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    Structured Review

    New England Biolabs ncoi
    mNET-seq Profiles for <t>PKM</t> Alternative Splicing after PTBP1 Depletion, Related to Figure 4 (A) PKM exons 8–11 are illustrated. Exon 9 (green) and exon 10 (orange) are mutually exclusive. PCR primers indicated as black triangles. RT-PCR products were digested with indicated exon-specific restriction enzyme <t>(NcoI</t> or PstI). (B) mNET-seq data around mutually exclusive exons 9 and 10 of PKM . mNET-seq/S5P signals at 3′ end of exon 9 and exon 10 are shown by green and orange arrows, respectively. Transcription direction, black arrow. (C) Western blot of PTBP1 and tubulin from siPTBP1-treated HeLa cells. (D) PKM RT-PCR products from PTBP1-depleted HeLa nuclear RNA were digested by NcoI. (E) mNET-seq/S5P data over mutually exclusive exons 9 and 10 of PKM from siLuc and siPTBP1-treated HeLa cells (top), followed by expanded view around 5′SS of introns 10 and 11. S5P-peaks at 3′ ends of exons, orange asterisks. Transcription direction, black arrows.
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    Images

    1) Product Images from "Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing"

    Article Title: Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing

    Journal: Cell

    doi: 10.1016/j.cell.2015.03.027

    mNET-seq Profiles for PKM Alternative Splicing after PTBP1 Depletion, Related to Figure 4 (A) PKM exons 8–11 are illustrated. Exon 9 (green) and exon 10 (orange) are mutually exclusive. PCR primers indicated as black triangles. RT-PCR products were digested with indicated exon-specific restriction enzyme (NcoI or PstI). (B) mNET-seq data around mutually exclusive exons 9 and 10 of PKM . mNET-seq/S5P signals at 3′ end of exon 9 and exon 10 are shown by green and orange arrows, respectively. Transcription direction, black arrow. (C) Western blot of PTBP1 and tubulin from siPTBP1-treated HeLa cells. (D) PKM RT-PCR products from PTBP1-depleted HeLa nuclear RNA were digested by NcoI. (E) mNET-seq/S5P data over mutually exclusive exons 9 and 10 of PKM from siLuc and siPTBP1-treated HeLa cells (top), followed by expanded view around 5′SS of introns 10 and 11. S5P-peaks at 3′ ends of exons, orange asterisks. Transcription direction, black arrows.
    Figure Legend Snippet: mNET-seq Profiles for PKM Alternative Splicing after PTBP1 Depletion, Related to Figure 4 (A) PKM exons 8–11 are illustrated. Exon 9 (green) and exon 10 (orange) are mutually exclusive. PCR primers indicated as black triangles. RT-PCR products were digested with indicated exon-specific restriction enzyme (NcoI or PstI). (B) mNET-seq data around mutually exclusive exons 9 and 10 of PKM . mNET-seq/S5P signals at 3′ end of exon 9 and exon 10 are shown by green and orange arrows, respectively. Transcription direction, black arrow. (C) Western blot of PTBP1 and tubulin from siPTBP1-treated HeLa cells. (D) PKM RT-PCR products from PTBP1-depleted HeLa nuclear RNA were digested by NcoI. (E) mNET-seq/S5P data over mutually exclusive exons 9 and 10 of PKM from siLuc and siPTBP1-treated HeLa cells (top), followed by expanded view around 5′SS of introns 10 and 11. S5P-peaks at 3′ ends of exons, orange asterisks. Transcription direction, black arrows.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    2) Product Images from "CRISPR/Cas9 Mutagenesis by Translocation of Cas9 Protein Into Plant Cells via the Agrobacterium Type IV Secretion System"

    Article Title: CRISPR/Cas9 Mutagenesis by Translocation of Cas9 Protein Into Plant Cells via the Agrobacterium Type IV Secretion System

