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    NcoI
    NcoI 5 000 units
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    Average 99 stars, based on 169 article reviews
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    Images

    1) Product Images from "A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription"

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1046

    A schematic diagram depicts the CTAB assay system “X” indicates an S -cdA, S -cdG, dA or dG, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites. Run-off RNA and truncated RNA resulting from transcription arrest at a lesion site are indicated. P1 represents a gene-specific primer used for reverse transcribing the run-off transcripts. Only the wild-type sequence of the RT-PCR products for the lesion-containing genome is shown. The RT-PCR products arising from the competitor genome are not depicted. The RT-PCR products were digested with two restriction enzymes (NcoI and AseI) and then subjected to PAGE or LC-MS/MS analyses. The cleavage sites of NcoI and AseI are designated with arrows.
    Figure Legend Snippet: A schematic diagram depicts the CTAB assay system “X” indicates an S -cdA, S -cdG, dA or dG, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites. Run-off RNA and truncated RNA resulting from transcription arrest at a lesion site are indicated. P1 represents a gene-specific primer used for reverse transcribing the run-off transcripts. Only the wild-type sequence of the RT-PCR products for the lesion-containing genome is shown. The RT-PCR products arising from the competitor genome are not depicted. The RT-PCR products were digested with two restriction enzymes (NcoI and AseI) and then subjected to PAGE or LC-MS/MS analyses. The cleavage sites of NcoI and AseI are designated with arrows.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    2) Product Images from "Fluorescence-based methods for measuring target interference by CRISPR-Cas systems"

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2018.10.027

    Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.
    Figure Legend Snippet: Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.

    Techniques Used: CRISPR, Sequencing, Mutagenesis, Labeling

    3) Product Images from "Mechanism of Action of a Distal NF-?B-Dependent Enhancer"

    Article Title: Mechanism of Action of a Distal NF-?B-Dependent Enhancer

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00271-06

    An interaction between the distal and proximal regulatory regions of the MCP - 1 gene forms following TNF stimulation. (A) A schematic representation of the 3C assay and the architecture of the MCP - 1 promoter with cis regulatory sites with selected restriction sites and primer positions (P1 through P4) is shown. In the 3C assay, cells were fixed with formaldehyde and chromatin was isolated and digested with NcoI. Following inactivation of the restriction enzyme, the sample was diluted and subjected to DNA ligation such that only intramolecular ligations would be preferred. The cross-links were then reversed, and the DNA was purified. PCR primers P1 and P2 were used to detect the formation of the novel ligated 3C product. Primers P3 and P4 were used to verify that similar levels of MCP - 1 DNA were present in the assay and that the DNA was amplifiable. Products from the 3C assay were examined on agarose gels stained with ethidium bromide. (B) NIH 3T3 cells treated in the absence (−) (lanes 1 to 4) or presence (+) (lanes 5 to 8) of TNF for 2 h were processed in the above 3C assay with and without formaldehyde (CH 2 O) or DNA ligase, as indicated, to control for specificity of the protocol. M, DNA marker. (C) Purified mouse genomic DNA was processed in the 3C assay and demonstrates that the unique PCR product cannot form with naked DNA templates. (D) KpnI cleaves the PCR product generated by the 3C assay, producing the anticipated DNA fragments as shown on the agarose gel. (E) PCR amplification of the 3C product and the non-3C product display similar relative efficiencies. PCR products generated from undigested genomic DNA (1,207 bp) and DNA from a 3C assay (713 bp) were quantitated by fluorometry and used as templates in PCRs to determine if there were major differences in their PCR efficiency. DNA templates (40 pg, 8 pg, and 1.6 pg) were amplified by primers P1 and P2 under identical PCR conditions. (F) Chromatin isolated from TNF-treated or control cells fixed with formaldehyde was subjected to NcoI digestion as indicated. Following deproteination, the DNA was purified and analyzed by PCR using the indicated primers. Because the ligation step of the 3C assay was not performed, P1/P2, P1/P6, and P2/P5 PCR products can only be observed when NcoI was omitted from the reaction, indicating that the chromatin is accessible to restriction digestion even in the absence of TNF.
    Figure Legend Snippet: An interaction between the distal and proximal regulatory regions of the MCP - 1 gene forms following TNF stimulation. (A) A schematic representation of the 3C assay and the architecture of the MCP - 1 promoter with cis regulatory sites with selected restriction sites and primer positions (P1 through P4) is shown. In the 3C assay, cells were fixed with formaldehyde and chromatin was isolated and digested with NcoI. Following inactivation of the restriction enzyme, the sample was diluted and subjected to DNA ligation such that only intramolecular ligations would be preferred. The cross-links were then reversed, and the DNA was purified. PCR primers P1 and P2 were used to detect the formation of the novel ligated 3C product. Primers P3 and P4 were used to verify that similar levels of MCP - 1 DNA were present in the assay and that the DNA was amplifiable. Products from the 3C assay were examined on agarose gels stained with ethidium bromide. (B) NIH 3T3 cells treated in the absence (−) (lanes 1 to 4) or presence (+) (lanes 5 to 8) of TNF for 2 h were processed in the above 3C assay with and without formaldehyde (CH 2 O) or DNA ligase, as indicated, to control for specificity of the protocol. M, DNA marker. (C) Purified mouse genomic DNA was processed in the 3C assay and demonstrates that the unique PCR product cannot form with naked DNA templates. (D) KpnI cleaves the PCR product generated by the 3C assay, producing the anticipated DNA fragments as shown on the agarose gel. (E) PCR amplification of the 3C product and the non-3C product display similar relative efficiencies. PCR products generated from undigested genomic DNA (1,207 bp) and DNA from a 3C assay (713 bp) were quantitated by fluorometry and used as templates in PCRs to determine if there were major differences in their PCR efficiency. DNA templates (40 pg, 8 pg, and 1.6 pg) were amplified by primers P1 and P2 under identical PCR conditions. (F) Chromatin isolated from TNF-treated or control cells fixed with formaldehyde was subjected to NcoI digestion as indicated. Following deproteination, the DNA was purified and analyzed by PCR using the indicated primers. Because the ligation step of the 3C assay was not performed, P1/P2, P1/P6, and P2/P5 PCR products can only be observed when NcoI was omitted from the reaction, indicating that the chromatin is accessible to restriction digestion even in the absence of TNF.

