xhoi  (New England Biolabs)


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    XhoI
    Description:
    XhoI 25 000 units
    Catalog Number:
    r0146l
    Price:
    290
    Size:
    25 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs xhoi
    XhoI
    XhoI 25 000 units
    https://www.bioz.com/result/xhoi/product/New England Biolabs
    Average 99 stars, based on 708 article reviews
    Price from $9.99 to $1999.99
    xhoi - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) 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

    2) Product Images from "A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability"

    Article Title: A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008217

    dmc1-E157D bypasses mei5 but not rad51 with respect to CO formation. (a) Southern blot analysis at the HIS4 :: LEU2 hotspot following digestion of genomic DNA from meiotic time course experiments with XhoI. Time points shown from left to right are: 0h, 6h, 8h, 10h, 24h. (b) Quantitation of 1D gels shown in (a) and meiotic progression data; black–wild-type, light blue –mei5 , purple –rad51 , red –dmc1-E157D , gray –dmc1-E157D mei5 , green –dmc1-E157D rad51 , yellow –dmc1-E157D mei5 rad51 . To score meiotic progression, ≥50 cells were scored per time point. Strains used in experiments in the order in which they appear in figure, left to right: DKB3698, DKB6320, DKB3710, DKB6342, DKB6300, DKB6393, DKB6412.
    Figure Legend Snippet: dmc1-E157D bypasses mei5 but not rad51 with respect to CO formation. (a) Southern blot analysis at the HIS4 :: LEU2 hotspot following digestion of genomic DNA from meiotic time course experiments with XhoI. Time points shown from left to right are: 0h, 6h, 8h, 10h, 24h. (b) Quantitation of 1D gels shown in (a) and meiotic progression data; black–wild-type, light blue –mei5 , purple –rad51 , red –dmc1-E157D , gray –dmc1-E157D mei5 , green –dmc1-E157D rad51 , yellow –dmc1-E157D mei5 rad51 . To score meiotic progression, ≥50 cells were scored per time point. Strains used in experiments in the order in which they appear in figure, left to right: DKB3698, DKB6320, DKB3710, DKB6342, DKB6300, DKB6393, DKB6412.

    Techniques Used: Southern Blot, Quantitation Assay

    3) Product Images from "A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta"

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta

    Journal: The Plant Cell

    doi:

    A Low-Copy Repeat Sequence Identifies Restriction Fragments Distal to the AVR-Pita Telomere That Are Deleted in Some Spontaneous Mutants. Genomic DNAs were digested with BglII for DNA gel blot analysis. Hybridization with the 10-kb XhoI fragment from cosmid A10H8 identified the 6.5-kb telomeric BglII fragment and an additional 4.0-kb fragment (arrows) that was altered in avr-pita − mutants. All avirulent laboratory strains inherited these two bands from the Chinese field isolate O-137 (lane 23) through the RFLP mapping strain 4224-7-8 (lane 2). The virulent mapping parent 6043 (lane 1) does not have these bands. Not shown are data consistent with this result but obtained by using SalI, EcoRI, and SacI. Except for lane 1, which contains DNA from the virulent parent 6043, each v lane contains DNA from a virulent mutant obtained from the first avirulent strain (A lanes) to its left. Lane 1, 6043 (v); lane 2, 4224-7-8 (A); lane 3, 4360-17-1 (A); lane 4, CP917 (v); lane 5, 4375-R-26 (A); lane 6, CP984 (v); lane 7, 4375-R-39 (A); lane 8, CP918 (v); lane 9, 4375-R-6 (A); lane 10, CP983 (v); lane 11, CP1614 (v); lane 12, CP1615 (v); lane 13, CP1631 (A); lane 14, CP1632 (v); lane 15, CP1634 (A); lane 16, CP1635 (v); lane 17, CP1637 (A); lane 18, CP1638 (v); lane 19, CP1640 (A); lane 20, CP1641 (v); lane 21, CP1643 (A); lane 22, CP1644 (v); and lane 23, O-137 (A). Markers at left identify the positions of λ HindIII DNA length standards, which are as follows (from the top): 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kb.
    Figure Legend Snippet: A Low-Copy Repeat Sequence Identifies Restriction Fragments Distal to the AVR-Pita Telomere That Are Deleted in Some Spontaneous Mutants. Genomic DNAs were digested with BglII for DNA gel blot analysis. Hybridization with the 10-kb XhoI fragment from cosmid A10H8 identified the 6.5-kb telomeric BglII fragment and an additional 4.0-kb fragment (arrows) that was altered in avr-pita − mutants. All avirulent laboratory strains inherited these two bands from the Chinese field isolate O-137 (lane 23) through the RFLP mapping strain 4224-7-8 (lane 2). The virulent mapping parent 6043 (lane 1) does not have these bands. Not shown are data consistent with this result but obtained by using SalI, EcoRI, and SacI. Except for lane 1, which contains DNA from the virulent parent 6043, each v lane contains DNA from a virulent mutant obtained from the first avirulent strain (A lanes) to its left. Lane 1, 6043 (v); lane 2, 4224-7-8 (A); lane 3, 4360-17-1 (A); lane 4, CP917 (v); lane 5, 4375-R-26 (A); lane 6, CP984 (v); lane 7, 4375-R-39 (A); lane 8, CP918 (v); lane 9, 4375-R-6 (A); lane 10, CP983 (v); lane 11, CP1614 (v); lane 12, CP1615 (v); lane 13, CP1631 (A); lane 14, CP1632 (v); lane 15, CP1634 (A); lane 16, CP1635 (v); lane 17, CP1637 (A); lane 18, CP1638 (v); lane 19, CP1640 (A); lane 20, CP1641 (v); lane 21, CP1643 (A); lane 22, CP1644 (v); and lane 23, O-137 (A). Markers at left identify the positions of λ HindIII DNA length standards, which are as follows (from the top): 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kb.

    Techniques Used: Sequencing, Western Blot, Hybridization, Mutagenesis

    4) Product Images from ""

    Article Title:

    Journal: Drug Metabolism and Disposition

    doi: 10.1124/dmd.114.059188

    Confirmation that CYP2F1 was inactivated and not expressed. (A) DNA sequence determined for WT and mutant CYP2F1 exon-10 region. The CYS codon (blue) and three additional nucleotides in WT CYP2F1 was replaced by an XhoI restriction site (red) and a loxP
    Figure Legend Snippet: Confirmation that CYP2F1 was inactivated and not expressed. (A) DNA sequence determined for WT and mutant CYP2F1 exon-10 region. The CYS codon (blue) and three additional nucleotides in WT CYP2F1 was replaced by an XhoI restriction site (red) and a loxP

    Techniques Used: Sequencing, Mutagenesis

    5) Product Images from "Widespread inhibition, antagonism, and synergy in mouse olfactory sensory neurons in vivo"

    Article Title: Widespread inhibition, antagonism, and synergy in mouse olfactory sensory neurons in vivo

    Journal: bioRxiv

    doi: 10.1101/803908

    OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). KpnI (K), SpeI (S), XhoI (X) and ApaI (A).
    Figure Legend Snippet: OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). KpnI (K), SpeI (S), XhoI (X) and ApaI (A).

    Techniques Used: Knock-Out, Mouse Assay, Transmission Assay, Southern Blot, Immunostaining, Mutagenesis

    6) Product Images from "Robust HIV-1 replication in the absence of integrase function"

    Article Title: Robust HIV-1 replication in the absence of integrase function

    Journal: bioRxiv

    doi: 10.1101/2020.03.18.997023

    Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.
    Figure Legend Snippet: Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.

    Techniques Used: Southern Blot, Binding Assay, Infection, Plasmid Preparation, Nucleic Acid Electrophoresis

    HTLV-1 Tax does not facilitate the illegitimate integration of IN- HIV-1. A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN- HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. C) Total 2LTR circle DNA was measured at the indicated timepoints by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN- HIV-1 in the presence or absence of Tax. D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells ± Tax infected with WT or IN- HIV-1. All data in A, C and D are normalized to WT infected Tax-cells at day 1, set to 1; n=3. Crosses indicate last day viable cells were detected. E) DNA from CEM-SS cells ± Tax expression, infected with WT or IN- HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR + linear unintegrated DNA (2.6-2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains ¼ of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. F ) Quantification of the bands (2LTR+1LTR+linear and 2LTR) in E expressed as a percentage of total HIV-1 DNA, which was set at 100%.
    Figure Legend Snippet: HTLV-1 Tax does not facilitate the illegitimate integration of IN- HIV-1. A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN- HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. C) Total 2LTR circle DNA was measured at the indicated timepoints by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN- HIV-1 in the presence or absence of Tax. D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells ± Tax infected with WT or IN- HIV-1. All data in A, C and D are normalized to WT infected Tax-cells at day 1, set to 1; n=3. Crosses indicate last day viable cells were detected. E) DNA from CEM-SS cells ± Tax expression, infected with WT or IN- HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR + linear unintegrated DNA (2.6-2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains ¼ of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. F ) Quantification of the bands (2LTR+1LTR+linear and 2LTR) in E expressed as a percentage of total HIV-1 DNA, which was set at 100%.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Expressing, Southern Blot

    7) Product Images from "Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11"

    Article Title: Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.123570

    Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.
    Figure Legend Snippet: Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.