    Journal: Frontiers in Genome Editing

    doi: 10.3389/fgeed.2020.00006

    Translocation of NCas9F and sgRNA complex for targeted mutagenesis in N. benthamiana . (A) Schematic representation of the translocation of the NCas9F-sgRNA complex from Agrobacterium cells to plant cells. The sgRNA expression cassette under the control of the bacterial expression promoters were cloned individually into the plasmid expressing NCas9F to express the NCas9F-sgRNA complex in Agrobacteria. (B) Targeted mutagenesis via sgRNA-PDS2 expressed from the three bacterial promoters pJ, pT, and pL. Genomic DNA extracted from the infiltrated leaf discs was predigested with NcoI and used as template for amplification of the 283 bp PDS target fragment. The resulting PCR products were digested with T7EI endonuclease. T7EI-digested fragments (arrows) were observed from tissue co-transformed by NCas9F protein and sgRNA-PDS2. (C) Sequence alignment of the mutations after translocation of the NCas9F-sgRNA-PDS2 complex. The PAM sequence is gray and the NcoI site is underlined. Insertions are marked in bold and deletions are indicated by dashes.
    Figure Legend Snippet: Translocation of NCas9F and sgRNA complex for targeted mutagenesis in N. benthamiana . (A) Schematic representation of the translocation of the NCas9F-sgRNA complex from Agrobacterium cells to plant cells. The sgRNA expression cassette under the control of the bacterial expression promoters were cloned individually into the plasmid expressing NCas9F to express the NCas9F-sgRNA complex in Agrobacteria. (B) Targeted mutagenesis via sgRNA-PDS2 expressed from the three bacterial promoters pJ, pT, and pL. Genomic DNA extracted from the infiltrated leaf discs was predigested with NcoI and used as template for amplification of the 283 bp PDS target fragment. The resulting PCR products were digested with T7EI endonuclease. T7EI-digested fragments (arrows) were observed from tissue co-transformed by NCas9F protein and sgRNA-PDS2. (C) Sequence alignment of the mutations after translocation of the NCas9F-sgRNA-PDS2 complex. The PAM sequence is gray and the NcoI site is underlined. Insertions are marked in bold and deletions are indicated by dashes.

    Techniques Used: Translocation Assay, Mutagenesis, Expressing, Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Transformation Assay, Sequencing