    Techniques Used: Isolation, DNA Ligation, Purification, Polymerase Chain Reaction, Staining, Marker, Generated, Agarose Gel Electrophoresis, Amplification, Ligation

    4) Product Images from "Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells"

    Article Title: Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139176

    Agarose gel electrophoresis analysis of 3 combinant expression plasmid. The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β 2 -AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.
    Figure Legend Snippet: Agarose gel electrophoresis analysis of 3 combinant expression plasmid. The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β 2 -AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.

    Techniques Used: Agarose Gel Electrophoresis, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Recombinant, Electrophoresis, Amplification

    5) 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

    6) Product Images from "β-nicotinamide mononucleotide (NMN) production in Escherichia coli"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30792-0

    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.
    Figure Legend 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.

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

    7) Product Images from "The antisense inhibition of accA suppressed luxS expression that is essential for quorum sensing, biofilm formation, and virulence"

    Article Title: The antisense inhibition of accA suppressed luxS expression that is essential for quorum sensing, biofilm formation, and virulence

    Journal: bioRxiv

    doi: 10.1101/747980

    Plasmid Assembly and the Gene Silencing of accA Plasmid Assembly and Gene Silencing the PCR products and the PTasRNA expression vector of the plasmid pHN1257 were digested with the restriction enzymes XhoI (upstream) and NcoI (downstream). After the restriction digestion, the antisense PCR product was ligated into the IPTG-PTasRNA inducible vector of pHN1257 at the multiple cloning site. doi: 10.3390/ijms15022773 This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/ ). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958881/figure/f6-ijms-15-02773/
    Figure Legend Snippet: Plasmid Assembly and the Gene Silencing of accA Plasmid Assembly and Gene Silencing the PCR products and the PTasRNA expression vector of the plasmid pHN1257 were digested with the restriction enzymes XhoI (upstream) and NcoI (downstream). After the restriction digestion, the antisense PCR product was ligated into the IPTG-PTasRNA inducible vector of pHN1257 at the multiple cloning site. doi: 10.3390/ijms15022773 This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/ ). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958881/figure/f6-ijms-15-02773/

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Expressing, Clone Assay

    8) Product Images from "Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363"

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

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    doi: 10.22034/APJCP.2017.18.3.783

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

    Techniques Used: Plasmid Preparation

    9) Product Images from "A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription"