    Techniques Used: Plasmid Preparation, Clone Assay

    8) Product Images from "Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen ▿O Fluxes of an Acidic Fen ▿ †"

    Article Title: Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen ▿O Fluxes of an Acidic Fen ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02256-09

    Comparative TRFLP analyses of narG (A to C) and nosZ (D to F) amplified from different soil layers of the acidic fen. PCR products were digested with CfoI (A), HaeIII (B), XhoI (C), BtgI (D), NlaIV (E), and PvuI and SacI (F). Mean values of three replicates
    Figure Legend Snippet: Comparative TRFLP analyses of narG (A to C) and nosZ (D to F) amplified from different soil layers of the acidic fen. PCR products were digested with CfoI (A), HaeIII (B), XhoI (C), BtgI (D), NlaIV (E), and PvuI and SacI (F). Mean values of three replicates

    Techniques Used: Terminal Restriction Fragment Length Polymorphism, Amplification, Polymerase Chain Reaction

    9) Product Images from "Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes"

    Article Title: Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-62703-968-0_14

    Example of assembly PCR primers and introduction of variation via degenerate codons. Each colored selection represents a single assembly primer; the sum of all primers is designed to produce the entire coding sequence shown. An NdeI restriction site (CATATG) has been added to the N-terminal end of the sequence, and an XhoI restriction site (CTCGAG) has been added to the C-terminal end. The magnified inset towards the C-terminal end of the sequence gives an example of introducing variation using the degenerate codon “RAA.” The “R” base designates the introduction of either a guanine (G) or adenine (A) base at that position, resulting in a translated protein sequence with either glutamic acid (E) or lysine (K)
    Figure Legend Snippet: Example of assembly PCR primers and introduction of variation via degenerate codons. Each colored selection represents a single assembly primer; the sum of all primers is designed to produce the entire coding sequence shown. An NdeI restriction site (CATATG) has been added to the N-terminal end of the sequence, and an XhoI restriction site (CTCGAG) has been added to the C-terminal end. The magnified inset towards the C-terminal end of the sequence gives an example of introducing variation using the degenerate codon “RAA.” The “R” base designates the introduction of either a guanine (G) or adenine (A) base at that position, resulting in a translated protein sequence with either glutamic acid (E) or lysine (K)

    Techniques Used: Polymerase Cycling Assembly, Selection, Sequencing

    10) Product Images from "Increased Genome Instability and Telomere Length in the elg1-Deficient Saccharomyces cerevisiae Mutant Are Regulated by S-Phase Checkpoints"

    Article Title: Increased Genome Instability and Telomere Length in the elg1-Deficient Saccharomyces cerevisiae Mutant Are Regulated by S-Phase Checkpoints

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.3.6.1557-1566.2004

    Telomere length was determined in different strains carrying the elg1 Δ mutation. Chromosomal DNAs from each strain were digested with XhoI and hybridized with the telomeric repeat probe that can detect the Y′ class of telomeres. (A) Mutations in telomere maintenance genes had synergistic effects on telomere size when combined with the elg1 Δ mutation. (B) S-phase checkpoint sensor gene mutations along with the elg1 Δ mutation generated different telomere size changes. (C) The elg1 Δ mutation in strains defective in S-phase checkpoint transducer genes also changed telomere length differently. (D) An additional mutation in the RAD24 gene in strains carrying elg1 Δ and genes functioning in the DNA replication checkpoint slightly decreased telomere size.
    Figure Legend Snippet: Telomere length was determined in different strains carrying the elg1 Δ mutation. Chromosomal DNAs from each strain were digested with XhoI and hybridized with the telomeric repeat probe that can detect the Y′ class of telomeres. (A) Mutations in telomere maintenance genes had synergistic effects on telomere size when combined with the elg1 Δ mutation. (B) S-phase checkpoint sensor gene mutations along with the elg1 Δ mutation generated different telomere size changes. (C) The elg1 Δ mutation in strains defective in S-phase checkpoint transducer genes also changed telomere length differently. (D) An additional mutation in the RAD24 gene in strains carrying elg1 Δ and genes functioning in the DNA replication checkpoint slightly decreased telomere size.

    Techniques Used: Mutagenesis, Generated

    11) Product Images from "Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit"

    Article Title: Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127321

    Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p
    Figure Legend Snippet: Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p

    Techniques Used: Activation Assay, Plasmid Preparation, Generated, Activity Assay

    12) Product Images from "Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats"

    Article Title: Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.908144

    NRG-1 recombinant plasmid was constructed by double-enzyme digestion and identified by PCR. ( A ) 8 clones (5–12) were selected randomly and identified by PCR, 1: blank control, 2: empty plasmid control, 3: positive control (GAPDH), 4: marker; ( B ) plasmid enzyme detachment, M: marker, after: after HindIII and XhoI double-enzyme digestion, before: before double-enzyme digestion.
    Figure Legend Snippet: NRG-1 recombinant plasmid was constructed by double-enzyme digestion and identified by PCR. ( A ) 8 clones (5–12) were selected randomly and identified by PCR, 1: blank control, 2: empty plasmid control, 3: positive control (GAPDH), 4: marker; ( B ) plasmid enzyme detachment, M: marker, after: after HindIII and XhoI double-enzyme digestion, before: before double-enzyme digestion.

    Techniques Used: Recombinant, Plasmid Preparation, Construct, Polymerase Chain Reaction, Clone Assay, Positive Control, Marker

    13) Product Images from "Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes"

    Article Title: Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-62703-968-0_14

    Example of assembly PCR primers and introduction of variation via degenerate codons. Each colored selection represents a single assembly primer; the sum of all primers is designed to produce the entire coding sequence shown. An NdeI restriction site (CATATG) has been added to the N-terminal end of the sequence, and an XhoI restriction site (CTCGAG) has been added to the C-terminal end. The magnified inset towards the C-terminal end of the sequence gives an example of introducing variation using the degenerate codon “RAA.” The “R” base designates the introduction of either a guanine (G) or adenine (A) base at that position, resulting in a translated protein sequence with either glutamic acid (E) or lysine (K)
    Figure Legend Snippet: Example of assembly PCR primers and introduction of variation via degenerate codons. Each colored selection represents a single assembly primer; the sum of all primers is designed to produce the entire coding sequence shown. An NdeI restriction site (CATATG) has been added to the N-terminal end of the sequence, and an XhoI restriction site (CTCGAG) has been added to the C-terminal end. The magnified inset towards the C-terminal end of the sequence gives an example of introducing variation using the degenerate codon “RAA.” The “R” base designates the introduction of either a guanine (G) or adenine (A) base at that position, resulting in a translated protein sequence with either glutamic acid (E) or lysine (K)

    Techniques Used: Polymerase Cycling Assembly, Selection, Sequencing

    14) Product Images from "Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers"

    Article Title: Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers

    Journal: Genome Research

    doi: 10.1101/gr.5681207

    Recombination breakpoint mapping using RAD markers in Drosophila . Polymorphic RAD markers between Oregon-R (OR) and Canton-S (CS) flies were identified on a genomic tiling path microarray. Black tick marks above the diagrammed second chromosome represent the genomic locations of EcoRI RAD tags with an average differential hybridization greater than twofold. Tick marks below the chromosome represent XhoI RAD tags with a differential hybridization > 2.3-fold. Polymorphic markers are found when the recombinant line (Rec.) is compared to the OR parental line on the left arm of the chromosome and when compared to the CS parental line on most of the right arm of the chromosome. Marker patterns indicative of CS inheritance are shown as blue regions of the chromosome, and OR inheritance as red. Arrows mark the recombination breakpoint. Large gaps in the tiling array are shown as white regions of the chromosome.
    Figure Legend Snippet: Recombination breakpoint mapping using RAD markers in Drosophila . Polymorphic RAD markers between Oregon-R (OR) and Canton-S (CS) flies were identified on a genomic tiling path microarray. Black tick marks above the diagrammed second chromosome represent the genomic locations of EcoRI RAD tags with an average differential hybridization greater than twofold. Tick marks below the chromosome represent XhoI RAD tags with a differential hybridization > 2.3-fold. Polymorphic markers are found when the recombinant line (Rec.) is compared to the OR parental line on the left arm of the chromosome and when compared to the CS parental line on most of the right arm of the chromosome. Marker patterns indicative of CS inheritance are shown as blue regions of the chromosome, and OR inheritance as red. Arrows mark the recombination breakpoint. Large gaps in the tiling array are shown as white regions of the chromosome.

