75c  (New England Biolabs)


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    MfeI HF
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    MfeI HF 2 500 units
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    r3589l
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    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    New England Biolabs 75c
    MfeI HF
    MfeI HF 2 500 units
    https://www.bioz.com/result/75c/product/New England Biolabs
    Average 85 stars, based on 16982 article reviews
    Price from $9.99 to $1999.99
    75c - by Bioz Stars, 2020-09
    85/100 stars

    Images

    1) Product Images from "Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection"

    Article Title: Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002781

    ORF75c − MuHV-4 shows defective immediate early gene expression. A. BHK-21 cells were infected (1000 genomes/cell) with wild-type (WT) or ORF75c − .4 (ORF75c − ) virions. DNA and RNA were recovered at 6 h and 24 h post-infection. RNA was reverse transcribed using ORF50, ORF73 and 18S rRNA-specific primers and cDNAs quantitated by real-time PCR. Controls without reverse transcriptase were all negative. Each sample was run in triplicate and mean copy numbers determined by comparison with known template dilutions. Viral genome numbers were quantitated by amplificating 100 ng DNA with MuHV-4 M2-specific primers and comparing with known template dilutions. B. BHK-21 cells were infected with ORF75c + (WT, rev.1) or ORF75c − (75c − .4, 75c − .7, 75c − .9) viruses (2 p.f.u./cell or an equivalent number of genomes), and 18 h later lysed in situ in agarose gels. UI = uninfected cells. Circular and linear genomes were distinguished by electrophoretic mobility and comparison with circular genomic BACs, which differ in size due to different numbers of terminal repeats. Viral genomes were identified by probing with a labelled terminal repeat fragment. The boxed area was exposed for a longer time to visualize ORF75c − genomes. C. BHK-21 cells were infected with BAC + ORF75c − MuHV-4 (1000 genomes/cell), then 24 h later super-infected with BAC − ORF50 − MuHV-4 (1000 genomes/cell), then 24 h later analyzed for BAC-based eGFP expression by flow cytometry. Each bar shows 20,000 cells. The data are from 1 of 3 equivalent experiments. The increase in eGFP expression with ORF50 − superinfection was highly significant (p
    Figure Legend Snippet: ORF75c − MuHV-4 shows defective immediate early gene expression. A. BHK-21 cells were infected (1000 genomes/cell) with wild-type (WT) or ORF75c − .4 (ORF75c − ) virions. DNA and RNA were recovered at 6 h and 24 h post-infection. RNA was reverse transcribed using ORF50, ORF73 and 18S rRNA-specific primers and cDNAs quantitated by real-time PCR. Controls without reverse transcriptase were all negative. Each sample was run in triplicate and mean copy numbers determined by comparison with known template dilutions. Viral genome numbers were quantitated by amplificating 100 ng DNA with MuHV-4 M2-specific primers and comparing with known template dilutions. B. BHK-21 cells were infected with ORF75c + (WT, rev.1) or ORF75c − (75c − .4, 75c − .7, 75c − .9) viruses (2 p.f.u./cell or an equivalent number of genomes), and 18 h later lysed in situ in agarose gels. UI = uninfected cells. Circular and linear genomes were distinguished by electrophoretic mobility and comparison with circular genomic BACs, which differ in size due to different numbers of terminal repeats. Viral genomes were identified by probing with a labelled terminal repeat fragment. The boxed area was exposed for a longer time to visualize ORF75c − genomes. C. BHK-21 cells were infected with BAC + ORF75c − MuHV-4 (1000 genomes/cell), then 24 h later super-infected with BAC − ORF50 − MuHV-4 (1000 genomes/cell), then 24 h later analyzed for BAC-based eGFP expression by flow cytometry. Each bar shows 20,000 cells. The data are from 1 of 3 equivalent experiments. The increase in eGFP expression with ORF50 − superinfection was highly significant (p

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, In Situ, BAC Assay, Flow Cytometry, Cytometry

