noti restriction enzymes  (New England Biolabs)


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    Structured Review

    New England Biolabs noti restriction enzymes
    Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with <t>BamHI/NotI</t> after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).
    Noti Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation"

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    Journal: bioRxiv

    doi: 10.1101/2020.07.02.184408

    Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).
    Figure Legend Snippet: Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).

    Techniques Used: BAC Assay, Binding Assay, Sequencing, Ligation, Amplification, Plasmid Preparation, Clone Assay

    Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.
    Figure Legend Snippet: Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.

    Techniques Used: Transfection, HAC Assay, BAC Assay, Real-time Polymerase Chain Reaction, Clone Assay, Fluorescence In Situ Hybridization, Staining, Southern Blot

    Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).
    Figure Legend Snippet: Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).

    Techniques Used: Transformation Assay, Plasmid Preparation, Transfection, Marker

    2) Product Images from "Transmission of Yersinia pseudotuberculosis in the Pork Production Chain from Farm to Slaughterhouse ▿"

    Article Title: Transmission of Yersinia pseudotuberculosis in the Pork Production Chain from Farm to Slaughterhouse ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02664-07

    Four different NotI profiles (lanes 1 to 4) of Yersinia pseudotuberculosis strains obtained. M designates midrange PFGE markers.
    Figure Legend Snippet: Four different NotI profiles (lanes 1 to 4) of Yersinia pseudotuberculosis strains obtained. M designates midrange PFGE markers.

    Techniques Used:

    3) Product Images from "EphA2 targeting peptide tethered bioreducible poly(cystamine bisacrylamide - diamino hexane) for the delivery of therapeutic pCMV-RAE-1? to pancreatic islets"

    Article Title: EphA2 targeting peptide tethered bioreducible poly(cystamine bisacrylamide - diamino hexane) for the delivery of therapeutic pCMV-RAE-1? to pancreatic islets

    Journal: Journal of Controlled Release

    doi: 10.1016/j.jconrel.2011.10.022

    Digestion of pCMV-RAE-1γ with KpnI and NotI.
    Figure Legend Snippet: Digestion of pCMV-RAE-1γ with KpnI and NotI.

    Techniques Used:

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    New England Biolabs noti restriction enzymes
    Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with <t>BamHI/NotI</t> after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).
    Noti 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 xhoi restriction enzymes
    Tg ( DnRest ) rescues hearing, balancing, and hair cells in Srrm4 bv/bv mice. (A) Verification of the composition of Tg ( DnRest ) bacterial artificial chromosome (BAC) construct using Southern blotting. Left: Schematic of Tg ( DnRest ) BAC depicting the backbone of the BAC vector (white box), the genomic DNA insert (circle), the DnREST-encoding sequence (red arrow), the mouse Myo7a gene (gray arrow), <t>XhoI</t> sites (X), <t>NotI</t> sites (N), and the size of the BAC plus DnRest in kilobases (kb). Middle: SYBR Gold staining of electrophoretically separated Tg ( DnRest ) fragments generated by NotI (N) and XhoI (X) digestion of the Tg ( DnRest ) BAC. Right: Southern blot of NotI (N)- and XhoI (X)-digested Tg ( DnRest ) BAC with a probe from the DnREST-encoding region. Expected positions (tick lines) and sizes (in kb) of Southern blot hybridization products are indicated (n = 2 independent assays). (B) Expression levels of a REST-binding site (RE1) containing reporter gene (RE1-TK-Firefly-luc) in HEK293 cells that were co-transfected with the RE1-TK-Firefly-luc gene, a control gene (TK-Renilla-luc), and a vector encoding Flag-DnREST, non-tagged DnREST, or no insert (vector). Schemes at the top show compositions of RE1-TK-Firefly-luc and TK-Renilla-luc. Luc, luciferase; TK, thymidine kinase gene promoter. Each symbol represents the value for an independent assay. (C) Immunofluorescence signals for MYO7A (red) and Flag-DnREST (green, negative control for Fig 1C ) in cochlear and utricular sections from a WT mouse (P1) (n = 2 mice). Scale bar, 20 μm. (D) F-actin–stained organ of Corti preparations from WT and Tg ( DnRest ) mice at P28 (n = 3 mice per genotype). The rows of inner hair cells (IHCs) (arrowheads) and outer hair cells (OHCs) (vertical lines) are indicated. Scale bar, 20 μm. (E) F-actin–stained utricular macula from WT and Tg ( DnRest ) mice at P28 (n = 3 mice per genotype). Scale bar, 20 μm. (F) Thresholds of broadband click-evoked auditory brainstem responses in WT, Srrm4 bv/bv , and Tg ( DnRest ); Srrm4 bv/bv mice at P120. Each symbol represents the value for a single mouse; blue lines indicate means (one-way ANOVA P
    Xhoi Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs single cutting restriction noti hf enzyme
    <t>Supercoiled</t> plasmid standards overestimate w Ls ftsZ copy numbers. ( A ) L. sigmodontis MF were counted (range 17 to 600) and DNA extracted for w Ls ftsZ qPCR. Quantification was done using circular ( N = 19) or <t>NotI</t> linearized ( N = 30) plasmids. Linearized plasmids resulted in reduced copy numbers (20.6 vs. 200 w Ls ftsZ /MF). Lines indicate median and interquartile range. ( B ) Full projection of confocal images showing 2 Litomosoides sigmodontis microfilariae stained with DAPI (magenta) and an anti-WSP (yellow) monoclonal antibody. Clusters of Wolbachia are located in the proximal part of the posterior half of MF. A single Wolbachia bacterium is 0.8–1 µm in length; thus, there are not hundreds of endobacteria in a single MF. MF were observed with a 63X objective on a Leica SP5. The scale bar = 10 µM. ( C ) Variation in w Ls ftsZ copy number per L. sigmodontis MF is greater in samples with few MF. DNA was extracted from 5 to 60 L. sigmodontis MF and used for w Ls ftsZ qPCR. Linear regression (blue line) and 95% confidence bars (red dotted lines) are shown. ( D ) The qPCR is reproducible and stable. Interassay reproducibility was tested on DNA extracted from 18 preparations, each with ( A ) 50 MF and ( B ) 20 MF, and ( C ) 12 preparations with 50 MF from L. sigmodontis to calculate w Ls ftsZ copies/MF. Lines indicate median and interquartile range
    Single Cutting Restriction Noti Hf Enzyme, 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 noti restriction site
    Removal of E N-glycosylation does not affect ZIKV replication in mammalian cell culture. (A) Schematic diagram of the ZIKV genome organization and the construction of the MR766 infectious clone. Three cDNA fragments designated A to C were synthesized and assembled into a low-copy-number PACYC177 vector with a <t>T7</t> promoter at the 5′ end and a <t>NotI</t> linearization site at the 3′ end. The viruses were rescued by transfection of the in vitro -transcribed RNA into HEK293T cells. (B) E protein expression in both wild-type (WT) and mutant virus (T156I) infectious clone-transfected cells was detected by immunofluorescence using anti-E 4G2 antibody (left). The right column is the bright-field image. The size bar indicates 50 μm. (C) Endoglycosidase analyses of E protein. WT and E-T156I viral particles were pelleted and treated with PNGase F overnight at room temperature. Enzyme-digested samples were blotted by anti-E monoclonal antibody 4G2. This is a representative example from three independent experiments. (D) Both the WT and E-T156I mutant virus showed similar plaque-forming efficiencies on Vero cells. (E) Virus samples in the supernatant of transfected HEK293T cells were collected at different time points, and the titers were tested by plaque assay on Vero cells. The titer of the ZIKV MR766 WT reached 6.8 × 10 6 PFU/ml at 24 h postelectroporation and peaked at 7.9 × 10 7 PFU/ml at 48 h ( n = 3). Error bars indicate SD. This is a representative example from three independent experiments. Both the WT virus and E-T156I mutant virus exhibited similar titers at all time points. (F) Multistep growth curve of WT and E-T156I mutant ZIKV MR766. C6/36 cells were infected by the WT and mutant viruses at an MOI of 0.01. Virus samples from the supernatant were collected at different time points and gauged by a plaque assay on Vero cells ( n = 3). Error bars indicate SD. The results are representative of three independent experiments.
    Noti Restriction Site, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).

    Journal: bioRxiv

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    doi: 10.1101/2020.07.02.184408

    Figure Lengend Snippet: Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).

    Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

    Techniques: BAC Assay, Binding Assay, Sequencing, Ligation, Amplification, Plasmid Preparation, Clone Assay

    Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.

    Journal: bioRxiv

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    doi: 10.1101/2020.07.02.184408

    Figure Lengend Snippet: Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.

    Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

    Techniques: Transfection, HAC Assay, BAC Assay, Real-time Polymerase Chain Reaction, Clone Assay, Fluorescence In Situ Hybridization, Staining, Southern Blot

    Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).

    Journal: bioRxiv

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    doi: 10.1101/2020.07.02.184408

    Figure Lengend Snippet: Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).

    Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

    Techniques: Transformation Assay, Plasmid Preparation, Transfection, Marker

    Tg ( DnRest ) rescues hearing, balancing, and hair cells in Srrm4 bv/bv mice. (A) Verification of the composition of Tg ( DnRest ) bacterial artificial chromosome (BAC) construct using Southern blotting. Left: Schematic of Tg ( DnRest ) BAC depicting the backbone of the BAC vector (white box), the genomic DNA insert (circle), the DnREST-encoding sequence (red arrow), the mouse Myo7a gene (gray arrow), XhoI sites (X), NotI sites (N), and the size of the BAC plus DnRest in kilobases (kb). Middle: SYBR Gold staining of electrophoretically separated Tg ( DnRest ) fragments generated by NotI (N) and XhoI (X) digestion of the Tg ( DnRest ) BAC. Right: Southern blot of NotI (N)- and XhoI (X)-digested Tg ( DnRest ) BAC with a probe from the DnREST-encoding region. Expected positions (tick lines) and sizes (in kb) of Southern blot hybridization products are indicated (n = 2 independent assays). (B) Expression levels of a REST-binding site (RE1) containing reporter gene (RE1-TK-Firefly-luc) in HEK293 cells that were co-transfected with the RE1-TK-Firefly-luc gene, a control gene (TK-Renilla-luc), and a vector encoding Flag-DnREST, non-tagged DnREST, or no insert (vector). Schemes at the top show compositions of RE1-TK-Firefly-luc and TK-Renilla-luc. Luc, luciferase; TK, thymidine kinase gene promoter. Each symbol represents the value for an independent assay. (C) Immunofluorescence signals for MYO7A (red) and Flag-DnREST (green, negative control for Fig 1C ) in cochlear and utricular sections from a WT mouse (P1) (n = 2 mice). Scale bar, 20 μm. (D) F-actin–stained organ of Corti preparations from WT and Tg ( DnRest ) mice at P28 (n = 3 mice per genotype). The rows of inner hair cells (IHCs) (arrowheads) and outer hair cells (OHCs) (vertical lines) are indicated. Scale bar, 20 μm. (E) F-actin–stained utricular macula from WT and Tg ( DnRest ) mice at P28 (n = 3 mice per genotype). Scale bar, 20 μm. (F) Thresholds of broadband click-evoked auditory brainstem responses in WT, Srrm4 bv/bv , and Tg ( DnRest ); Srrm4 bv/bv mice at P120. Each symbol represents the value for a single mouse; blue lines indicate means (one-way ANOVA P

    Journal: Life Science Alliance

    Article Title: Inhibition of a transcriptional repressor rescues hearing in a splicing factor–deficient mouse

    doi: 10.26508/lsa.202000841

    Figure Lengend Snippet: Tg ( DnRest ) rescues hearing, balancing, and hair cells in Srrm4 bv/bv mice. (A) Verification of the composition of Tg ( DnRest ) bacterial artificial chromosome (BAC) construct using Southern blotting. Left: Schematic of Tg ( DnRest ) BAC depicting the backbone of the BAC vector (white box), the genomic DNA insert (circle), the DnREST-encoding sequence (red arrow), the mouse Myo7a gene (gray arrow), XhoI sites (X), NotI sites (N), and the size of the BAC plus DnRest in kilobases (kb). Middle: SYBR Gold staining of electrophoretically separated Tg ( DnRest ) fragments generated by NotI (N) and XhoI (X) digestion of the Tg ( DnRest ) BAC. Right: Southern blot of NotI (N)- and XhoI (X)-digested Tg ( DnRest ) BAC with a probe from the DnREST-encoding region. Expected positions (tick lines) and sizes (in kb) of Southern blot hybridization products are indicated (n = 2 independent assays). (B) Expression levels of a REST-binding site (RE1) containing reporter gene (RE1-TK-Firefly-luc) in HEK293 cells that were co-transfected with the RE1-TK-Firefly-luc gene, a control gene (TK-Renilla-luc), and a vector encoding Flag-DnREST, non-tagged DnREST, or no insert (vector). Schemes at the top show compositions of RE1-TK-Firefly-luc and TK-Renilla-luc. Luc, luciferase; TK, thymidine kinase gene promoter. Each symbol represents the value for an independent assay. (C) Immunofluorescence signals for MYO7A (red) and Flag-DnREST (green, negative control for Fig 1C ) in cochlear and utricular sections from a WT mouse (P1) (n = 2 mice). Scale bar, 20 μm. (D) F-actin–stained organ of Corti preparations from WT and Tg ( DnRest ) mice at P28 (n = 3 mice per genotype). The rows of inner hair cells (IHCs) (arrowheads) and outer hair cells (OHCs) (vertical lines) are indicated. Scale bar, 20 μm. (E) F-actin–stained utricular macula from WT and Tg ( DnRest ) mice at P28 (n = 3 mice per genotype). Scale bar, 20 μm. (F) Thresholds of broadband click-evoked auditory brainstem responses in WT, Srrm4 bv/bv , and Tg ( DnRest ); Srrm4 bv/bv mice at P120. Each symbol represents the value for a single mouse; blue lines indicate means (one-way ANOVA P