    Techniques Used: Microarray, Hybridization, Recombinant, Marker

    15) Product Images from "A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity"

    Article Title: A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr597

    Comparison of activity versus toxicity profiles of TALEN and ZFN. ( a ) Designer nuclease-mediated gene disruption. The HEK293-based reporter cells harbor an integrated dsEGFP gene that contains an inverted heterodimeric AvrBs4/AvrBs3 target sequence separated by 13-bp spacer in the 5′-end of the open reading frame. The position of the diagnostic XhoI site is indicated. For internal reference, a binding site for I-SceI was placed downstream of the TALEN target site. The graph shows the percentage of EGFP-negative cells 5 days after transfection with the nuclease expression vectors (TALEN, ZFN or I-SceI). ( b ) Molecular characterization. Genomic DNA was extracted 5 days after transfection and PCR amplicons encompassing the target sites were used as templates for digestion with XhoI. An arrow indicates the position of the XhoI-resistant DNA fragment. The numbers below designate the percentages of XhoI-resistant PCR fragments (note background level of ~6%). ( c ) Activity versus toxicity. The EGFP reporter cells were transfected with increasing amounts (1–600 ng) of nuclease expression vectors (TALEN, ZFN or I-SceI) and a mCherry-encoding plasmid. The percentage of EGFP and mCherry-positive cells was determined by flow cytometry after 2 and 5 days. The graphs display gene disruptions activities (EGFP-negative cells at Day 2; top) and nuclease-associated cytotoxicities (fraction of mCherry-positive cells at Day 5 as compared to Day 2 after transfection; bottom), relative to cells transfected with a mock plasmid. Statistically significant differences in toxicities between TALEN and ZFN are indicated by * P
    Figure Legend Snippet: Comparison of activity versus toxicity profiles of TALEN and ZFN. ( a ) Designer nuclease-mediated gene disruption. The HEK293-based reporter cells harbor an integrated dsEGFP gene that contains an inverted heterodimeric AvrBs4/AvrBs3 target sequence separated by 13-bp spacer in the 5′-end of the open reading frame. The position of the diagnostic XhoI site is indicated. For internal reference, a binding site for I-SceI was placed downstream of the TALEN target site. The graph shows the percentage of EGFP-negative cells 5 days after transfection with the nuclease expression vectors (TALEN, ZFN or I-SceI). ( b ) Molecular characterization. Genomic DNA was extracted 5 days after transfection and PCR amplicons encompassing the target sites were used as templates for digestion with XhoI. An arrow indicates the position of the XhoI-resistant DNA fragment. The numbers below designate the percentages of XhoI-resistant PCR fragments (note background level of ~6%). ( c ) Activity versus toxicity. The EGFP reporter cells were transfected with increasing amounts (1–600 ng) of nuclease expression vectors (TALEN, ZFN or I-SceI) and a mCherry-encoding plasmid. The percentage of EGFP and mCherry-positive cells was determined by flow cytometry after 2 and 5 days. The graphs display gene disruptions activities (EGFP-negative cells at Day 2; top) and nuclease-associated cytotoxicities (fraction of mCherry-positive cells at Day 5 as compared to Day 2 after transfection; bottom), relative to cells transfected with a mock plasmid. Statistically significant differences in toxicities between TALEN and ZFN are indicated by * P

    Techniques Used: Activity Assay, Sequencing, Diagnostic Assay, Binding Assay, Transfection, Expressing, Polymerase Chain Reaction, Plasmid Preparation, Flow Cytometry, Cytometry

    16) Product Images from "Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing"

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing

    Journal: Nature methods

    doi: 10.1038/nmeth.2408

    BLESS workflow and specificity. ( a ) DSBs are ligated in situ to a proximal linker (red arch) covalently linked to biotin (orange oval) (1), gDNA is extracted and fragmented (2), and labeled fragments are captured on streptavidin beads (gray ovals) (3). A distal linker (blue arch) is then ligated to the free extremity of captured fragments (4), and fragments are released by linker digestion with I-SceI (5). Released fragments are amplified by PCR using linker-specific primers (6), and sequenced (7). ( b ) Structure of linkers. Both proximal (P) and distal (D) linkers share an XhoI site (yellow), the I-SceI endonuclease minimal recognition site (non-highlighted letters), and a seven-thymine loop (bold). Each linker contains a specific barcode sequence marking the ligation site (orange and brown). The proximal linker is biotinylated (orange oval). ( c ) Proportion of fragments with proximal (P) and distal (D) barcodes in single-end (SE) and pair-end (PE) Illumina sequencing experiments. Mean ± s.d. is shown.
    Figure Legend Snippet: BLESS workflow and specificity. ( a ) DSBs are ligated in situ to a proximal linker (red arch) covalently linked to biotin (orange oval) (1), gDNA is extracted and fragmented (2), and labeled fragments are captured on streptavidin beads (gray ovals) (3). A distal linker (blue arch) is then ligated to the free extremity of captured fragments (4), and fragments are released by linker digestion with I-SceI (5). Released fragments are amplified by PCR using linker-specific primers (6), and sequenced (7). ( b ) Structure of linkers. Both proximal (P) and distal (D) linkers share an XhoI site (yellow), the I-SceI endonuclease minimal recognition site (non-highlighted letters), and a seven-thymine loop (bold). Each linker contains a specific barcode sequence marking the ligation site (orange and brown). The proximal linker is biotinylated (orange oval). ( c ) Proportion of fragments with proximal (P) and distal (D) barcodes in single-end (SE) and pair-end (PE) Illumina sequencing experiments. Mean ± s.d. is shown.

    Techniques Used: In Situ, Labeling, Amplification, Polymerase Chain Reaction, Sequencing, Ligation

    17) Product Images from "Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters"

    Article Title: Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt731

    Duplexes carrying damaged sites used in this study. Oligonucleotides carrying oG, hU and U. The modified bases are indicated in bold. oG was separated by 3 bp from hU located on the complementary strand in MDS/+1, MDS/−5. Uracil was positioned at +1 position relatively to hU in MDS/+1 and at −5 position in MDS/−5. The control U/U oligonucleotide consists of two bistranded Us separated by 5 bp. Undamaged and MDS-containing 56-mers duplexes harbor EcoRI and XhoI restriction sites at each extremity as indicated.
    Figure Legend Snippet: Duplexes carrying damaged sites used in this study. Oligonucleotides carrying oG, hU and U. The modified bases are indicated in bold. oG was separated by 3 bp from hU located on the complementary strand in MDS/+1, MDS/−5. Uracil was positioned at +1 position relatively to hU in MDS/+1 and at −5 position in MDS/−5. The control U/U oligonucleotide consists of two bistranded Us separated by 5 bp. Undamaged and MDS-containing 56-mers duplexes harbor EcoRI and XhoI restriction sites at each extremity as indicated.

    Techniques Used: Modification

    18) Product Images from "Genome-Wide Mapping of DNA Strand Breaks"

    Article Title: Genome-Wide Mapping of DNA Strand Breaks

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017353

    dDIP enrichment of yeast telomeric DNA evaluated by Southern blot. Extracted yeast DNA was first digested by XhoI or PstI, two enzymes cutting once in the conserved telomere proximal Y' repeat element giving a≈1.2 kb and ≈1.0 kb terminal restriction fragment respectively. A probe covering part of the telomeric Y' fragment including the terminal 0.35 kb TG1-3 repeats was used to reveal the capture of telomeric DNA. To evaluate the telomeres immunoprecipitation efficiency by the dDIP technique, 30%, 40% and 50% of the input DNA before immunoprecipitation was applied to the gel. N+, DNA from uninduced cells end-labeled with dATP, biotin-dATP and TdT. N-, DNA from uninduced cells end-labeled with dATP, biotin-dATP without TdT.
    Figure Legend Snippet: dDIP enrichment of yeast telomeric DNA evaluated by Southern blot. Extracted yeast DNA was first digested by XhoI or PstI, two enzymes cutting once in the conserved telomere proximal Y' repeat element giving a≈1.2 kb and ≈1.0 kb terminal restriction fragment respectively. A probe covering part of the telomeric Y' fragment including the terminal 0.35 kb TG1-3 repeats was used to reveal the capture of telomeric DNA. To evaluate the telomeres immunoprecipitation efficiency by the dDIP technique, 30%, 40% and 50% of the input DNA before immunoprecipitation was applied to the gel. N+, DNA from uninduced cells end-labeled with dATP, biotin-dATP and TdT. N-, DNA from uninduced cells end-labeled with dATP, biotin-dATP without TdT.

    Techniques Used: Southern Blot, Immunoprecipitation, Labeling

    19) Product Images from "Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena"

    Article Title: Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena

    Journal: Genes

    doi: 10.3390/genes9040179

    Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.
    Figure Legend Snippet: Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.

    Techniques Used: Mutagenesis, Plasmid Preparation, Homologous Recombination, Southern Blot, Expressing, Molecular Weight, Western Blot

    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).
    Figure Legend Snippet: Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Techniques Used: Knock-Out, Plasmid Preparation, Homologous Recombination, Southern Blot, Molecular Weight

    20) Product Images from "High-throughput screening of soluble recombinant proteins"

    Article Title: High-throughput screening of soluble recombinant proteins

    Journal: Protein Science : A Publication of the Protein Society

    doi:

    Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.
    Figure Legend Snippet: Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Ligation

    21) Product Images from "Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1"