    No evidence for ORF75c deficiency compromising virion morphogenesis. A. Wild-type (WT), ORF75c − (75c − .4, 75c − .7, 75c − .9) and revertant (rev.1, rev.2) virus stocks were normalized by genome content, denatured in Laemmli's buffer and immunoblotted for the ORF17 capsid component with mAb 150-7D1, for gB (C-terminal furin cleavage product) with mAb MG-4D11, and for thymidine kinase (TK) with mAb CS-4A5. B. Virus stocks were denatured, resolved by SDS-PAGE and analyzed by Coomassie staining. The positions of bovine albumin (alb, 63 kDa), the ORF25 capsid component (160 kDa) and the ORFs 75a/b/c (150 kDa) are shown. C. BHK-21 cells were infected with wild-type (WT) MuHV-4 (1 p.f.u./cell, 18 h) or ORF75c − .4 (ORF75c − ) MuHV-4 to an equivalent infection level based on viral eGFP expression, then processed for transmission electron microscopy. The scale bars show 1 µM in the overview images. nuc = nucleus, cyt = cytoplasm. The boxed regions in the overview images correspond to the zoomed images 1–4. The arrows show examples of mature virions.
    Figure Legend Snippet: No evidence for ORF75c deficiency compromising virion morphogenesis. A. Wild-type (WT), ORF75c − (75c − .4, 75c − .7, 75c − .9) and revertant (rev.1, rev.2) virus stocks were normalized by genome content, denatured in Laemmli's buffer and immunoblotted for the ORF17 capsid component with mAb 150-7D1, for gB (C-terminal furin cleavage product) with mAb MG-4D11, and for thymidine kinase (TK) with mAb CS-4A5. B. Virus stocks were denatured, resolved by SDS-PAGE and analyzed by Coomassie staining. The positions of bovine albumin (alb, 63 kDa), the ORF25 capsid component (160 kDa) and the ORFs 75a/b/c (150 kDa) are shown. C. BHK-21 cells were infected with wild-type (WT) MuHV-4 (1 p.f.u./cell, 18 h) or ORF75c − .4 (ORF75c − ) MuHV-4 to an equivalent infection level based on viral eGFP expression, then processed for transmission electron microscopy. The scale bars show 1 µM in the overview images. nuc = nucleus, cyt = cytoplasm. The boxed regions in the overview images correspond to the zoomed images 1–4. The arrows show examples of mature virions.

    Techniques Used: SDS Page, Staining, Infection, Expressing, Transmission Assay, Electron Microscopy

    Generation of ORF75c − MuHV-4 mutants. A. Schematic diagram of the ORF75c genomic locus and the introduced stop oligo mutation. B. Southern blot of viral DNA recovered from wild-type (WT), ORF75c mutant (75c − .4, 75c − .7, 75c − .9) and 75c − .4 revertant (75c + rev.1) virus stocks. The viral DNA was digested with Eco RI and probed with the labelled Bam HI-H genomic fragment shown in A. There is an invariant band of 5186 bp. The wild-type 7776 bp band shifts to 7048 bp in the mutant. The small mutant band of 728 bp is not visible on this gel. C. Two ORF75c − mutants were compared with wild-type and revertant viruses for infectivity. The viral genome content of each stock was determined by real-time PCR, and equivalent genome numbers used to infected BHK-21 cells (18 h, 37°C). The number of infected cells was determined by flow cytometry of eGFP expression from the MuHV-4 BAC cassette. D. BHK-21 cells were infected with ORF75c − (ORF75c − .4, ORF75c − .7) or ORF75c + (wild-type, revertant.1) viruses at 10 genomes/cell - equivalent for the wild-type and revertant to 0.01 p.f.u./cell. The spread of infection with time was assayed by flow cytometry of viral eGFP expression.
    Figure Legend Snippet: Generation of ORF75c − MuHV-4 mutants. A. Schematic diagram of the ORF75c genomic locus and the introduced stop oligo mutation. B. Southern blot of viral DNA recovered from wild-type (WT), ORF75c mutant (75c − .4, 75c − .7, 75c − .9) and 75c − .4 revertant (75c + rev.1) virus stocks. The viral DNA was digested with Eco RI and probed with the labelled Bam HI-H genomic fragment shown in A. There is an invariant band of 5186 bp. The wild-type 7776 bp band shifts to 7048 bp in the mutant. The small mutant band of 728 bp is not visible on this gel. C. Two ORF75c − mutants were compared with wild-type and revertant viruses for infectivity. The viral genome content of each stock was determined by real-time PCR, and equivalent genome numbers used to infected BHK-21 cells (18 h, 37°C). The number of infected cells was determined by flow cytometry of eGFP expression from the MuHV-4 BAC cassette. D. BHK-21 cells were infected with ORF75c − (ORF75c − .4, ORF75c − .7) or ORF75c + (wild-type, revertant.1) viruses at 10 genomes/cell - equivalent for the wild-type and revertant to 0.01 p.f.u./cell. The spread of infection with time was assayed by flow cytometry of viral eGFP expression.