    NCas9F translocation and TRV-based targeted mutagenesis in N. benthamiana . (A) Schematic presentation of the TRV-based sgRNA expression and NCas9F translocation. (B) The PDS2 target fragment was amplified from N. benthamiana genomic DNA, isolated from seven biological replicates (1–7) and one control sample without sgRNA (C). The resulting 283 bp PCR products were digested with T7EI endonuclease. T7EI-digested fragments (arrows) were observed from tissue co-transformed with NCas9F protein and TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (C) Purified 404 bp PCR products were digested with NcoI to determine loss of the targeted restriction site. NcoI-resistant fragments (arrow) were observed from tissue co-transformed with NCas9F protein and TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (D) Alignment of the mutations after transformation with the NCas9F protein and TRV encoding the sgRNA-PDS cassette. (E) The PDS2 target fragment was amplified from N. benthamiana genomic DNA, isolated from two biological NCas9F replicates (with VirF translocation signal), five biological NCas9 replicates (without translocation signal) and 1 untransformed control sample (C). The 404 bp PCR products were digested with NcoI. NcoI-resistant fragments (arrow) were observed from tissue co-transformed with NCas9F protein and TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (F) TRV was replicated in the wild-type N. benthamiana plants and the extracted sap containing viral RNA was applied to the N. benthamiana plants that had been infiltrated with Agrobacteria expressing NCas9F, 1 day earlier. The PDS2 target fragment was amplified from the genomic DNA, isolated from eight biological replicates (1–8) and 1 control sample without sgRNA (C) and subjected to NcoI digestion. NcoI-resistant fragments (arrow) were observed from tissue co-transformed with NCas9F protein and enriched TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (G) Sequence analysis of mutations in NcoI-resistant PCR products shown in (F) . The PAM sequence is gray and the NcoI site is underlined. Deletions are indicated by dashes.
    Figure Legend Snippet: NCas9F translocation and TRV-based targeted mutagenesis in N. benthamiana . (A) Schematic presentation of the TRV-based sgRNA expression and NCas9F translocation. (B) The PDS2 target fragment was amplified from N. benthamiana genomic DNA, isolated from seven biological replicates (1–7) and one control sample without sgRNA (C). The resulting 283 bp PCR products were digested with T7EI endonuclease. T7EI-digested fragments (arrows) were observed from tissue co-transformed with NCas9F protein and TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (C) Purified 404 bp PCR products were digested with NcoI to determine loss of the targeted restriction site. NcoI-resistant fragments (arrow) were observed from tissue co-transformed with NCas9F protein and TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (D) Alignment of the mutations after transformation with the NCas9F protein and TRV encoding the sgRNA-PDS cassette. (E) The PDS2 target fragment was amplified from N. benthamiana genomic DNA, isolated from two biological NCas9F replicates (with VirF translocation signal), five biological NCas9 replicates (without translocation signal) and 1 untransformed control sample (C). The 404 bp PCR products were digested with NcoI. NcoI-resistant fragments (arrow) were observed from tissue co-transformed with NCas9F protein and TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (F) TRV was replicated in the wild-type N. benthamiana plants and the extracted sap containing viral RNA was applied to the N. benthamiana plants that had been infiltrated with Agrobacteria expressing NCas9F, 1 day earlier. The PDS2 target fragment was amplified from the genomic DNA, isolated from eight biological replicates (1–8) and 1 control sample without sgRNA (C) and subjected to NcoI digestion. NcoI-resistant fragments (arrow) were observed from tissue co-transformed with NCas9F protein and enriched TRV encoding the sgRNA-PDS2 cassette. % indels is indicated. (G) Sequence analysis of mutations in NcoI-resistant PCR products shown in (F) . The PAM sequence is gray and the NcoI site is underlined. Deletions are indicated by dashes.

    Techniques Used: Translocation Assay, Mutagenesis, Expressing, Amplification, Isolation, Polymerase Chain Reaction, Transformation Assay, Purification, Sequencing

    3) Product Images from "Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium"

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00227-15

    Maps of pNZC and inserts used in this study. A chloride-inducible promoter (CIP) was inserted between BglII and NcoI. P gadR is a constitutive promoter controlling the production of the activator protein GadR. lacZ and AMP expression are controlled by the chloride-inducible promoter P gad (activated by GadR). lacZ and Bac are inserted between cut sites NcoI and SpeI in pNZC to create pNZCL and pNZCA3.
    Figure Legend Snippet: Maps of pNZC and inserts used in this study. A chloride-inducible promoter (CIP) was inserted between BglII and NcoI. P gadR is a constitutive promoter controlling the production of the activator protein GadR. lacZ and AMP expression are controlled by the chloride-inducible promoter P gad (activated by GadR). lacZ and Bac are inserted between cut sites NcoI and SpeI in pNZC to create pNZCL and pNZCA3.

    Techniques Used: Expressing, BAC Assay

    4) Product Images from "Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology"