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1046

    A schematic diagram depicts the CTAB assay system “X” indicates an S -cdA, S -cdG, dA or dG, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites. Run-off RNA and truncated RNA resulting from transcription arrest at a lesion site are indicated. P1 represents a gene-specific primer used for reverse transcribing the run-off transcripts. Only the wild-type sequence of the RT-PCR products for the lesion-containing genome is shown. The RT-PCR products arising from the competitor genome are not depicted. The RT-PCR products were digested with two restriction enzymes (NcoI and AseI) and then subjected to PAGE or LC-MS/MS analyses. The cleavage sites of NcoI and AseI are designated with arrows.
    Figure Legend Snippet: A schematic diagram depicts the CTAB assay system “X” indicates an S -cdA, S -cdG, dA or dG, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites. Run-off RNA and truncated RNA resulting from transcription arrest at a lesion site are indicated. P1 represents a gene-specific primer used for reverse transcribing the run-off transcripts. Only the wild-type sequence of the RT-PCR products for the lesion-containing genome is shown. The RT-PCR products arising from the competitor genome are not depicted. The RT-PCR products were digested with two restriction enzymes (NcoI and AseI) and then subjected to PAGE or LC-MS/MS analyses. The cleavage sites of NcoI and AseI are designated with arrows.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    10) 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

    11) Product Images from "High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells"

    Article Title: High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-52

    Development of vectors for transient antibody expression in HEK293 cell lines. A ) Illustration of the structure of scFv-Fc and scFv-Fc-RNase constructs. B ) The vector pCMV-hIgG1Fc-XP based on pCMV/myc/ER backbone which contains a neomycin phosphotransferase (neo) selection marker under control of a simian virus (SV) 40 promoter (with SV40 ori) and poly A (pA) signal. The antibody gene expression cassette consists of a modified untranslated 5’ region and ribosomal binding site (modified KOZAK sequence), mouse heavy Ig chain leader comprising two exons (E1 and 2, light blue) interrupted by one intron (I) for stabilized expression, followed by a spacer sequence (grey) flanked unique NcoI/NotI sites for single step in frame cloning and fusion with following constant hinge, CH2 and CH3 regions of human IgG1 Fc gene fragment (dark blue). The gene expression cassette is driven by a CMV promoter and terminated by bovine growth hormone (bGH) poly adenylation signal (pA). C ) pCMX2.5-hIgG1Fc-XP has a modified hinge without genetic upper hinge of the IgG1 heavy chain and optimized translation termination sequence at the 3’ of the Fc gene fragment. D ) pCMV-neo - and pCMV-oriP1 test vectors containing CD30 specific scFv-Fc or scFv-Fc-RNase were generated from the parental pCMV vectors [ 11 ] by deleting the complete neo expression cassette or replacing it by a short EBV oriP variant. E ) The expression vector pCSE2.5-hIgG1Fc-XP was newly generated and comprises all advantages for transient expression of the previous vectors including the optimized hinge and stop sequences of pCMX2.5, no selection marker expression cassette, minimal SV40 ori and short EBV oriP for episomal replication in SV40-large T antigen or EBNA1 positive mammalian expression cell lines.
    Figure Legend Snippet: Development of vectors for transient antibody expression in HEK293 cell lines. A ) Illustration of the structure of scFv-Fc and scFv-Fc-RNase constructs. B ) The vector pCMV-hIgG1Fc-XP based on pCMV/myc/ER backbone which contains a neomycin phosphotransferase (neo) selection marker under control of a simian virus (SV) 40 promoter (with SV40 ori) and poly A (pA) signal. The antibody gene expression cassette consists of a modified untranslated 5’ region and ribosomal binding site (modified KOZAK sequence), mouse heavy Ig chain leader comprising two exons (E1 and 2, light blue) interrupted by one intron (I) for stabilized expression, followed by a spacer sequence (grey) flanked unique NcoI/NotI sites for single step in frame cloning and fusion with following constant hinge, CH2 and CH3 regions of human IgG1 Fc gene fragment (dark blue). The gene expression cassette is driven by a CMV promoter and terminated by bovine growth hormone (bGH) poly adenylation signal (pA). C ) pCMX2.5-hIgG1Fc-XP has a modified hinge without genetic upper hinge of the IgG1 heavy chain and optimized translation termination sequence at the 3’ of the Fc gene fragment. D ) pCMV-neo - and pCMV-oriP1 test vectors containing CD30 specific scFv-Fc or scFv-Fc-RNase were generated from the parental pCMV vectors [ 11 ] by deleting the complete neo expression cassette or replacing it by a short EBV oriP variant. E ) The expression vector pCSE2.5-hIgG1Fc-XP was newly generated and comprises all advantages for transient expression of the previous vectors including the optimized hinge and stop sequences of pCMX2.5, no selection marker expression cassette, minimal SV40 ori and short EBV oriP for episomal replication in SV40-large T antigen or EBNA1 positive mammalian expression cell lines.

    Techniques Used: Expressing, Construct, Plasmid Preparation, Selection, Marker, Modification, Binding Assay, Sequencing, Clone Assay, Generated, Variant Assay

    12) 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

    13) 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

    14) 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

    15) Product Images from "β-nicotinamide mononucleotide (NMN) production in Escherichia coli"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30792-0

    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.
    Figure Legend 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.