    Article Title: Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1

    Journal: Genes & Development

    doi: 10.1101/gad.310995.117

    The nucleosome exclusion reaction counteracts DNA synthesis-coupled chromatin assembly. ( A ) Schematic diagram of the primer extension assay. A 92-nucleotide (nt) primer carrying either no mismatch or an A:C mismatch is annealed on a single-stranded pMM1. Upon incubation in NPE, complementary DNA is synthesized depending on the primer, converting the substrate into covalently closed circular DNA. ( B ) The requirements of canonical MMR factors for primer extension-coupled mismatch correction. The primer extension assay was performed in mock-treated, Mlh1-depleted (ΔMlh1), Msh2-depleted (ΔMsh2), or Msh2/Mlh1 doubly depleted (ΔMsh2ΔMlh1) NPE. The ratio of XhoI-sensitive molecules that correspond to the C-to-T repair products is plotted in a graph. Mean ± one SD is shown. n = 4. P -values were calculated by the unpaired t , for the details of quantification. ( C ) Nucleosome exclusion on the primer extension products. The products described in B were separated by agarose gel without any treatment (lanes 2 – 5 ), after digestion of incomplete intermediates by S1 nuclease and ExoV (lanes 6 – 9 ), or after digestion of C-to-T repair products and incomplete intermediates by XhoI, S1 nuclease, and λ exonuclease (lanes 10 – 13 ). (ss) ssDNA; (IM) primer extension intermediates. ( D ) The assay presented in C was repeated in NPE depleted of Mlh1 and HIRA (lanes 3 , 4 ) or Mlh1, HIRA, and Smarcad1 (lanes 5 – 8 ) supplemented with either buffer (lanes 3 – 6 ) or recombinant Smarcad1 (lanes 7 , 8 ). The linking number of each band relative to the open circular or relaxed DNA (oc/r) position (ΔL) is indicated at the right of the gel. ( E ) The ratio of the plasmids of the indicated ΔL in D was quantified and is presented as a graph. Mean ± one SD is shown. n = 3.
    Figure Legend Snippet: The nucleosome exclusion reaction counteracts DNA synthesis-coupled chromatin assembly. ( A ) Schematic diagram of the primer extension assay. A 92-nucleotide (nt) primer carrying either no mismatch or an A:C mismatch is annealed on a single-stranded pMM1. Upon incubation in NPE, complementary DNA is synthesized depending on the primer, converting the substrate into covalently closed circular DNA. ( B ) The requirements of canonical MMR factors for primer extension-coupled mismatch correction. The primer extension assay was performed in mock-treated, Mlh1-depleted (ΔMlh1), Msh2-depleted (ΔMsh2), or Msh2/Mlh1 doubly depleted (ΔMsh2ΔMlh1) NPE. The ratio of XhoI-sensitive molecules that correspond to the C-to-T repair products is plotted in a graph. Mean ± one SD is shown. n = 4. P -values were calculated by the unpaired t , for the details of quantification. ( C ) Nucleosome exclusion on the primer extension products. The products described in B were separated by agarose gel without any treatment (lanes 2 – 5 ), after digestion of incomplete intermediates by S1 nuclease and ExoV (lanes 6 – 9 ), or after digestion of C-to-T repair products and incomplete intermediates by XhoI, S1 nuclease, and λ exonuclease (lanes 10 – 13 ). (ss) ssDNA; (IM) primer extension intermediates. ( D ) The assay presented in C was repeated in NPE depleted of Mlh1 and HIRA (lanes 3 , 4 ) or Mlh1, HIRA, and Smarcad1 (lanes 5 – 8 ) supplemented with either buffer (lanes 3 – 6 ) or recombinant Smarcad1 (lanes 7 , 8 ). The linking number of each band relative to the open circular or relaxed DNA (oc/r) position (ΔL) is indicated at the right of the gel. ( E ) The ratio of the plasmids of the indicated ΔL in D was quantified and is presented as a graph. Mean ± one SD is shown. n = 3.

    Techniques Used: DNA Synthesis, Primer Extension Assay, Incubation, Synthesized, Agarose Gel Electrophoresis, Recombinant

    22) 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

    23) Product Images from "Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats"

    Article Title: Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.908144

    NRG-1 recombinant plasmid was constructed by double-enzyme digestion and identified by PCR. ( A ) 8 clones (5–12) were selected randomly and identified by PCR, 1: blank control, 2: empty plasmid control, 3: positive control (GAPDH), 4: marker; ( B ) plasmid enzyme detachment, M: marker, after: after HindIII and XhoI double-enzyme digestion, before: before double-enzyme digestion.
    Figure Legend Snippet: NRG-1 recombinant plasmid was constructed by double-enzyme digestion and identified by PCR. ( A ) 8 clones (5–12) were selected randomly and identified by PCR, 1: blank control, 2: empty plasmid control, 3: positive control (GAPDH), 4: marker; ( B ) plasmid enzyme detachment, M: marker, after: after HindIII and XhoI double-enzyme digestion, before: before double-enzyme digestion.

    Techniques Used: Recombinant, Plasmid Preparation, Construct, Polymerase Chain Reaction, Clone Assay, Positive Control, Marker

    24) Product Images from "Soluble L1CAM promotes breast cancer cell adhesion and migration in vitro, but not invasion"

    Article Title: Soluble L1CAM promotes breast cancer cell adhesion and migration in vitro, but not invasion

    Journal: Cancer Cell International

    doi: 10.1186/1475-2867-10-34

    Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).
    Figure Legend Snippet: Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).

    Techniques Used: Expressing, Multiple Displacement Amplification, Plasmid Preparation, Amplification, Construct, Stable Transfection, Immunostaining, FACS, Infection, TCA Precipitation, Western Blot

    25) Product Images from "Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins"

    Article Title: Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00943-15

    Hypomorphic telomerase does not show a synthetic phenotype with mrc1 AQ rad9 Δ mutation. (A) Southern blot analysis of terminal XhoI restriction fragments. DNA was probed with an α- 32 P-labeled 5′-(TGTGGG) 4 -3′ Y′ telomere-specific probe on samples from the indicated passages. (B and C) Streaks (B) and serial dilutions (C) of the hypomorphic TLC1 allele, tlc1-11 . The tlc1-11 mutation did not create synthetic lethality with mrc1 AQ rad9 Δ mutations. In order to ensure shortened telomeres, all the tlc1-11 haploid strains were obtained by sporulation of a homozygous tlc1-11/tlc1-11 diploid, which was heterozygous for all the other mutations. (D) MRC1 is phosphorylated as telomeres become critically short. A Western blot of protein extracts was made from the same cultures used in panels A and E and probed with 9E10 anti-myc antibody. The control was WT protein extract treated with 200 mM hydroxyurea for 2 h. *, phosphorylation of Mrc1-myc13 after the indicated passages. (E) Cell cycle phase assignments made from FACS data on samples at 40 (P1) and 60 (P2) generations by plotting the DNA content (CellQuest Pro), followed by curve fitting and quantification of DNA peaks into G 1 , S, and G 2 /M categories (Flow-Jo).
    Figure Legend Snippet: Hypomorphic telomerase does not show a synthetic phenotype with mrc1 AQ rad9 Δ mutation. (A) Southern blot analysis of terminal XhoI restriction fragments. DNA was probed with an α- 32 P-labeled 5′-(TGTGGG) 4 -3′ Y′ telomere-specific probe on samples from the indicated passages. (B and C) Streaks (B) and serial dilutions (C) of the hypomorphic TLC1 allele, tlc1-11 . The tlc1-11 mutation did not create synthetic lethality with mrc1 AQ rad9 Δ mutations. In order to ensure shortened telomeres, all the tlc1-11 haploid strains were obtained by sporulation of a homozygous tlc1-11/tlc1-11 diploid, which was heterozygous for all the other mutations. (D) MRC1 is phosphorylated as telomeres become critically short. A Western blot of protein extracts was made from the same cultures used in panels A and E and probed with 9E10 anti-myc antibody. The control was WT protein extract treated with 200 mM hydroxyurea for 2 h. *, phosphorylation of Mrc1-myc13 after the indicated passages. (E) Cell cycle phase assignments made from FACS data on samples at 40 (P1) and 60 (P2) generations by plotting the DNA content (CellQuest Pro), followed by curve fitting and quantification of DNA peaks into G 1 , S, and G 2 /M categories (Flow-Jo).

    Techniques Used: Mutagenesis, Southern Blot, Labeling, Western Blot, FACS, Flow Cytometry

    The ETI mrc1 AQ rad9 Δ synthetic phenotype and sm1 Δ rescue cannot be explained by changing telomere lengths or accelerated senescence. Shown is Southern blot analysis of terminal XhoI restriction fragments. DNA was probed with an α- 32 P-labeled 5′-(TGTGGG) 4 -3′ Y′-specific probe. The lowest band represents the DNA fragment containing the terminal telomeric repeats. Telomere lengths for the first (1) and second (2) passages after sporulation of a diploid heterozygous strain are represented for all the genotypes. *, grid lines are added to reveal the inherent “smile” in the gel and to allow better comparison of lanes.
    Figure Legend Snippet: The ETI mrc1 AQ rad9 Δ synthetic phenotype and sm1 Δ rescue cannot be explained by changing telomere lengths or accelerated senescence. Shown is Southern blot analysis of terminal XhoI restriction fragments. DNA was probed with an α- 32 P-labeled 5′-(TGTGGG) 4 -3′ Y′-specific probe. The lowest band represents the DNA fragment containing the terminal telomeric repeats. Telomere lengths for the first (1) and second (2) passages after sporulation of a diploid heterozygous strain are represented for all the genotypes. *, grid lines are added to reveal the inherent “smile” in the gel and to allow better comparison of lanes.

    Techniques Used: Southern Blot, Labeling

    26) Product Images from "Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained *"

    Article Title: Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.024612

    Schematic representation of the pBACgus-70 transfer vector construct. The coding sequence of the human hsp72 gene was cloned into baculovirus transfer plasmid pBACgus-2cp between HindIII and XhoI restriction sites to form the pBACgus-70 transfer vector.
    Figure Legend Snippet: Schematic representation of the pBACgus-70 transfer vector construct. The coding sequence of the human hsp72 gene was cloned into baculovirus transfer plasmid pBACgus-2cp between HindIII and XhoI restriction sites to form the pBACgus-70 transfer vector.