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology

    Journal: Orphanet Journal of Rare Diseases

    doi: 10.1186/1750-1172-9-56

    Relative DNA methylation levels at germline gene promoters in whole blood and buccal swabs from ICF patients. Methylation analysis in whole blood (A) and buccal swabs (B) were assessed by Methylation-Sensitive Restriction Assay, followed by qRT-PCR amplification of the AciI digested product with primers flanking at least two AciI sites within the promoter CpG island. A non-cutter NcoI control digest served to normalize data that are presented as a percentage of methylation relative to the control digest. ICF subtypes 1, 2, and X are indicated and separated by dotted lines. For the X-linked gene TEX11, female patients are indicated as black bars. Raw data used to built this Figure can be found in Additional file 8 . WB and BS are control whole blood DNA and buccal swabs from healthy donor, respectively. Error bars represent standard error.
    Figure Legend Snippet: Relative DNA methylation levels at germline gene promoters in whole blood and buccal swabs from ICF patients. Methylation analysis in whole blood (A) and buccal swabs (B) were assessed by Methylation-Sensitive Restriction Assay, followed by qRT-PCR amplification of the AciI digested product with primers flanking at least two AciI sites within the promoter CpG island. A non-cutter NcoI control digest served to normalize data that are presented as a percentage of methylation relative to the control digest. ICF subtypes 1, 2, and X are indicated and separated by dotted lines. For the X-linked gene TEX11, female patients are indicated as black bars. Raw data used to built this Figure can be found in Additional file 8 . WB and BS are control whole blood DNA and buccal swabs from healthy donor, respectively. Error bars represent standard error.

    Techniques Used: DNA Methylation Assay, Methylation, Restriction Assay, Quantitative RT-PCR, Amplification, Western Blot

    5) Product Images from "Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands"

    Article Title: Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands

    Journal: Scientific Reports

    doi: 10.1038/srep20657

    Detection of strand-specific nicking activity of TALE nickase. ( A ) Illustration of the expected digestion patterns of DNA products following in vitro cleavage with TALENs and TALE nickase. ( B ) In vitro assessment of cleavage activity of TALENs and TALE nickase under non-denaturing and denaturing conditions. The percentages of the cleaved products (indicated by black arrows) are shown at the bottom of each lane. ( C ) Detection of gene mutation in BFFs treated with TALENs and TALE nickase using surveyor nuclease assay by NcoI . (Left) NcoI digestion of PCR products analyzed using PAGE. Arrows indicate the uncleaved products. (Right) Statistical results of NHEJ efficiency in the cells treated with nuclease and nickase based on the result of NcoI digestion of PCR products. ( D,E ) Flow cytometric analysis of cellular γH2AX expression. ( D ) Percentage of γH2AX-positive cells analyzed using flow cytometry. After flow cytometric analysis of cellular γH2AX expression levels, a gate was drawn (black line in the figures) to encompass about 1% of BFFs of negative control. ( E ) Statistical results of cellular γH2AX expression. The columns indicate the fraction of γH2AX-positive cells of the transfected cells. Columns in ( C , E ) represent the average of three experiments. *Statistical significance increases compared with the no TALEN sample ( P
    Figure Legend Snippet: Detection of strand-specific nicking activity of TALE nickase. ( A ) Illustration of the expected digestion patterns of DNA products following in vitro cleavage with TALENs and TALE nickase. ( B ) In vitro assessment of cleavage activity of TALENs and TALE nickase under non-denaturing and denaturing conditions. The percentages of the cleaved products (indicated by black arrows) are shown at the bottom of each lane. ( C ) Detection of gene mutation in BFFs treated with TALENs and TALE nickase using surveyor nuclease assay by NcoI . (Left) NcoI digestion of PCR products analyzed using PAGE. Arrows indicate the uncleaved products. (Right) Statistical results of NHEJ efficiency in the cells treated with nuclease and nickase based on the result of NcoI digestion of PCR products. ( D,E ) Flow cytometric analysis of cellular γH2AX expression. ( D ) Percentage of γH2AX-positive cells analyzed using flow cytometry. After flow cytometric analysis of cellular γH2AX expression levels, a gate was drawn (black line in the figures) to encompass about 1% of BFFs of negative control. ( E ) Statistical results of cellular γH2AX expression. The columns indicate the fraction of γH2AX-positive cells of the transfected cells. Columns in ( C , E ) represent the average of three experiments. *Statistical significance increases compared with the no TALEN sample ( P

    Techniques Used: Activity Assay, In Vitro, TALENs, Mutagenesis, Nuclease Assay, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Non-Homologous End Joining, Flow Cytometry, Expressing, Cytometry, Negative Control, Transfection