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

    16) 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

    17) Product Images from "The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics"

    Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.03439004

    Concept of the asymmetric directional T-vector. ( A ) Construction of pGEX-4T3-PRESAT and direct cloning of PCR product in pGEX-4T3-PRESAT. ( B ) The schematic representation of the ORF selection method using potential restriction enzyme site. The figure illustrates the case in which NcoI is chosen as the second restriction enzyme for selection. The rear PCR primer is designed with 5′-GG at the 5′ end, so that only the ligated plasmid with insert in the reverse orientation will have the NcoI site at the TA-cloning position. For NdeI selection, the rear primer with 5′-ATG is used instead of the 5′-GG primer.
    Figure Legend Snippet: Concept of the asymmetric directional T-vector. ( A ) Construction of pGEX-4T3-PRESAT and direct cloning of PCR product in pGEX-4T3-PRESAT. ( B ) The schematic representation of the ORF selection method using potential restriction enzyme site. The figure illustrates the case in which NcoI is chosen as the second restriction enzyme for selection. The rear PCR primer is designed with 5′-GG at the 5′ end, so that only the ligated plasmid with insert in the reverse orientation will have the NcoI site at the TA-cloning position. For NdeI selection, the rear primer with 5′-ATG is used instead of the 5′-GG primer.

    Techniques Used: Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Selection, TA Cloning

    18) Product Images from "The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism"

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0914845107

    Protein and transcript expression patterns of pyruvate kinase M1/M2 isoforms in cells and tissues. ( A ) Total adult-mouse organ/tissue homogenates were used for Western blotting with the indicated antibodies. rM1 and rM2: Flag-tagged purified recombinant human PK isoforms. ( B ) Total cell lysates of five human cancer-cell lines were used for Western blotting with the indicated antibodies. ( C ) Primers annealing to exon 8 and exon 11, respectively, were used to amplify mouse or human PK-M transcripts. The alternative exons that encode the distinctive segments of PK-M1 and PK-M2 are indicated in ( black ) and ( gray ), respectively. To distinguish between PK-M1 (exon 9 included) and PK-M2 (exon 10 included) isoforms, the PCR products were cleaved with NcoI, PstI, or both. There is an additional NcoI site (*) 11 bp away from the 3′ end of mouse exon 11. ( D ) Mouse organs were freshly dissected and perfused with saline. Total RNA was analyzed by radioactive RT-PCR followed by digestion with NcoI (N), PstI (P), or both enzymes (NP), plus an uncut control (U). Numbered bands are as follows: 1: Uncut M1 (502 bp); 2: uncut M2 (502 bp); 2*: M2 cleaved with NcoI in exon 11 (491 bp); 3: Pst1-cleaved M2 5’ fragment (286 bp); 4: NcoI-cleaved M1 5′ fragment (245 bp); 5: NcoI-cleaved M1 3’ fragment (240 bp); 6: PstI-cleaved M2 3’ fragment (216 bp); 7: PstI + NcoI-cleaved M2 3’ fragment (205 bp). The %M1 was quantified from band 1 (M1) and bands 3 and 6 (M2) in each P lane. ( E ) RT-PCR and restriction digest analysis of total RNA from the indicated human cell lines. The bands are numbered as for the mouse RT-PCR products, but the sizes are different because of the positions of the primers; the sizes are as follows: 1: 398 bp; 2: 398 bp; 3: 185 bp; 4: 144 bp; 5: 248 bp; 6: 213 bp. Note that the PK-M1 bands in the P and U lanes migrate slightly above the PK-M2 bands, which is also the case for the mouse PK-M1 transcripts.
    Figure Legend Snippet: Protein and transcript expression patterns of pyruvate kinase M1/M2 isoforms in cells and tissues. ( A ) Total adult-mouse organ/tissue homogenates were used for Western blotting with the indicated antibodies. rM1 and rM2: Flag-tagged purified recombinant human PK isoforms. ( B ) Total cell lysates of five human cancer-cell lines were used for Western blotting with the indicated antibodies. ( C ) Primers annealing to exon 8 and exon 11, respectively, were used to amplify mouse or human PK-M transcripts. The alternative exons that encode the distinctive segments of PK-M1 and PK-M2 are indicated in ( black ) and ( gray ), respectively. To distinguish between PK-M1 (exon 9 included) and PK-M2 (exon 10 included) isoforms, the PCR products were cleaved with NcoI, PstI, or both. There is an additional NcoI site (*) 11 bp away from the 3′ end of mouse exon 11. ( D ) Mouse organs were freshly dissected and perfused with saline. Total RNA was analyzed by radioactive RT-PCR followed by digestion with NcoI (N), PstI (P), or both enzymes (NP), plus an uncut control (U). Numbered bands are as follows: 1: Uncut M1 (502 bp); 2: uncut M2 (502 bp); 2*: M2 cleaved with NcoI in exon 11 (491 bp); 3: Pst1-cleaved M2 5’ fragment (286 bp); 4: NcoI-cleaved M1 5′ fragment (245 bp); 5: NcoI-cleaved M1 3’ fragment (240 bp); 6: PstI-cleaved M2 3’ fragment (216 bp); 7: PstI + NcoI-cleaved M2 3’ fragment (205 bp). The %M1 was quantified from band 1 (M1) and bands 3 and 6 (M2) in each P lane. ( E ) RT-PCR and restriction digest analysis of total RNA from the indicated human cell lines. The bands are numbered as for the mouse RT-PCR products, but the sizes are different because of the positions of the primers; the sizes are as follows: 1: 398 bp; 2: 398 bp; 3: 185 bp; 4: 144 bp; 5: 248 bp; 6: 213 bp. Note that the PK-M1 bands in the P and U lanes migrate slightly above the PK-M2 bands, which is also the case for the mouse PK-M1 transcripts.

