calculating the KH1/GAPDH ratio. Similar results were obtained when normalizing with DNA polymerase as housekeeping gene. Exposures were cropped from original images available as supplementary material.

    Journal: Scientific Reports

    Article Title: Growth arrested live-attenuated Leishmania infantum KHARON1 null mutants display cytokinesis defect and protective immunity in mice

    doi: 10.1038/s41598-018-30076-7

    Figure Lengend Snippet: Confirmation of KH1 deletion in L. infantum . ( a ) Comparison of the copy number of KH1 gene in the genomic DNA of the mutant lines. The copy number was determined by quantitative real-time PCR relative to the housekeeping genes GAPDH , using the comparative C T method (2 −ΔΔCT Method). ( b ) Schematic representation of KH1 locus before and after the cassettes integration. The cleavage sites of Nde I and Pvu II restriction enzymes and the length of the generated fragments were represented. ( c , d ) Southern blot analysis performed with 10 µg of genomic DNA of L. infantum wild-type (WT), the mutants Δ Likh1 + , Δ Likh1 and Δ Likh1 [pSP72αZEOα/ KH1 ] were digested with Nde I and Pvu II restriction enzymes. The membranes were hybridized with 32P -labelled probes, that reconize the KH1 5′UTR ( c ) or Kh1 gene ( d ). The normalization was performed by calculating the KH1/GAPDH ratio. Similar results were obtained when normalizing with DNA polymerase as housekeeping gene. Exposures were cropped from original images available as supplementary material.

    Article Snippet: About 10 µg of genomic DNA from L. infantum WT, Δ Likh1 + , Δ Likh1 and Δ Likh1 [pSP72αZEOα/ KH1 ] were digested with restriction enzymes Pvu II and Nde I (Promega, Madison, WI, USA).

    Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Generated, Southern Blot

    Cloning, expression, and purification of rPCNA. (A) Specific PCR of PCNA. Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; NdeI- and BamHI-digested pTZ57R/T-PCNA. (C)

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

    doi: 10.1128/AAC.01847-13

    Figure Lengend Snippet: Cloning, expression, and purification of rPCNA. (A) Specific PCR of PCNA. Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; NdeI- and BamHI-digested pTZ57R/T-PCNA. (C)

    Article Snippet: PCNA was further subcloned at the NdeI and BamHI sites in the bacterial expression vector pET28a (Novagen).

    Techniques: Clone Assay, Expressing, Purification, Polymerase Chain Reaction, Amplification

    Design and cloning of SDF1-ELP. (A) Cloning of SDF-ELP was done using a peT25B+ expression vector. SDF1 was fused to ELP using the XbaI and NdeI restriction sites. The plasmid with SDF1-ELP was mutated to put a 6X Histidine tag in frame with the protein

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: The development and characterization of SDF1α-elastin-like-peptide nanoparticles for wound healing

    doi: 10.1016/j.jconrel.2016.04.020

    Figure Lengend Snippet: Design and cloning of SDF1-ELP. (A) Cloning of SDF-ELP was done using a peT25B+ expression vector. SDF1 was fused to ELP using the XbaI and NdeI restriction sites. The plasmid with SDF1-ELP was mutated to put a 6X Histidine tag in frame with the protein

    Article Snippet: The NdeI and XbaI restriction enzymes and DNA ligation kit used for cloning were obtained from Thermo Fisher Scientific.

    Techniques: Clone Assay, Expressing, Plasmid Preparation