    6) Product Images from "Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for Diagnostic and Therapeutic Applications"

    Article Title: Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for Diagnostic and Therapeutic Applications

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-103

    Reconstruction and amplification of rlukS-PV and rlukF-PV from protein expression system. A. Gene map of pET-21d(+)-lukF-PV (Reconstructed using VECTOR NTI software). The insert ( rlukF-PV ) locates within the NcoI and AvaI RDE sites, technically between the T7 promoter and terminator, all confirmed by sequencing. NOTE: The VECTOR NTI prefers the non-specific nuclease (AvaI), which recognises the degenerate sequence (CYCGRG), over the specific XhoI used in the cloning experiments which specifically recognises the sequence (CTCGAG) engineered into the primers used for recombination. B. Amplification of rlukS-PV and rlukF-PV from the expression system. Lane 1, rlukS-PV amplified from expression E. coli BL21(DE3)-pET-21d(+)-lukS-PV; Lane 2, rlukF-PV amplified from expression E. coli BL21(DE3)-pET-21d(+)-lukF-PV; Lane 3, Molecular grade water used as negative PCR control; Lane 4, 100 bp DNA ladder.
    Figure Legend Snippet: Reconstruction and amplification of rlukS-PV and rlukF-PV from protein expression system. A. Gene map of pET-21d(+)-lukF-PV (Reconstructed using VECTOR NTI software). The insert ( rlukF-PV ) locates within the NcoI and AvaI RDE sites, technically between the T7 promoter and terminator, all confirmed by sequencing. NOTE: The VECTOR NTI prefers the non-specific nuclease (AvaI), which recognises the degenerate sequence (CYCGRG), over the specific XhoI used in the cloning experiments which specifically recognises the sequence (CTCGAG) engineered into the primers used for recombination. B. Amplification of rlukS-PV and rlukF-PV from the expression system. Lane 1, rlukS-PV amplified from expression E. coli BL21(DE3)-pET-21d(+)-lukS-PV; Lane 2, rlukF-PV amplified from expression E. coli BL21(DE3)-pET-21d(+)-lukF-PV; Lane 3, Molecular grade water used as negative PCR control; Lane 4, 100 bp DNA ladder.

    Techniques Used: Amplification, Expressing, Positron Emission Tomography, Plasmid Preparation, Software, Sequencing, Clone Assay, Polymerase Chain Reaction

    Start-up amplification and restriction digests of rlukS-PV and rlukF-PV. A. PCR amplification of rlukS-PV and rlukF-PV from S. aureus MW2 template genomic DNA. Lane L, 100 bp DNA Marker (NEB, UK); Lanes 1 and 2, rlukS-PV, 868 bp; Lane 3, Molecular grade water used as PCR negative control; Lanes 4 and 5 rlukF-PV, 919 bp. B. Double digests ( NcoI and XhoI ) of minipreps of the intermediate host E. coli 5α-pGEMT-Easy-luk-PV to release the insert DNA fragments. Lane 1, Undigested pGEMT-Easy-rlukF-PV; Lane 2, Digested pGEMT-Easy-rlukF-PV showing the cleaved insert rlukF-PV below the 1.0 kb mark; Lane 3, Duplicate of lane 1; Lane 4, Duplicate of lane 2; Lane 5, Undigested pGEMT-Easy-rlukS-PV; Lane 6, Digested pGEMT-Easy-rlukS-PV showing the cleaved insert rlukS-PV, below the 1.0 kb mark; Lane 7, Duplicate of lane 5; Lane 8, Duplicate of lane 6; Lane 9, Undigested pET expression vector; Lane 10, pET vector cut with NcoI and XhoI (the next home of the cleaved insert DNA fragments); Lane 11, Molecular grade water; Lane L, 1 kb DNA Marker (NEB, UK).
    Figure Legend Snippet: Start-up amplification and restriction digests of rlukS-PV and rlukF-PV. A. PCR amplification of rlukS-PV and rlukF-PV from S. aureus MW2 template genomic DNA. Lane L, 100 bp DNA Marker (NEB, UK); Lanes 1 and 2, rlukS-PV, 868 bp; Lane 3, Molecular grade water used as PCR negative control; Lanes 4 and 5 rlukF-PV, 919 bp. B. Double digests ( NcoI and XhoI ) of minipreps of the intermediate host E. coli 5α-pGEMT-Easy-luk-PV to release the insert DNA fragments. Lane 1, Undigested pGEMT-Easy-rlukF-PV; Lane 2, Digested pGEMT-Easy-rlukF-PV showing the cleaved insert rlukF-PV below the 1.0 kb mark; Lane 3, Duplicate of lane 1; Lane 4, Duplicate of lane 2; Lane 5, Undigested pGEMT-Easy-rlukS-PV; Lane 6, Digested pGEMT-Easy-rlukS-PV showing the cleaved insert rlukS-PV, below the 1.0 kb mark; Lane 7, Duplicate of lane 5; Lane 8, Duplicate of lane 6; Lane 9, Undigested pET expression vector; Lane 10, pET vector cut with NcoI and XhoI (the next home of the cleaved insert DNA fragments); Lane 11, Molecular grade water; Lane L, 1 kb DNA Marker (NEB, UK).