    Techniques Used: Plasmid Preparation, Construct, Sequencing, Clone Assay

    27) Product Images from "An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein"

    Article Title: An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043527

    Construction of plasmid pRLuc42R. The DmpR (1.692 kb) and its promoter (Pr) and operator (Po) were cloned from Pseudomonas sp. CF600. DNA segment was digested with XhoI and Hind III and introduced upstream of the luciferase gene in the pGL3 basic expression vector. The arrows indicate the transcription or processing direction for genes. (a) Digested and gel purified pGL3 vector and Gel Purified DmpR+Po Promoter for Ligation (b) Double digestion of pRLuc42R. (c) Vector drawing of pRLuc42R Fig (a) I-500 bp ladder, II-2377 bp (insert), III-4818 bp (vector) Fig (b) I-500 bp ladder, II-Double digested pRLuc42R.
    Figure Legend Snippet: Construction of plasmid pRLuc42R. The DmpR (1.692 kb) and its promoter (Pr) and operator (Po) were cloned from Pseudomonas sp. CF600. DNA segment was digested with XhoI and Hind III and introduced upstream of the luciferase gene in the pGL3 basic expression vector. The arrows indicate the transcription or processing direction for genes. (a) Digested and gel purified pGL3 vector and Gel Purified DmpR+Po Promoter for Ligation (b) Double digestion of pRLuc42R. (c) Vector drawing of pRLuc42R Fig (a) I-500 bp ladder, II-2377 bp (insert), III-4818 bp (vector) Fig (b) I-500 bp ladder, II-Double digested pRLuc42R.

    Techniques Used: Plasmid Preparation, Clone Assay, Luciferase, Expressing, Purification, Ligation

    28) 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

    29) Product Images from "Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells"

    Article Title: Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089546

    Schematic representation of restriction sites in 646-PCR amplicon. Specific restriction sites were used to distinguish between embryonic OCT4A transcript and different pseudogenes. Red arrows show restriction sites. ApaI restriction site is present only in embryonic OCT4A and can be used to distinguish embryonic form from all six pseudogenes; after restriction, a 146 bp and 500 bp long fragments are produced. HinfI digestion results in several smaller fragments among which the 434 bp fragment is specific only for OCT4-pg1. BglI digests only OCT4-pg3 into two fragments of 412 bp and 232 bp. XhoI does not digest OCT4-pg4.
    Figure Legend Snippet: Schematic representation of restriction sites in 646-PCR amplicon. Specific restriction sites were used to distinguish between embryonic OCT4A transcript and different pseudogenes. Red arrows show restriction sites. ApaI restriction site is present only in embryonic OCT4A and can be used to distinguish embryonic form from all six pseudogenes; after restriction, a 146 bp and 500 bp long fragments are produced. HinfI digestion results in several smaller fragments among which the 434 bp fragment is specific only for OCT4-pg1. BglI digests only OCT4-pg3 into two fragments of 412 bp and 232 bp. XhoI does not digest OCT4-pg4.

    Techniques Used: Polymerase Chain Reaction, Amplification, Produced

    30) Product Images from "RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation"

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp780

    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Figure Legend Snippet: Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Techniques Used: Southern Blot, Staining, Produced, Generated

    31) Product Images from "Optimizing the design of protein nanoparticles as carriers for vaccine applications"

    Article Title: Optimizing the design of protein nanoparticles as carriers for vaccine applications

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    doi: 10.1016/j.nano.2015.05.003

    (a) PDB search for protein structures with inter-helical angles similar to the mode of the nanoparticle followed by superposition of the helices of the different pdb-structures (blue and red) onto the pentamer (green) and trimer (blue) helices of the monomer of the peptide nanoparticle. The residues with angles similar to the angle between the helices of the pentamer and the trimer in the peptide nanoparticle were selected (gray region). ( b) Schematics of the molecular biology strategy for inserting the new linker region: the original construct with pentameric (green) and trimeric (blue) helices is double digested with restriction enzymes ApaI and XhoI. ( c) Linker oligonucleotides selected from panel a are ligated into double digested vector (panel b ) using T4 DNA ligase generating new plasmid which codes for the genetically modified single polypeptide chain.
    Figure Legend Snippet: (a) PDB search for protein structures with inter-helical angles similar to the mode of the nanoparticle followed by superposition of the helices of the different pdb-structures (blue and red) onto the pentamer (green) and trimer (blue) helices of the monomer of the peptide nanoparticle. The residues with angles similar to the angle between the helices of the pentamer and the trimer in the peptide nanoparticle were selected (gray region). ( b) Schematics of the molecular biology strategy for inserting the new linker region: the original construct with pentameric (green) and trimeric (blue) helices is double digested with restriction enzymes ApaI and XhoI. ( c) Linker oligonucleotides selected from panel a are ligated into double digested vector (panel b ) using T4 DNA ligase generating new plasmid which codes for the genetically modified single polypeptide chain.

    Techniques Used: Construct, Plasmid Preparation, Genetically Modified

    32) Product Images from "Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena"

    Article Title: Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena

    Journal: Genes

    doi: 10.3390/genes9040179

    Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.
    Figure Legend Snippet: Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.

    Techniques Used: Mutagenesis, Plasmid Preparation, Homologous Recombination, Southern Blot, Expressing, Molecular Weight, Western Blot

    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).
    Figure Legend Snippet: Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Techniques Used: Knock-Out, Plasmid Preparation, Homologous Recombination, Southern Blot, Molecular Weight

    33) Product Images from "Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice"

    Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI127277

    Generation and initial characterization of knockin mice harboring a Drp1S600A mutation . ( A ) Diagram of the domain structure of Drp1, illustrating the S600 site at the juncture of the VD and GED. PIM1, serine/threonine protein kinase Pim-1; CaN, calcineurin; PP2A, protein phosphatase 2A; PGAM5, phosphoglycerate mutase family member 5. ( B ) Structure of the Drp1-targeting locus, the Drp1- targeting construct, and the conditional allele after homologous recombination. The ScaI and XhoI sites used in Southern blot analysis and the location of the 3′ and 5′ probes are indicated. FRT, flippase recognition target (yellow triangles); FLP, flippase. ( C ) Gross appearance of WT, homozygous, and heterozygous Drp1S600A-knockin mice, and PCR sequence from genomic DNA showing mutation of the allele in the genome. ( D ) Southern blot analysis of ScaI and XhoI digested genomic DNA from mice of the indicated genotypes, showing the WT (12.7-kb) and mutant (5.8-kb) bands (upper panel). PCR genotyping of Drp1S600A heterozygosity and homozygosity (lower panel). Mutant and WT products are shown. ( E ) Cartesian allelic discrimination plot shows the relative levels of the Drp1S600A-mutant fluorescence signal for each sample plotted on the y axis and the WT signal on the x axis. Homozygous Drp1S600A (red dots), homozygous WT (blue dots), and heterozygous (violet dots) samples are shown. The no-template control is depicted by the black circle. ( F ) Western blot of Drp1 protein from mice of the three S600-mutant genotypes and densitometric quantification of Drp1 normalized to GAPDH protein expression. ( G ) Albumin/creatinine ratio (ACR) analysis of WT, heterozygous, and homozygous Drp1S600A-knockin mice at 20 weeks of age. ( H ) Mitochondrial AR from podocytes from mice of the three Drp1S600A genotypes as determined from TEM images. P
    Figure Legend Snippet: Generation and initial characterization of knockin mice harboring a Drp1S600A mutation . ( A ) Diagram of the domain structure of Drp1, illustrating the S600 site at the juncture of the VD and GED. PIM1, serine/threonine protein kinase Pim-1; CaN, calcineurin; PP2A, protein phosphatase 2A; PGAM5, phosphoglycerate mutase family member 5. ( B ) Structure of the Drp1-targeting locus, the Drp1- targeting construct, and the conditional allele after homologous recombination. The ScaI and XhoI sites used in Southern blot analysis and the location of the 3′ and 5′ probes are indicated. FRT, flippase recognition target (yellow triangles); FLP, flippase. ( C ) Gross appearance of WT, homozygous, and heterozygous Drp1S600A-knockin mice, and PCR sequence from genomic DNA showing mutation of the allele in the genome. ( D ) Southern blot analysis of ScaI and XhoI digested genomic DNA from mice of the indicated genotypes, showing the WT (12.7-kb) and mutant (5.8-kb) bands (upper panel). PCR genotyping of Drp1S600A heterozygosity and homozygosity (lower panel). Mutant and WT products are shown. ( E ) Cartesian allelic discrimination plot shows the relative levels of the Drp1S600A-mutant fluorescence signal for each sample plotted on the y axis and the WT signal on the x axis. Homozygous Drp1S600A (red dots), homozygous WT (blue dots), and heterozygous (violet dots) samples are shown. The no-template control is depicted by the black circle. ( F ) Western blot of Drp1 protein from mice of the three S600-mutant genotypes and densitometric quantification of Drp1 normalized to GAPDH protein expression. ( G ) Albumin/creatinine ratio (ACR) analysis of WT, heterozygous, and homozygous Drp1S600A-knockin mice at 20 weeks of age. ( H ) Mitochondrial AR from podocytes from mice of the three Drp1S600A genotypes as determined from TEM images. P

    Techniques Used: Knock-In, Mouse Assay, Mutagenesis, Construct, Homologous Recombination, Southern Blot, Polymerase Chain Reaction, Sequencing, Fluorescence, Western Blot, Expressing, Transmission Electron Microscopy