    Techniques Used: Expressing, Western Blot, Purification, Recombinant, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    19) 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

    20) Product Images from "Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV ▿Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV ▿ ‡"

    Article Title: Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV ▿Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV ▿ ‡

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01188-06

    Targeted disruption of the murine SNEV locus. (A) Homologous recombination of the targeting construct with the wild-type locus leads to a loss-of-function targeted allele. Small boxes indicate exons; the start and stop codons lie in the first and last exons, respectively. The first six exons were replaced by a lacZ reporter (LacZ) and a neomycin resistance cassette (Neo), both containing poly(A) signals and the latter also carrying a Rous sarcoma virus promoter. The DTA cassette was used as a negative selection marker. Restriction sites for NcoI (n) and XhoI (x) and PCR screening primer binding sites (short arrows) are shown. (B) Nested PCR screening of representative ES cell clones yielded a 1,546-bp product, if the locus was correctly targeted. m, marker lane; ntc, no template negative control; mock, mock vector positive control. (C) Southern blotting using a radioactively labeled probe was performed to confirm the correct integration of the targeting construct. Digestion with restriction enzyme NcoI or XhoI yields fragments of 2.9 and 5.5 kbp for the targeted allele and 2.2 and 2.1 kbp for the wild-type allele, respectively. (D) Genotyping of mice and embryos by a three-primer PCR yielded a product of 1,750 bp for the knockout allele and one of 1,431 bp for the wild-type allele. (E) Genotyping of blastocysts by nested PCR. Amplification with wild-type primers (WT-PCR) yielded a 1,111-bp product for the wild-type allele. With neomycin-specific sense primers, a product of 1,554 bp was amplified from the targeted allele (knockout [KO]-PCR).
    Figure Legend Snippet: Targeted disruption of the murine SNEV locus. (A) Homologous recombination of the targeting construct with the wild-type locus leads to a loss-of-function targeted allele. Small boxes indicate exons; the start and stop codons lie in the first and last exons, respectively. The first six exons were replaced by a lacZ reporter (LacZ) and a neomycin resistance cassette (Neo), both containing poly(A) signals and the latter also carrying a Rous sarcoma virus promoter. The DTA cassette was used as a negative selection marker. Restriction sites for NcoI (n) and XhoI (x) and PCR screening primer binding sites (short arrows) are shown. (B) Nested PCR screening of representative ES cell clones yielded a 1,546-bp product, if the locus was correctly targeted. m, marker lane; ntc, no template negative control; mock, mock vector positive control. (C) Southern blotting using a radioactively labeled probe was performed to confirm the correct integration of the targeting construct. Digestion with restriction enzyme NcoI or XhoI yields fragments of 2.9 and 5.5 kbp for the targeted allele and 2.2 and 2.1 kbp for the wild-type allele, respectively. (D) Genotyping of mice and embryos by a three-primer PCR yielded a product of 1,750 bp for the knockout allele and one of 1,431 bp for the wild-type allele. (E) Genotyping of blastocysts by nested PCR. Amplification with wild-type primers (WT-PCR) yielded a 1,111-bp product for the wild-type allele. With neomycin-specific sense primers, a product of 1,554 bp was amplified from the targeted allele (knockout [KO]-PCR).