    Techniques Used: Amplification, Polymerase Chain Reaction, Marker, Negative Control, Positron Emission Tomography, Expressing, Plasmid Preparation

    7) Product Images from "Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways"

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1252

    Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P
    Figure Legend Snippet: Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P

    Techniques Used: Homologous Recombination, Functional Assay, Plasmid Preparation, Expressing, FACS, Transfection, Southern Blot, Titration, In Vivo, Labeling, In Vitro, Purification, Radioactivity

    8) Product Images from "Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)"

    Article Title: Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)

    Journal: Jundishapur Journal of Natural Pharmaceutical Products

    doi:

    T-DNA Region of pCAMINFα-2b. LB and RB: Left and Right Borders, HYG(R): Hygromycin Selectable Marker, CaMV35s: Cauliflower Mosaic Virus Promoter, NcoI and BstEII: Restriction Sites and NOS: Nopaline Synthase Terminator.
    Figure Legend Snippet: T-DNA Region of pCAMINFα-2b. LB and RB: Left and Right Borders, HYG(R): Hygromycin Selectable Marker, CaMV35s: Cauliflower Mosaic Virus Promoter, NcoI and BstEII: Restriction Sites and NOS: Nopaline Synthase Terminator.

    Techniques Used: Marker

    9) Product Images from "Selection of human single domain antibodies (sdAb) against thymidine kinase 1 and their incorporation into sdAb-Fc antibody constructs for potential use in cancer therapy"

    Article Title: Selection of human single domain antibodies (sdAb) against thymidine kinase 1 and their incorporation into sdAb-Fc antibody constructs for potential use in cancer therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0264822

    Amplification and ligation of anti-TK1-dAb fragments into the pET-scFv-T expression vector. A) a representative image of a PCR showing amplification of anti-TK1 dAb fragments. B) Restriction enzyme analysis with NcoI and NotI enzymes of pET-anti-TK1-dAb constructs. C) Map of a pET-anti-TK1-dAb construct. Fragments are ligated into the pET-scFv plasmid using NcoI and NotI restriction sites. D) Alignment of the 4-H-TK1_A1 and 4-H-TK1_D1 sdAb sequences shows that the differences of the sdAbs are in their CDRs.
    Figure Legend Snippet: Amplification and ligation of anti-TK1-dAb fragments into the pET-scFv-T expression vector. A) a representative image of a PCR showing amplification of anti-TK1 dAb fragments. B) Restriction enzyme analysis with NcoI and NotI enzymes of pET-anti-TK1-dAb constructs. C) Map of a pET-anti-TK1-dAb construct. Fragments are ligated into the pET-scFv plasmid using NcoI and NotI restriction sites. D) Alignment of the 4-H-TK1_A1 and 4-H-TK1_D1 sdAb sequences shows that the differences of the sdAbs are in their CDRs.