    Article Snippet: Southern blotting of Tg (DnRest )-harboring BAC DNABAC DNA was isolated from bacteria using the Large-Construct Kit (QIAGEN) and incubated with NotI and XhoI restriction enzymes (New England Biolabs).

    Techniques: Mouse Assay, BAC Assay, Construct, Southern Blot, Plasmid Preparation, Sequencing, Staining, Generated, Hybridization, Expressing, Binding Assay, Transfection, Luciferase, Immunofluorescence, Negative Control

    Supercoiled plasmid standards overestimate w Ls ftsZ copy numbers. ( A ) L. sigmodontis MF were counted (range 17 to 600) and DNA extracted for w Ls ftsZ qPCR. Quantification was done using circular ( N = 19) or NotI linearized ( N = 30) plasmids. Linearized plasmids resulted in reduced copy numbers (20.6 vs. 200 w Ls ftsZ /MF). Lines indicate median and interquartile range. ( B ) Full projection of confocal images showing 2 Litomosoides sigmodontis microfilariae stained with DAPI (magenta) and an anti-WSP (yellow) monoclonal antibody. Clusters of Wolbachia are located in the proximal part of the posterior half of MF. A single Wolbachia bacterium is 0.8–1 µm in length; thus, there are not hundreds of endobacteria in a single MF. MF were observed with a 63X objective on a Leica SP5. The scale bar = 10 µM. ( C ) Variation in w Ls ftsZ copy number per L. sigmodontis MF is greater in samples with few MF. DNA was extracted from 5 to 60 L. sigmodontis MF and used for w Ls ftsZ qPCR. Linear regression (blue line) and 95% confidence bars (red dotted lines) are shown. ( D ) The qPCR is reproducible and stable. Interassay reproducibility was tested on DNA extracted from 18 preparations, each with ( A ) 50 MF and ( B ) 20 MF, and ( C ) 12 preparations with 50 MF from L. sigmodontis to calculate w Ls ftsZ copies/MF. Lines indicate median and interquartile range

    Journal: Parasitology Research

    Article Title: A qPCR to quantify Wolbachia from few Onchocerca volvulus microfilariae as a surrogate for adult worm histology in clinical trials of antiwolbachial drugs

    doi: 10.1007/s00436-021-07411-5

    Figure Lengend Snippet: Supercoiled plasmid standards overestimate w Ls ftsZ copy numbers. ( A ) L. sigmodontis MF were counted (range 17 to 600) and DNA extracted for w Ls ftsZ qPCR. Quantification was done using circular ( N = 19) or NotI linearized ( N = 30) plasmids. Linearized plasmids resulted in reduced copy numbers (20.6 vs. 200 w Ls ftsZ /MF). Lines indicate median and interquartile range. ( B ) Full projection of confocal images showing 2 Litomosoides sigmodontis microfilariae stained with DAPI (magenta) and an anti-WSP (yellow) monoclonal antibody. Clusters of Wolbachia are located in the proximal part of the posterior half of MF. A single Wolbachia bacterium is 0.8–1 µm in length; thus, there are not hundreds of endobacteria in a single MF. MF were observed with a 63X objective on a Leica SP5. The scale bar = 10 µM. ( C ) Variation in w Ls ftsZ copy number per L. sigmodontis MF is greater in samples with few MF. DNA was extracted from 5 to 60 L. sigmodontis MF and used for w Ls ftsZ qPCR. Linear regression (blue line) and 95% confidence bars (red dotted lines) are shown. ( D ) The qPCR is reproducible and stable. Interassay reproducibility was tested on DNA extracted from 18 preparations, each with ( A ) 50 MF and ( B ) 20 MF, and ( C ) 12 preparations with 50 MF from L. sigmodontis to calculate w Ls ftsZ copies/MF. Lines indicate median and interquartile range

    Article Snippet: Plasmid standards containing the w OvftsZ sequence were tested in two different conformations: as supercoiled plasmids and after linearization with the single-cutting restriction NotI-HF enzyme according to the manufacturer’s protocol (NEB, Ipswich, MA).

    Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Staining

    Removal of E N-glycosylation does not affect ZIKV replication in mammalian cell culture. (A) Schematic diagram of the ZIKV genome organization and the construction of the MR766 infectious clone. Three cDNA fragments designated A to C were synthesized and assembled into a low-copy-number PACYC177 vector with a T7 promoter at the 5′ end and a NotI linearization site at the 3′ end. The viruses were rescued by transfection of the in vitro -transcribed RNA into HEK293T cells. (B) E protein expression in both wild-type (WT) and mutant virus (T156I) infectious clone-transfected cells was detected by immunofluorescence using anti-E 4G2 antibody (left). The right column is the bright-field image. The size bar indicates 50 μm. (C) Endoglycosidase analyses of E protein. WT and E-T156I viral particles were pelleted and treated with PNGase F overnight at room temperature. Enzyme-digested samples were blotted by anti-E monoclonal antibody 4G2. This is a representative example from three independent experiments. (D) Both the WT and E-T156I mutant virus showed similar plaque-forming efficiencies on Vero cells. (E) Virus samples in the supernatant of transfected HEK293T cells were collected at different time points, and the titers were tested by plaque assay on Vero cells. The titer of the ZIKV MR766 WT reached 6.8 × 10 6 PFU/ml at 24 h postelectroporation and peaked at 7.9 × 10 7 PFU/ml at 48 h ( n = 3). Error bars indicate SD. This is a representative example from three independent experiments. Both the WT virus and E-T156I mutant virus exhibited similar titers at all time points. (F) Multistep growth curve of WT and E-T156I mutant ZIKV MR766. C6/36 cells were infected by the WT and mutant viruses at an MOI of 0.01. Virus samples from the supernatant were collected at different time points and gauged by a plaque assay on Vero cells ( n = 3). Error bars indicate SD. The results are representative of three independent experiments.

    Journal: mBio

    Article Title: N-glycosylation of Viral E Protein Is the Determinant for Vector Midgut Invasion by Flaviviruses

    doi: 10.1128/mBio.00046-18

    Figure Lengend Snippet: Removal of E N-glycosylation does not affect ZIKV replication in mammalian cell culture. (A) Schematic diagram of the ZIKV genome organization and the construction of the MR766 infectious clone. Three cDNA fragments designated A to C were synthesized and assembled into a low-copy-number PACYC177 vector with a T7 promoter at the 5′ end and a NotI linearization site at the 3′ end. The viruses were rescued by transfection of the in vitro -transcribed RNA into HEK293T cells. (B) E protein expression in both wild-type (WT) and mutant virus (T156I) infectious clone-transfected cells was detected by immunofluorescence using anti-E 4G2 antibody (left). The right column is the bright-field image. The size bar indicates 50 μm. (C) Endoglycosidase analyses of E protein. WT and E-T156I viral particles were pelleted and treated with PNGase F overnight at room temperature. Enzyme-digested samples were blotted by anti-E monoclonal antibody 4G2. This is a representative example from three independent experiments. (D) Both the WT and E-T156I mutant virus showed similar plaque-forming efficiencies on Vero cells. (E) Virus samples in the supernatant of transfected HEK293T cells were collected at different time points, and the titers were tested by plaque assay on Vero cells. The titer of the ZIKV MR766 WT reached 6.8 × 10 6 PFU/ml at 24 h postelectroporation and peaked at 7.9 × 10 7 PFU/ml at 48 h ( n = 3). Error bars indicate SD. This is a representative example from three independent experiments. Both the WT virus and E-T156I mutant virus exhibited similar titers at all time points. (F) Multistep growth curve of WT and E-T156I mutant ZIKV MR766. C6/36 cells were infected by the WT and mutant viruses at an MOI of 0.01. Virus samples from the supernatant were collected at different time points and gauged by a plaque assay on Vero cells ( n = 3). Error bars indicate SD. The results are representative of three independent experiments.

    Article Snippet: All of the segments were further assembled into low-copy-number vector PACYC177 with a T7 promoter in front of the 5′ terminus and a NotI restriction site following the 3′ end using NEBuilder Hi-Fi DNA assembly master mix (NEB Biolabs).

    Techniques: Cell Culture, Synthesized, Low Copy Number, Plasmid Preparation, Transfection, In Vitro, Expressing, Mutagenesis, Immunofluorescence, Plaque Assay, Infection