    34) Product Images from "STAR: a simple TAL effector assembly reaction using isothermal assembly"

    Article Title: STAR: a simple TAL effector assembly reaction using isothermal assembly

    Journal: Scientific Reports

    doi: 10.1038/srep33209

    Full-length 16mer TALEs in destination vectors are generated in a second Gibson reaction. ( a ) Schematic of fully assembled TALE into destination vector showing both the primer binding sites for colony PCR and XhoI/NheI restriction sites for digest of TALE-TFs. ( b ) Colony PCR of bacterial colonies expressing a typical TALE-TF reaction. Clones expressing the fully assembled TALE-TF show amplicons of about 1.7 kb length (↓– clone potentially expressing full-length DNA binding domain; * – clones shown in panel c). ( c ) Clones identified in colony PCR are further characterised by restriction digest yielding a 2.4 kb fragment for fully assembled TALE-TFs (* – sequence-verified clones expressing full-length TALE-TF).
    Figure Legend Snippet: Full-length 16mer TALEs in destination vectors are generated in a second Gibson reaction. ( a ) Schematic of fully assembled TALE into destination vector showing both the primer binding sites for colony PCR and XhoI/NheI restriction sites for digest of TALE-TFs. ( b ) Colony PCR of bacterial colonies expressing a typical TALE-TF reaction. Clones expressing the fully assembled TALE-TF show amplicons of about 1.7 kb length (↓– clone potentially expressing full-length DNA binding domain; * – clones shown in panel c). ( c ) Clones identified in colony PCR are further characterised by restriction digest yielding a 2.4 kb fragment for fully assembled TALE-TFs (* – sequence-verified clones expressing full-length TALE-TF).

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

    35) Product Images from "Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli"

    Article Title: Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli

    Journal: mBio

    doi: 10.1128/mBio.01443-16

    Linear dimer recombination assay. (A) The linear dimer substrate is depicted, with genes and mutations indicated. Each of the two tet mutations generates XhoI restriction sites. Normal PstI sites are located in the bla genes. The dimer is made linear by digestion at a unique BamHI site; the star indicates the location of a second defective BamHI site. (B) The 5′-to-3′ dsDNA exonuclease, either λ Exo or Rac RecE, degrades the 5′ strands of the linear dsDNA to reveal 3′ overhangs to which the λ Beta or Rac RecT recombinase binds. (C) The recombinase anneals the internal complementary ssDNA regions to form a circular monomeric plasmid intermediate with 3′ ssDNA tails. Host functions presumably remove the long single-stranded 3′ ends, with DNA ligase sealing the resulting nicks.
    Figure Legend Snippet: Linear dimer recombination assay. (A) The linear dimer substrate is depicted, with genes and mutations indicated. Each of the two tet mutations generates XhoI restriction sites. Normal PstI sites are located in the bla genes. The dimer is made linear by digestion at a unique BamHI site; the star indicates the location of a second defective BamHI site. (B) The 5′-to-3′ dsDNA exonuclease, either λ Exo or Rac RecE, degrades the 5′ strands of the linear dsDNA to reveal 3′ overhangs to which the λ Beta or Rac RecT recombinase binds. (C) The recombinase anneals the internal complementary ssDNA regions to form a circular monomeric plasmid intermediate with 3′ ssDNA tails. Host functions presumably remove the long single-stranded 3′ ends, with DNA ligase sealing the resulting nicks.

    Techniques Used: Recombination Assay, Plasmid Preparation

    36) Product Images from "Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters"

    Article Title: Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt731

    Duplexes carrying damaged sites used in this study. Oligonucleotides carrying oG, hU and U. The modified bases are indicated in bold. oG was separated by 3 bp from hU located on the complementary strand in MDS/+1, MDS/−5. Uracil was positioned at +1 position relatively to hU in MDS/+1 and at −5 position in MDS/−5. The control U/U oligonucleotide consists of two bistranded Us separated by 5 bp. Undamaged and MDS-containing 56-mers duplexes harbor EcoRI and XhoI restriction sites at each extremity as indicated.
    Figure Legend Snippet: Duplexes carrying damaged sites used in this study. Oligonucleotides carrying oG, hU and U. The modified bases are indicated in bold. oG was separated by 3 bp from hU located on the complementary strand in MDS/+1, MDS/−5. Uracil was positioned at +1 position relatively to hU in MDS/+1 and at −5 position in MDS/−5. The control U/U oligonucleotide consists of two bistranded Us separated by 5 bp. Undamaged and MDS-containing 56-mers duplexes harbor EcoRI and XhoI restriction sites at each extremity as indicated.

    Techniques Used: Modification

    37) Product Images from "Analyzing the Branch Migration Activities of Eukaryotic Proteins"

    Article Title: Analyzing the Branch Migration Activities of Eukaryotic Proteins

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2010.02.010

    Production of joint molecules used to test the branch migration activity of proteins. (A) Joint molecules with a 3′ displaced ssDNA tail are produced by RAD51 using gapped DNA and pBS II K (+) dsDNA linearized with XhoI. (B) Joint molecules with
    Figure Legend Snippet: Production of joint molecules used to test the branch migration activity of proteins. (A) Joint molecules with a 3′ displaced ssDNA tail are produced by RAD51 using gapped DNA and pBS II K (+) dsDNA linearized with XhoI. (B) Joint molecules with

    Techniques Used: Migration, Activity Assay, Produced

    The scheme used to produce gapped DNA. i) pBS II K (+) plasmid DNA is digested with XhoI and AlwNI. ii) The large DNA fragment from this digest is purified by gel electrophoresis in agarose gels, and then iii) annealed to circular ssDNA to generate gapped
    Figure Legend Snippet: The scheme used to produce gapped DNA. i) pBS II K (+) plasmid DNA is digested with XhoI and AlwNI. ii) The large DNA fragment from this digest is purified by gel electrophoresis in agarose gels, and then iii) annealed to circular ssDNA to generate gapped

    Techniques Used: Plasmid Preparation, Purification, Nucleic Acid Electrophoresis

    Illustration of the 0.8% agarose gel used to purify the large (2065 bp) dsDNA fragment of pBS II K (+) following digestion with XhoI and AlwNI. After electrophoresis, lanes A, C, and E are excised from the gel and stained with ethidium bromide (dashed
    Figure Legend Snippet: Illustration of the 0.8% agarose gel used to purify the large (2065 bp) dsDNA fragment of pBS II K (+) following digestion with XhoI and AlwNI. After electrophoresis, lanes A, C, and E are excised from the gel and stained with ethidium bromide (dashed

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Staining

    38) Product Images from "Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System"

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030851

    Experimental design for SDF and cell clone generation. A) SDF sequence is homologous to the entire wild type eGFP coding sequence. SDF-PCR-WT, 876 bp long was generated by PCR amplification with primer pair 1F/1R ( Table 1 ). SDF-DIG-WT, 752 bp long, was obtained by HindIII and XhoI digestion of pCR-2.1 vector. C/T transition, responsible of fluorescence switching off, is showed. B) Sequencing analysis showing wild type (WT; top panel) and mutated (Mut; bottom panel) pCEP4-eGFP in C1 and D1 cell clones, respectively. Arrows indicate the modified base (C→T). C) FACS density plot of C1 (WT; top) and D1 (Mut; bottom) respectively. D) pCEP4-eGFP copy number determination for each cell clone.
    Figure Legend Snippet: Experimental design for SDF and cell clone generation. A) SDF sequence is homologous to the entire wild type eGFP coding sequence. SDF-PCR-WT, 876 bp long was generated by PCR amplification with primer pair 1F/1R ( Table 1 ). SDF-DIG-WT, 752 bp long, was obtained by HindIII and XhoI digestion of pCR-2.1 vector. C/T transition, responsible of fluorescence switching off, is showed. B) Sequencing analysis showing wild type (WT; top panel) and mutated (Mut; bottom panel) pCEP4-eGFP in C1 and D1 cell clones, respectively. Arrows indicate the modified base (C→T). C) FACS density plot of C1 (WT; top) and D1 (Mut; bottom) respectively. D) pCEP4-eGFP copy number determination for each cell clone.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Generated, Amplification, Plasmid Preparation, Fluorescence, Clone Assay, Modification, FACS

    39) Product Images from "Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length †"

    Article Title: Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.3.896-906.2005

    Oligonucleotide-modified plasmid assay. (A) Plasmid modification scheme. pTW423 is digested with BglII and XhoI and purified, removing the polyterminator. Annealed oligonucleotides are then ligated onto the BglII and XhoI ends, restoring the ADE2 coding sequence. Precise in-frame repair of the break yields Ade + colonies. (B) Primer extension assay to determine the oligonucleotide ligation efficiency. Annealed oligonucleotides (Oligos) were added to the ligation reaction mixture at concentrations of 50, 100, 500, 1,000, and 5,000-fold molar excess over the concentration of the linearized plasmid (indicated by the thickness of the triangle over the lanes). Primer extension was performed after ligation as described in Materials and Methods.
    Figure Legend Snippet: Oligonucleotide-modified plasmid assay. (A) Plasmid modification scheme. pTW423 is digested with BglII and XhoI and purified, removing the polyterminator. Annealed oligonucleotides are then ligated onto the BglII and XhoI ends, restoring the ADE2 coding sequence. Precise in-frame repair of the break yields Ade + colonies. (B) Primer extension assay to determine the oligonucleotide ligation efficiency. Annealed oligonucleotides (Oligos) were added to the ligation reaction mixture at concentrations of 50, 100, 500, 1,000, and 5,000-fold molar excess over the concentration of the linearized plasmid (indicated by the thickness of the triangle over the lanes). Primer extension was performed after ligation as described in Materials and Methods.