    Techniques Used: Amplification, Ligation, Positron Emission Tomography, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Construct

    10) Product Images from "Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis ▿"

    Article Title: Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00988-07

    PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with NcoI (lane 4) or SmaI (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).
    Figure Legend Snippet: PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with NcoI (lane 4) or SmaI (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).

    Techniques Used: Southern Blot, Staining, Western Blot, Plasmid Preparation, Marker

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    New England Biolabs ncoi restriction enzymes
    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with <t>NcoI</t> and <t>XbaI</t> restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.
    Ncoi Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs restriction enzymes ncoi
    The Double Digestion Patterns of the pNZ8148-HPV16-optiE7 Shuttle Plasmid via <t>NcoI</t> and <t>SacI</t> Restriction Endonuclease
    Restriction Enzymes Ncoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ncoi
    PAGE analysis for determining the effects of DNA lesions on transcription. ( a ) Sample processing for restriction digestion using <t>NcoI</t> and <t>SfaNI</t> and postlabeling assay (p* indicates a 32 P-labeled phosphate group). The recognition sequences for restriction
    Ncoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Journal: Scientific Reports

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

    doi: 10.1038/s41598-018-30792-0

    Figure Lengend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Article Snippet: For the simultaneous expression of Nampt and PRPP synthetase a bicistronic vector was constructed by PCR amplifying the baPrs sequence (Q5 High-Fidelity 2X Master Mix, NEB) from pUC57-Kan plasmid with M13 forward and reverse primers, double digestion with XbaI and NcoI restriction enzymes (NEB) of PCR product and pET28a-hdNAdV plasmid, followed by the separation of the desired DNA fragments on 1.5% agarose gel electrophoresis , purification from gel (using Wizard SV Gel and PCR Clean-Up System, Promega, Madison, USA) of desired bands (pET28a-hdNadV and baPrs) followed by ligation with T4 DNA ligase (NEB) (as shown in Fig. ).

    Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay

    The Double Digestion Patterns of the pNZ8148-HPV16-optiE7 Shuttle Plasmid via NcoI and SacI Restriction Endonuclease

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    Article Title: Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363

    doi: 10.22034/APJCP.2017.18.3.783

    Figure Lengend Snippet: The Double Digestion Patterns of the pNZ8148-HPV16-optiE7 Shuttle Plasmid via NcoI and SacI Restriction Endonuclease

    Article Snippet: Construction of shuttle vector The optimized E7 gene, encoding the E7 oncoprotein from HPV 16, was obtained as a 291 bp DNA fragment by digesting plasmid PMD18 with restriction enzymes NcoI and SacI (New England Biolabs).

    Techniques: Plasmid Preparation

    mNET-seq Profiles for PKM Alternative Splicing after PTBP1 Depletion, Related to Figure 4 (A) PKM exons 8–11 are illustrated. Exon 9 (green) and exon 10 (orange) are mutually exclusive. PCR primers indicated as black triangles. RT-PCR products were digested with indicated exon-specific restriction enzyme (NcoI or PstI). (B) mNET-seq data around mutually exclusive exons 9 and 10 of PKM . mNET-seq/S5P signals at 3′ end of exon 9 and exon 10 are shown by green and orange arrows, respectively. Transcription direction, black arrow. (C) Western blot of PTBP1 and tubulin from siPTBP1-treated HeLa cells. (D) PKM RT-PCR products from PTBP1-depleted HeLa nuclear RNA were digested by NcoI. (E) mNET-seq/S5P data over mutually exclusive exons 9 and 10 of PKM from siLuc and siPTBP1-treated HeLa cells (top), followed by expanded view around 5′SS of introns 10 and 11. S5P-peaks at 3′ ends of exons, orange asterisks. Transcription direction, black arrows.