    Techniques Used: Mutagenesis, Southern Blot, Infection, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing, BAC Assay

    2) Product Images from "The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice"

    Article Title: The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice

    Journal: eLife

    doi: 10.7554/eLife.18197

    Generation of TBC1D3 transgenic mouse. ( A ) Schematic structure of the construct used for generating TBC1D3 transgenic mice. The expression of TBC1D3 was under the control of promoter composed of the second intron enhancer of rat nestin gene and the minimum promoter of heat shock protein 68 (hsp68) ( Kawaguchi et al., 2001 ). P1 and P2, primers used for genotyping. ( B ) Relative copy numbers of TBC1D3 genes inserted in the genome of transgenic mouse founders (10#, 14#, 51#) were determined by real-time PCR using the tail genomic DNA as template. The number of inserted TBC1D3 gene was normalized to that of glcoe4 (n = 8 mice, mean = 3.15, SEM = 0.47 for 10#; n = 2 mice, mean = 0.54, SEM = 0.03 for 14#; n = 4, mean = 0.46, SEM = 0.03 for 51#). ( C ) Protein levels of TBC1D3 in brains of E12.5 TG mouse lines (L10, L51), from founders 10# or 51#, were measured by immunoblot (left panel) and quantified (right panel). GAPDH was used as the internal control (L10: n = 4 mice, mean = 2.92, SEM = 0.72; L51: n = 5 mice, mean = 1.00, SEM = 0.08). ( D ) Immunostaining for the expression of TBC1D3 in E12.5 TG (L10) mouse brain. Note the magnified area (D1) showing cytosol distribution of TBC1D3. Scale bars, 200 μm ( D ) and 20 μm (D1). ( E ) TBC1D3 protein levels in TG mice (L10) at indicated developmental stages. Shown in the histogram is mean of 2 mice in each stage with GAPDH as control. DOI: http://dx.doi.org/10.7554/eLife.18197.019
    Figure Legend Snippet: Generation of TBC1D3 transgenic mouse. ( A ) Schematic structure of the construct used for generating TBC1D3 transgenic mice. The expression of TBC1D3 was under the control of promoter composed of the second intron enhancer of rat nestin gene and the minimum promoter of heat shock protein 68 (hsp68) ( Kawaguchi et al., 2001 ). P1 and P2, primers used for genotyping. ( B ) Relative copy numbers of TBC1D3 genes inserted in the genome of transgenic mouse founders (10#, 14#, 51#) were determined by real-time PCR using the tail genomic DNA as template. The number of inserted TBC1D3 gene was normalized to that of glcoe4 (n = 8 mice, mean = 3.15, SEM = 0.47 for 10#; n = 2 mice, mean = 0.54, SEM = 0.03 for 14#; n = 4, mean = 0.46, SEM = 0.03 for 51#). ( C ) Protein levels of TBC1D3 in brains of E12.5 TG mouse lines (L10, L51), from founders 10# or 51#, were measured by immunoblot (left panel) and quantified (right panel). GAPDH was used as the internal control (L10: n = 4 mice, mean = 2.92, SEM = 0.72; L51: n = 5 mice, mean = 1.00, SEM = 0.08). ( D ) Immunostaining for the expression of TBC1D3 in E12.5 TG (L10) mouse brain. Note the magnified area (D1) showing cytosol distribution of TBC1D3. Scale bars, 200 μm ( D ) and 20 μm (D1). ( E ) TBC1D3 protein levels in TG mice (L10) at indicated developmental stages. Shown in the histogram is mean of 2 mice in each stage with GAPDH as control. DOI: http://dx.doi.org/10.7554/eLife.18197.019

    Techniques Used: Transgenic Assay, Construct, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Immunostaining

    3) Product Images from "The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus"

    Article Title: The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus

    Journal: Journal of Bacteriology

    doi:

    Cleavage of plasmid DNA prior to electroporation into M. xanthus affects the frequency and nature of recombination events between the plasmid and chromosome. Shown to approximate scale in the upper left is the structure of plasmid pAY1074, which is Km
    Figure Legend Snippet: Cleavage of plasmid DNA prior to electroporation into M. xanthus affects the frequency and nature of recombination events between the plasmid and chromosome. Shown to approximate scale in the upper left is the structure of plasmid pAY1074, which is Km

    Techniques Used: Plasmid Preparation, Electroporation

    4) Product Images from "Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end"