    Techniques Used: Modification, Plasmid Preparation, Purification, Sequencing, Primer Extension Assay, Ligation, Concentration Assay

    40) Product Images from "DNA electrophoresis in a nanofence array"

    Article Title: DNA electrophoresis in a nanofence array

    Journal: Lab on a chip

    doi: 10.1039/c2lc00016d

    Evolution of the bands during DNA electrophoresis at 10 V/cm. Multiple scans of the separation channel are acquired at different times during the run, making it possible to monitor the continuous separation of λ and the XhoI λ-digest.
    Figure Legend Snippet: Evolution of the bands during DNA electrophoresis at 10 V/cm. Multiple scans of the separation channel are acquired at different times during the run, making it possible to monitor the continuous separation of λ and the XhoI λ-digest.

    Techniques Used: Nucleic Acid Electrophoresis

    Related Articles

    Polymerase Chain Reaction:

    Article Title:
    Article Snippet: .. Restriction enzyme analysis of PCR products containing the CYP2B6 exon-9 region was performed by incubating 2 μ g DNA with 100 U XhoI (NEB) overnight. .. Total RNA was isolated using Trizol (Invitrogen).

    Article Title: A partial form of recessive STAT1 deficiency in humans
    Article Snippet: .. PCR products and empty pSPL3 plasmids (provided by Ralph Burkhardt, The Rockefeller University) were digested with Xho I and BamH 1 (New England BioLabs). .. Plasmids were then dephosphorylated, and 50 ng of purified plasmids and 250 ng of purified PCR products were ligated with T4 ligase (New England BioLabs).

    Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies
    Article Snippet: .. The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions. .. The pCL6-2AEGwo lentiviral backbone vector, a kind gift from Dr. Zanxian Xia, School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China, contains both a “ribosome-skip” fragment encoding the 2A-like peptide APVKQTLNFDLLKLAGDVESNPGP and an in-frame eGFP fluorescent coding sequence downstream of a multiple cloning site.

    Incubation:

    Article Title: Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers
    Article Snippet: .. Linear DNA (l-DNA) Purified c-DNA was linearized using the restriction enzyme, Xho I (New England Biolabs; Pickering, ON) for pORF9-hTNFRS11b or Cla I (Invitrogen; Burlington, ON) for pEGFP-N2, Restriction digestion were set up with 5 μg of DNA per 50 μL of reaction volume containing 3 units of enzyme and incubated at 37°C for 16 hours. .. The enzyme was then heat-inactivated by incubating the mixture at 65°C for 10 min. Digested DNA was purified using QIAEX II Gel Extraction Kit (Qiagen, Mississauga, ON).

    Agarose Gel Electrophoresis:

    Article Title: Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site
    Article Snippet: .. The DNA samples were digested with 10 U of Xho I for 2 h at 37 °C (New England BioLabs) and electrophoresed in a 0.8% agarose gel in 0.5X TBE. .. Results Since the BAC cloning vector pBeloBAc11 was commercially available from New England BioLabs, I decided to modify this plasmid to increase its utility for genomics studies.

    Plasmid Preparation:

    Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies
    Article Snippet: .. The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions. .. The pCL6-2AEGwo lentiviral backbone vector, a kind gift from Dr. Zanxian Xia, School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China, contains both a “ribosome-skip” fragment encoding the 2A-like peptide APVKQTLNFDLLKLAGDVESNPGP and an in-frame eGFP fluorescent coding sequence downstream of a multiple cloning site.

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs). .. The ExoIII-digested DNAs were treated with S1 nuclease (Sigma) followed by a fill-in reaction with the Klenow fragment of DNA polymerase I, ligation, and transformation into DH5αMCR cells (Gibco BRL).

    Purification:

    Article Title: Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers
    Article Snippet: .. Linear DNA (l-DNA) Purified c-DNA was linearized using the restriction enzyme, Xho I (New England Biolabs; Pickering, ON) for pORF9-hTNFRS11b or Cla I (Invitrogen; Burlington, ON) for pEGFP-N2, Restriction digestion were set up with 5 μg of DNA per 50 μL of reaction volume containing 3 units of enzyme and incubated at 37°C for 16 hours. .. The enzyme was then heat-inactivated by incubating the mixture at 65°C for 10 min. Digested DNA was purified using QIAEX II Gel Extraction Kit (Qiagen, Mississauga, ON).

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    New England Biolabs xhoi restriction sites underlined
    Effect of CD22ΔE12 vs. CD22 WT on clonogenicity and self-renewal rate of ALL cells. [A1] Depicted is a representative 1% agarose gel showing the PCR products of FL CD22 and CD22ΔE12–14. [A.2] Subcloning of the CD22 FL or CD22ΔE12–14 genes into the <t>lentiviral</t> pCL6-2AEGwo vector through NheI and <t>XhoI</t> restriction sites was confirmed through restriction enzyme digestion and DNA sequencing. Depicted is a representative 1% agarose gel showing that the generated lentiviral constructs have the correct size FL CD22 (2.5-kb) or CD22ΔE12–14 (2.2-kb) inserts. [B.1] In order to confirm that the lentiviral vectors can be used to achieve expression of FL and truncated CD22 in human cells, 293T cells were transfected with lentiviral constructs for FL CD22, CD22ΔE12–14, as well as pCL6-2AEGwo lentiviral vector without any subcloned CD22. 48 h post transfection cells were examined for FL CD22 and CD22ΔE12–14 mRNA by RT-PCR using the P7, P9, and P10 primer pairs. The P7 primer set was used to amplify a 182-bp region (c.2180–c.2361) of the CD22 cDNA extending from Exon 11 to Exon 13 and spanning the entire Exon 12. The P9 primer set was used amplify a 160-bp region (c.2304–c.2463) of the CD22 cDNA extending from Exon 12 to Exon 14 and spanning the entire Exon 13. The P10 primer set was used to amplify a 213-bp region (c.433–c.645) of Exon 4 of the CD22 cDNA present in both wildtype CD22 and CD22ΔE12–14 mRNA species. As expected all primer sets yielded PCR products in cells transfected with the FL CD22 vector and only the P10 primer set yielded a PCR product in cells transfected with the CD22ΔE12–14 vector. [B.2 and B3] The increased expression levels of the full-length and truncated proteins in transduced 293-T cells (depicted in B.2) and ALL-1 cells (depicted in B.3) were confirmed to be similar by Western blot analysis done at 96 h post-transduction. [C D] ALL cell lines DAUDI (Burkitt's leukemia/B-ALL) (panels C1 C.2) and ALL1 (BCR-ABL + B-precursor ALL) (panel C3) were transduced (trans) with wildtype and mutant human CD22 genes using the pCL6-2AEGwo lentiviral vector and then assayed for colony formation in semi-solid methylcellulose cultures without additional stroma support or cytokines. Colony formation was examined using an inverted Nikon Eclipse TS100 microscope with an Epifluorescence attachment. Images were taken using a Digital Sight DS-2MBW Nikon camera (System magnification: 100× or 200 × as indicated). Green fluorescence of colonies resulting from GFP expression confirms successful transduction of the cell lines. [D] Depicted are bar graphs comparing the mean colony numbers in cultures of untransduced and transduced cells.
    Xhoi Restriction Sites Underlined, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1030 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of CD22ΔE12 vs. CD22 WT on clonogenicity and self-renewal rate of ALL cells. [A1] Depicted is a representative 1% agarose gel showing the PCR products of FL CD22 and CD22ΔE12–14. [A.2] Subcloning of the CD22 FL or CD22ΔE12–14 genes into the lentiviral pCL6-2AEGwo vector through NheI and XhoI restriction sites was confirmed through restriction enzyme digestion and DNA sequencing. Depicted is a representative 1% agarose gel showing that the generated lentiviral constructs have the correct size FL CD22 (2.5-kb) or CD22ΔE12–14 (2.2-kb) inserts. [B.1] In order to confirm that the lentiviral vectors can be used to achieve expression of FL and truncated CD22 in human cells, 293T cells were transfected with lentiviral constructs for FL CD22, CD22ΔE12–14, as well as pCL6-2AEGwo lentiviral vector without any subcloned CD22. 48 h post transfection cells were examined for FL CD22 and CD22ΔE12–14 mRNA by RT-PCR using the P7, P9, and P10 primer pairs. The P7 primer set was used to amplify a 182-bp region (c.2180–c.2361) of the CD22 cDNA extending from Exon 11 to Exon 13 and spanning the entire Exon 12. The P9 primer set was used amplify a 160-bp region (c.2304–c.2463) of the CD22 cDNA extending from Exon 12 to Exon 14 and spanning the entire Exon 13. The P10 primer set was used to amplify a 213-bp region (c.433–c.645) of Exon 4 of the CD22 cDNA present in both wildtype CD22 and CD22ΔE12–14 mRNA species. As expected all primer sets yielded PCR products in cells transfected with the FL CD22 vector and only the P10 primer set yielded a PCR product in cells transfected with the CD22ΔE12–14 vector. [B.2 and B3] The increased expression levels of the full-length and truncated proteins in transduced 293-T cells (depicted in B.2) and ALL-1 cells (depicted in B.3) were confirmed to be similar by Western blot analysis done at 96 h post-transduction. [C D] ALL cell lines DAUDI (Burkitt's leukemia/B-ALL) (panels C1 C.2) and ALL1 (BCR-ABL + B-precursor ALL) (panel C3) were transduced (trans) with wildtype and mutant human CD22 genes using the pCL6-2AEGwo lentiviral vector and then assayed for colony formation in semi-solid methylcellulose cultures without additional stroma support or cytokines. Colony formation was examined using an inverted Nikon Eclipse TS100 microscope with an Epifluorescence attachment. Images were taken using a Digital Sight DS-2MBW Nikon camera (System magnification: 100× or 200 × as indicated). Green fluorescence of colonies resulting from GFP expression confirms successful transduction of the cell lines. [D] Depicted are bar graphs comparing the mean colony numbers in cultures of untransduced and transduced cells.