    Journal: Cell

    Article Title: Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing

    doi: 10.1016/j.cell.2015.03.027

    Figure Lengend Snippet: mNET-seq Profiles for PKM Alternative Splicing after PTBP1 Depletion, Related to Figure 4 (A) PKM exons 8–11 are illustrated. Exon 9 (green) and exon 10 (orange) are mutually exclusive. PCR primers indicated as black triangles. RT-PCR products were digested with indicated exon-specific restriction enzyme (NcoI or PstI). (B) mNET-seq data around mutually exclusive exons 9 and 10 of PKM . mNET-seq/S5P signals at 3′ end of exon 9 and exon 10 are shown by green and orange arrows, respectively. Transcription direction, black arrow. (C) Western blot of PTBP1 and tubulin from siPTBP1-treated HeLa cells. (D) PKM RT-PCR products from PTBP1-depleted HeLa nuclear RNA were digested by NcoI. (E) mNET-seq/S5P data over mutually exclusive exons 9 and 10 of PKM from siLuc and siPTBP1-treated HeLa cells (top), followed by expanded view around 5′SS of introns 10 and 11. S5P-peaks at 3′ ends of exons, orange asterisks. Transcription direction, black arrows.

    Article Snippet: BRD2_ex4_FW: 5′-CAAAATTATAAAACAGCCTATGGACATG-3′ BRD2_ex5_RV: 5′-TTTTCCAGCGTTTGTGCCATTAGGA-3′ BZW1_ex3_FW: 5′-TACCGTCGATATGCAGAAACA-3′ BZW1_ex4_RV: 5′-GAGCAAATGCTTGCATGGTCT-3′ PKMex8_Fw: 5′-GATGGAGCCGACTGCATCATG-3′, PKMex11_Rv: 5′-ATTCCGGGTCACAGCAATGAT-3′ For PKM, PCR products were digested by either NcoI (NEB) or PstI (NEB) for 6 hr.

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    PAGE analysis for determining the effects of DNA lesions on transcription. ( a ) Sample processing for restriction digestion using NcoI and SfaNI and postlabeling assay (p* indicates a 32 P-labeled phosphate group). The recognition sequences for restriction

    Journal: Nature protocols

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo

    doi: 10.1038/nprot.2015.094

    Figure Lengend Snippet: PAGE analysis for determining the effects of DNA lesions on transcription. ( a ) Sample processing for restriction digestion using NcoI and SfaNI and postlabeling assay (p* indicates a 32 P-labeled phosphate group). The recognition sequences for restriction

    Article Snippet: SfaNI (New England BioLabs, cat. no. R0172S) NcoI (New England BioLabs, cat. no. R0193S) NotI (New England BioLabs, cat. no. R0189S) T7 RNA polymerase, supplied with 5× transcription buffer (Promega, cat. no. P2075) 100 mM DTT (Promega, cat. no. P1171) 100 mM rATP (Promega, cat. no. E6011) 100 mM rUTP (Promega, cat. no. E6021) 100 mM rGTP (Promega, cat. no. E6031) 100 mM rCTP (Promega, cat. no. E6041) RNase inhibitor (New England BioLabs, cat. no. M0307S) HeLaScribe nuclear extract in vitro transcription system, supplied with HeLa nuclear extract, 1× transcription buffer and HeLa extract stop solution (Promega, cat. no. E3110) pGEM-T vector (Promega, cat. no. A3600) Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668-019) DMEM (Life Technologies, cat. no. 11995-073) FBS (Life Technologies, cat. no. 16000-044) Penicillin-streptomycin solution (American Type Culture Collection, cat. no. 30-2300) 0.25% (wt/vol) trypsin-EDTA (Life Technologies, cat. no. 25200-056) 10× PBS solution (VWR, cat. no. 97064-158) E.Z.N.A.

    Techniques: Polyacrylamide Gel Electrophoresis, Postlabeling Assay, Labeling