    Techniques Used: Homologous Recombination, Construct, Selection, Marker, Polymerase Chain Reaction, Binding Assay, Nested PCR, Clone Assay, Negative Control, Plasmid Preparation, Positive Control, Southern Blot, Labeling, Mouse Assay, Knock-Out, Amplification

    21) 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

    22) Product Images from "Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy"

    Article Title: Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2019-000299

    Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are four restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3′LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5′ overhangs: 5′-CATG-3′, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3′LTR and the 3′LTR/distal transgene junction, indicated by continuous arrows. This is followed by nested PCR, indicated by dotted arrows, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3′ end and a 5′overhang (5′-ATG-3′). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3′ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3′LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a low annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3′LTR primer and the longer cassette strand.
    Figure Legend Snippet: Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are four restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3′LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5′ overhangs: 5′-CATG-3′, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3′LTR and the 3′LTR/distal transgene junction, indicated by continuous arrows. This is followed by nested PCR, indicated by dotted arrows, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3′ end and a 5′overhang (5′-ATG-3′). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3′ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3′LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a low annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3′LTR primer and the longer cassette strand.

    Techniques Used: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Inverse PCR, Nested PCR, Ligation, Blocking Assay

    23) Product Images from "Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds"

    Article Title: Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-10-183

    Southern blot analysis of GS2 genes in Medicago truncatula . A. Schematic representation of BAC mth2-53e90 indicating the position of MtGS2a (AC148968-43) and MtGS2b (AC 1448968-42) and the restriction sites relevant for the southern analysis. The position of the probe used for the Southern analysis is also indicated. B. Southern hybridization of M. truncatula J5 genomic DNA and BAC mth2-53e90 DNA. 20 μg of genomic DNA and 5 μg of BAC DNA were digested with BgLII (1), NcoI (2) and EcoRV (3) and probed with a 260 bp DNA fragment corresponding to part of the 5'UTR and coding sequence of GS2a cDNA.
    Figure Legend Snippet: Southern blot analysis of GS2 genes in Medicago truncatula . A. Schematic representation of BAC mth2-53e90 indicating the position of MtGS2a (AC148968-43) and MtGS2b (AC 1448968-42) and the restriction sites relevant for the southern analysis. The position of the probe used for the Southern analysis is also indicated. B. Southern hybridization of M. truncatula J5 genomic DNA and BAC mth2-53e90 DNA. 20 μg of genomic DNA and 5 μg of BAC DNA were digested with BgLII (1), NcoI (2) and EcoRV (3) and probed with a 260 bp DNA fragment corresponding to part of the 5'UTR and coding sequence of GS2a cDNA.

    Techniques Used: Southern Blot, BAC Assay, Hybridization, Sequencing

    24) Product Images from "Effects of Tet-mediated Oxidation Products of 5-Methylcytosine on DNA Transcription in vitro and in Mammalian Cells"

    Article Title: Effects of Tet-mediated Oxidation Products of 5-Methylcytosine on DNA Transcription in vitro and in Mammalian Cells

    Journal: Scientific Reports

    doi: 10.1038/srep07052

    Experimental outline. (a) Schematic diagrams showing the procedures for the construction of the plasmids harboring a site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC. (b) CTAB assay for assessing the impact of the oxidized 5-mC derivatives on DNA transcription. “X” indicates 5-HmC, 5-FoC or 5-CaC, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The +1 transcription start sites are indicated by arrowheads. Oxidized 5-mC derivative-bearing or unmodified control plasmids were mixed individually with the competitor genome as DNA templates for in vitro or in vivo transcription. Although truncated RNA may be produced when transcription arrests at or near a lesion site, only run-off RNA is shown and used for RT-PCR. Among the RT-PCR products, only the wild-type sequence arising from the cytosine derivative-containing vector is shown. The arrows indicate the cleavage sites of Nt.BstNBI, NcoI and SfaNI. The last two enzymes were used to digest the RT-PCR products for subsequent PAGE and LC-MS/MS analyses.
    Figure Legend Snippet: Experimental outline. (a) Schematic diagrams showing the procedures for the construction of the plasmids harboring a site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC. (b) CTAB assay for assessing the impact of the oxidized 5-mC derivatives on DNA transcription. “X” indicates 5-HmC, 5-FoC or 5-CaC, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The +1 transcription start sites are indicated by arrowheads. Oxidized 5-mC derivative-bearing or unmodified control plasmids were mixed individually with the competitor genome as DNA templates for in vitro or in vivo transcription. Although truncated RNA may be produced when transcription arrests at or near a lesion site, only run-off RNA is shown and used for RT-PCR. Among the RT-PCR products, only the wild-type sequence arising from the cytosine derivative-containing vector is shown. The arrows indicate the cleavage sites of Nt.BstNBI, NcoI and SfaNI. The last two enzymes were used to digest the RT-PCR products for subsequent PAGE and LC-MS/MS analyses.