    Journal: EBioMedicine

    Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies

    doi: 10.1016/j.ebiom.2014.10.013

    Figure Lengend Snippet: Effect of CD22ΔE12 vs. CD22 WT on clonogenicity and self-renewal rate of ALL cells. [A1] Depicted is a representative 1% agarose gel showing the PCR products of FL CD22 and CD22ΔE12–14. [A.2] Subcloning of the CD22 FL or CD22ΔE12–14 genes into the lentiviral pCL6-2AEGwo vector through NheI and XhoI restriction sites was confirmed through restriction enzyme digestion and DNA sequencing. Depicted is a representative 1% agarose gel showing that the generated lentiviral constructs have the correct size FL CD22 (2.5-kb) or CD22ΔE12–14 (2.2-kb) inserts. [B.1] In order to confirm that the lentiviral vectors can be used to achieve expression of FL and truncated CD22 in human cells, 293T cells were transfected with lentiviral constructs for FL CD22, CD22ΔE12–14, as well as pCL6-2AEGwo lentiviral vector without any subcloned CD22. 48 h post transfection cells were examined for FL CD22 and CD22ΔE12–14 mRNA by RT-PCR using the P7, P9, and P10 primer pairs. The P7 primer set was used to amplify a 182-bp region (c.2180–c.2361) of the CD22 cDNA extending from Exon 11 to Exon 13 and spanning the entire Exon 12. The P9 primer set was used amplify a 160-bp region (c.2304–c.2463) of the CD22 cDNA extending from Exon 12 to Exon 14 and spanning the entire Exon 13. The P10 primer set was used to amplify a 213-bp region (c.433–c.645) of Exon 4 of the CD22 cDNA present in both wildtype CD22 and CD22ΔE12–14 mRNA species. As expected all primer sets yielded PCR products in cells transfected with the FL CD22 vector and only the P10 primer set yielded a PCR product in cells transfected with the CD22ΔE12–14 vector. [B.2 and B3] The increased expression levels of the full-length and truncated proteins in transduced 293-T cells (depicted in B.2) and ALL-1 cells (depicted in B.3) were confirmed to be similar by Western blot analysis done at 96 h post-transduction. [C D] ALL cell lines DAUDI (Burkitt's leukemia/B-ALL) (panels C1 C.2) and ALL1 (BCR-ABL + B-precursor ALL) (panel C3) were transduced (trans) with wildtype and mutant human CD22 genes using the pCL6-2AEGwo lentiviral vector and then assayed for colony formation in semi-solid methylcellulose cultures without additional stroma support or cytokines. Colony formation was examined using an inverted Nikon Eclipse TS100 microscope with an Epifluorescence attachment. Images were taken using a Digital Sight DS-2MBW Nikon camera (System magnification: 100× or 200 × as indicated). Green fluorescence of colonies resulting from GFP expression confirms successful transduction of the cell lines. [D] Depicted are bar graphs comparing the mean colony numbers in cultures of untransduced and transduced cells.

    Article Snippet: The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Subcloning, Plasmid Preparation, DNA Sequencing, Generated, Construct, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transduction, Mutagenesis, Microscopy, Fluorescence

    A Low-Copy Repeat Sequence Identifies Restriction Fragments Distal to the AVR-Pita Telomere That Are Deleted in Some Spontaneous Mutants. Genomic DNAs were digested with BglII for DNA gel blot analysis. Hybridization with the 10-kb XhoI fragment from cosmid A10H8 identified the 6.5-kb telomeric BglII fragment and an additional 4.0-kb fragment (arrows) that was altered in avr-pita − mutants. All avirulent laboratory strains inherited these two bands from the Chinese field isolate O-137 (lane 23) through the RFLP mapping strain 4224-7-8 (lane 2). The virulent mapping parent 6043 (lane 1) does not have these bands. Not shown are data consistent with this result but obtained by using SalI, EcoRI, and SacI. Except for lane 1, which contains DNA from the virulent parent 6043, each v lane contains DNA from a virulent mutant obtained from the first avirulent strain (A lanes) to its left. Lane 1, 6043 (v); lane 2, 4224-7-8 (A); lane 3, 4360-17-1 (A); lane 4, CP917 (v); lane 5, 4375-R-26 (A); lane 6, CP984 (v); lane 7, 4375-R-39 (A); lane 8, CP918 (v); lane 9, 4375-R-6 (A); lane 10, CP983 (v); lane 11, CP1614 (v); lane 12, CP1615 (v); lane 13, CP1631 (A); lane 14, CP1632 (v); lane 15, CP1634 (A); lane 16, CP1635 (v); lane 17, CP1637 (A); lane 18, CP1638 (v); lane 19, CP1640 (A); lane 20, CP1641 (v); lane 21, CP1643 (A); lane 22, CP1644 (v); and lane 23, O-137 (A). Markers at left identify the positions of λ HindIII DNA length standards, which are as follows (from the top): 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kb.

    Journal: The Plant Cell

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta

    doi:

    Figure Lengend Snippet: A Low-Copy Repeat Sequence Identifies Restriction Fragments Distal to the AVR-Pita Telomere That Are Deleted in Some Spontaneous Mutants. Genomic DNAs were digested with BglII for DNA gel blot analysis. Hybridization with the 10-kb XhoI fragment from cosmid A10H8 identified the 6.5-kb telomeric BglII fragment and an additional 4.0-kb fragment (arrows) that was altered in avr-pita − mutants. All avirulent laboratory strains inherited these two bands from the Chinese field isolate O-137 (lane 23) through the RFLP mapping strain 4224-7-8 (lane 2). The virulent mapping parent 6043 (lane 1) does not have these bands. Not shown are data consistent with this result but obtained by using SalI, EcoRI, and SacI. Except for lane 1, which contains DNA from the virulent parent 6043, each v lane contains DNA from a virulent mutant obtained from the first avirulent strain (A lanes) to its left. Lane 1, 6043 (v); lane 2, 4224-7-8 (A); lane 3, 4360-17-1 (A); lane 4, CP917 (v); lane 5, 4375-R-26 (A); lane 6, CP984 (v); lane 7, 4375-R-39 (A); lane 8, CP918 (v); lane 9, 4375-R-6 (A); lane 10, CP983 (v); lane 11, CP1614 (v); lane 12, CP1615 (v); lane 13, CP1631 (A); lane 14, CP1632 (v); lane 15, CP1634 (A); lane 16, CP1635 (v); lane 17, CP1637 (A); lane 18, CP1638 (v); lane 19, CP1640 (A); lane 20, CP1641 (v); lane 21, CP1643 (A); lane 22, CP1644 (v); and lane 23, O-137 (A). Markers at left identify the positions of λ HindIII DNA length standards, which are as follows (from the top): 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kb.

    Article Snippet: Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs).

    Techniques: Sequencing, Western Blot, Hybridization, Mutagenesis

    Confirmation that CYP2F1 was inactivated and not expressed. (A) DNA sequence determined for WT and mutant CYP2F1 exon-10 region. The CYS codon (blue) and three additional nucleotides in WT CYP2F1 was replaced by an XhoI restriction site (red) and a loxP

    Journal: Drug Metabolism and Disposition

    Article Title:

    doi: 10.1124/dmd.114.059188

    Figure Lengend Snippet: Confirmation that CYP2F1 was inactivated and not expressed. (A) DNA sequence determined for WT and mutant CYP2F1 exon-10 region. The CYS codon (blue) and three additional nucleotides in WT CYP2F1 was replaced by an XhoI restriction site (red) and a loxP

    Article Snippet: Restriction enzyme analysis of PCR products containing the CYP2B6 exon-9 region was performed by incubating 2 μ g DNA with 100 U XhoI (NEB) overnight.

    Techniques: Sequencing, Mutagenesis

    Structure of the DNA molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.

    Journal: BMC Biotechnology

    Article Title: Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers

    doi: 10.1186/1472-6750-8-23

    Figure Lengend Snippet: Structure of the DNA molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.

    Article Snippet: Linear DNA (l-DNA) Purified c-DNA was linearized using the restriction enzyme, Xho I (New England Biolabs; Pickering, ON) for pORF9-hTNFRS11b or Cla I (Invitrogen; Burlington, ON) for pEGFP-N2, Restriction digestion were set up with 5 μg of DNA per 50 μL of reaction volume containing 3 units of enzyme and incubated at 37°C for 16 hours.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Generated, Amplification, Transfection, Sequencing