    Techniques Used: In Vitro, In Vivo, Produced, Reverse Transcription Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    25) 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

    26) Product Images from "Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA"

    Article Title: Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.23224

    S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.
    Figure Legend Snippet: S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Molecular Weight

    27) Product Images from "Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform"

    Article Title: Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform

    Journal: bioRxiv

    doi: 10.1101/833897

    Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are 4 restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3’LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5’ overhangs: 5’-CATG-3’, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3’LTR and the 3’LTR/distal transgene junction. This is followed by nested PCR, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3’ end and a 5’overhang (5’-ATG-3’). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3’ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3’LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a high annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3’LTR primer and the longer cassette strand.
    Figure Legend Snippet: Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are 4 restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3’LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5’ overhangs: 5’-CATG-3’, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3’LTR and the 3’LTR/distal transgene junction. This is followed by nested PCR, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3’ end and a 5’overhang (5’-ATG-3’). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3’ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3’LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a high annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3’LTR primer and the longer cassette strand.

    Techniques Used: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Inverse PCR, Nested PCR, Ligation, Blocking Assay

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    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
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    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
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    A schematic diagram depicts the CTAB assay system “X” indicates an S -cdA, S <t>-cdG,</t> dA or dG, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites. Run-off RNA and truncated RNA resulting from transcription arrest at a lesion site are indicated. P1 represents a gene-specific primer used for reverse transcribing the run-off transcripts. Only the wild-type sequence of the RT-PCR products for the lesion-containing genome is shown. The RT-PCR products arising from the competitor genome are not depicted. The RT-PCR products were digested with two restriction enzymes <t>(NcoI</t> and AseI) and then subjected to PAGE or LC-MS/MS analyses. The cleavage sites of NcoI and AseI are designated with arrows.
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    A schematic diagram depicts the CTAB assay system “X” indicates an S -cdA, S -cdG, dA or dG, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites. Run-off RNA and truncated RNA resulting from transcription arrest at a lesion site are indicated. P1 represents a gene-specific primer used for reverse transcribing the run-off transcripts. Only the wild-type sequence of the RT-PCR products for the lesion-containing genome is shown. The RT-PCR products arising from the competitor genome are not depicted. The RT-PCR products were digested with two restriction enzymes (NcoI and AseI) and then subjected to PAGE or LC-MS/MS analyses. The cleavage sites of NcoI and AseI are designated with arrows.

    Journal: Nature chemical biology

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription

    doi: 10.1038/nchembio.1046

    Figure Lengend Snippet: A schematic diagram depicts the CTAB assay system “X” indicates an S -cdA, S -cdG, dA or dG, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites. Run-off RNA and truncated RNA resulting from transcription arrest at a lesion site are indicated. P1 represents a gene-specific primer used for reverse transcribing the run-off transcripts. Only the wild-type sequence of the RT-PCR products for the lesion-containing genome is shown. The RT-PCR products arising from the competitor genome are not depicted. The RT-PCR products were digested with two restriction enzymes (NcoI and AseI) and then subjected to PAGE or LC-MS/MS analyses. The cleavage sites of NcoI and AseI are designated with arrows.

    Article Snippet: LC-MS/MS analysis In order to identify the transcription products of S -cdA and S -cdG using LC-MS/MS, RT-PCR products were treated with 50 U NcoI and 20 U shrimp alkaline phosphatase in 250 μL NEB buffer 3 at 37°C for 2 h, followed by heating at 80°C for 20 min. To the resulting solution was added 50 U of AseI, and the reaction mixture was incubated at 37°C for 1 h, followed by extraction once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v).

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.

    Journal: Methods in enzymology

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    doi: 10.1016/bs.mie.2018.10.027

    Figure Lengend Snippet: Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.

    Article Snippet: T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega)

    Techniques: CRISPR, Sequencing, Mutagenesis, Labeling

    An interaction between the distal and proximal regulatory regions of the MCP - 1 gene forms following TNF stimulation. (A) A schematic representation of the 3C assay and the architecture of the MCP - 1 promoter with cis regulatory sites with selected restriction sites and primer positions (P1 through P4) is shown. In the 3C assay, cells were fixed with formaldehyde and chromatin was isolated and digested with NcoI. Following inactivation of the restriction enzyme, the sample was diluted and subjected to DNA ligation such that only intramolecular ligations would be preferred. The cross-links were then reversed, and the DNA was purified. PCR primers P1 and P2 were used to detect the formation of the novel ligated 3C product. Primers P3 and P4 were used to verify that similar levels of MCP - 1 DNA were present in the assay and that the DNA was amplifiable. Products from the 3C assay were examined on agarose gels stained with ethidium bromide. (B) NIH 3T3 cells treated in the absence (−) (lanes 1 to 4) or presence (+) (lanes 5 to 8) of TNF for 2 h were processed in the above 3C assay with and without formaldehyde (CH 2 O) or DNA ligase, as indicated, to control for specificity of the protocol. M, DNA marker. (C) Purified mouse genomic DNA was processed in the 3C assay and demonstrates that the unique PCR product cannot form with naked DNA templates. (D) KpnI cleaves the PCR product generated by the 3C assay, producing the anticipated DNA fragments as shown on the agarose gel. (E) PCR amplification of the 3C product and the non-3C product display similar relative efficiencies. PCR products generated from undigested genomic DNA (1,207 bp) and DNA from a 3C assay (713 bp) were quantitated by fluorometry and used as templates in PCRs to determine if there were major differences in their PCR efficiency. DNA templates (40 pg, 8 pg, and 1.6 pg) were amplified by primers P1 and P2 under identical PCR conditions. (F) Chromatin isolated from TNF-treated or control cells fixed with formaldehyde was subjected to NcoI digestion as indicated. Following deproteination, the DNA was purified and analyzed by PCR using the indicated primers. Because the ligation step of the 3C assay was not performed, P1/P2, P1/P6, and P2/P5 PCR products can only be observed when NcoI was omitted from the reaction, indicating that the chromatin is accessible to restriction digestion even in the absence of TNF.

    Journal: Molecular and Cellular Biology

    Article Title: Mechanism of Action of a Distal NF-?B-Dependent Enhancer

    doi: 10.1128/MCB.00271-06

    Figure Lengend Snippet: An interaction between the distal and proximal regulatory regions of the MCP - 1 gene forms following TNF stimulation. (A) A schematic representation of the 3C assay and the architecture of the MCP - 1 promoter with cis regulatory sites with selected restriction sites and primer positions (P1 through P4) is shown. In the 3C assay, cells were fixed with formaldehyde and chromatin was isolated and digested with NcoI. Following inactivation of the restriction enzyme, the sample was diluted and subjected to DNA ligation such that only intramolecular ligations would be preferred. The cross-links were then reversed, and the DNA was purified. PCR primers P1 and P2 were used to detect the formation of the novel ligated 3C product. Primers P3 and P4 were used to verify that similar levels of MCP - 1 DNA were present in the assay and that the DNA was amplifiable. Products from the 3C assay were examined on agarose gels stained with ethidium bromide. (B) NIH 3T3 cells treated in the absence (−) (lanes 1 to 4) or presence (+) (lanes 5 to 8) of TNF for 2 h were processed in the above 3C assay with and without formaldehyde (CH 2 O) or DNA ligase, as indicated, to control for specificity of the protocol. M, DNA marker. (C) Purified mouse genomic DNA was processed in the 3C assay and demonstrates that the unique PCR product cannot form with naked DNA templates. (D) KpnI cleaves the PCR product generated by the 3C assay, producing the anticipated DNA fragments as shown on the agarose gel. (E) PCR amplification of the 3C product and the non-3C product display similar relative efficiencies. PCR products generated from undigested genomic DNA (1,207 bp) and DNA from a 3C assay (713 bp) were quantitated by fluorometry and used as templates in PCRs to determine if there were major differences in their PCR efficiency. DNA templates (40 pg, 8 pg, and 1.6 pg) were amplified by primers P1 and P2 under identical PCR conditions. (F) Chromatin isolated from TNF-treated or control cells fixed with formaldehyde was subjected to NcoI digestion as indicated. Following deproteination, the DNA was purified and analyzed by PCR using the indicated primers. Because the ligation step of the 3C assay was not performed, P1/P2, P1/P6, and P2/P5 PCR products can only be observed when NcoI was omitted from the reaction, indicating that the chromatin is accessible to restriction digestion even in the absence of TNF.

    Article Snippet: The cross-linked DNA was digested overnight with 500 to 800 units NcoI restriction enzyme (New England Biolabs, Inc.).

    Techniques: Isolation, DNA Ligation, Purification, Polymerase Chain Reaction, Staining, Marker, Generated, Agarose Gel Electrophoresis, Amplification, Ligation

    Agarose gel electrophoresis analysis of 3 combinant expression plasmid. The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β 2 -AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.

    Journal: PLoS ONE

    Article Title: Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells

    doi: 10.1371/journal.pone.0139176

    Figure Lengend Snippet: Agarose gel electrophoresis analysis of 3 combinant expression plasmid. The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β 2 -AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.

    Article Snippet: The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA).

    Techniques: Agarose Gel Electrophoresis, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Recombinant, Electrophoresis, Amplification