    Article Title: Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end

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

    doi: 10.1073/pnas.1203118109

    Real-time traces for supercoil relaxation by topo IB (0.5 pN, ΔLk = -30). ( A ) A 1 μm-diameter bead; relaxation leads to bead velocity of 21 ± 1 μm/s. ( B ) A 2.8 μm-diameter
    Figure Legend Snippet: Real-time traces for supercoil relaxation by topo IB (0.5 pN, ΔLk = -30). ( A ) A 1 μm-diameter bead; relaxation leads to bead velocity of 21 ± 1 μm/s. ( B ) A 2.8 μm-diameter

    Techniques Used:

    Experiment setup and supercoil relaxation assay. ( A ) A supercoiled dsDNA is tethered between a surface and a paramagnetic bead. As plectonemic supercoils are introduced, DNA extension is reduced. ( B ) When supercoiling is relaxed, an increase in DNA extension
    Figure Legend Snippet: Experiment setup and supercoil relaxation assay. ( A ) A supercoiled dsDNA is tethered between a surface and a paramagnetic bead. As plectonemic supercoils are introduced, DNA extension is reduced. ( B ) When supercoiling is relaxed, an increase in DNA extension

    Techniques Used:

    Religation times (“open” times) and corresponding rates for Bxb1 integrase depends on bead size. Following double-strand cleavage and supercoil relaxation, attP –Bxb1 integrase– attB –CT synapses usually religate,
    Figure Legend Snippet: Religation times (“open” times) and corresponding rates for Bxb1 integrase depends on bead size. Following double-strand cleavage and supercoil relaxation, attP –Bxb1 integrase– attB –CT synapses usually religate,

    Techniques Used:

    Dependence of supercoil relaxation velocity on the inverse of effective bead diameter (0.5 pN, ΔLk = -30). For each enzyme, a well-defined slope (in units of μm 2 /s) was obtained from a two-parameter linear fit to
    Figure Legend Snippet: Dependence of supercoil relaxation velocity on the inverse of effective bead diameter (0.5 pN, ΔLk = -30). For each enzyme, a well-defined slope (in units of μm 2 /s) was obtained from a two-parameter linear fit to

    Techniques Used:

    5) Product Images from "Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni"

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095084

    Adaptation of the pRRC gene delivery and expression system to harbor hygromycin B or apramycin resistance, and testing of genome-integrated markers for detrimental effects of resistance genes. (A) Schematic of pRRC, which inserts into any of 3 rRNA clusters in the genome by homologous recombination. (B) Inverse PCR amplification of pRRC with primers 5705 ( Kpn I) and 5706 deleted the chloramphenicol resistance gene but conserved the Campylobacter -optimized cat promoter. (C) The inverse PCR product was digested with Kpn I and Xba I, and ligated to similarly digested aph (7″) or aac (3)IV from pAC1H or pAC1A to create pRRH and pRRA respectively (only pRRH is shown). (D) Restriction digest analysis confirmed the function of all introduced sites. (E) The resistance markers from pRRK, pRRC, pRRH and pRRA were inserted into the C. jejuni 81–176 genome, and each resulting strain was analyzed for microaerobic growth and survival in shaken Mueller-Hinton (MH) broth by counting CFU over 48 hours at both 42°C (left panel) and 37°C (right panel). (F) To determine if the introduction of either marker contributed any fitness cost that could affect competitiveness against wild-type or the other marked strains, a competition assay was performed. Equal numbers of wild-type marked with hygromycin, apramycin, chloramphenicol and kanamycin resistance markers were co-cultured with unmarked wild-type in shaking MH broth at 37°C under microaerobic conditions. CFU were assessed by plating a dilution series on MH agar. (G) CFU were further assessed from the co-culture by plating on MH only (the total CFU, same data as in F) or MH supplemented with each antibiotic, representing the number of bacteria resistant to each antibiotic.
    Figure Legend Snippet: Adaptation of the pRRC gene delivery and expression system to harbor hygromycin B or apramycin resistance, and testing of genome-integrated markers for detrimental effects of resistance genes. (A) Schematic of pRRC, which inserts into any of 3 rRNA clusters in the genome by homologous recombination. (B) Inverse PCR amplification of pRRC with primers 5705 ( Kpn I) and 5706 deleted the chloramphenicol resistance gene but conserved the Campylobacter -optimized cat promoter. (C) The inverse PCR product was digested with Kpn I and Xba I, and ligated to similarly digested aph (7″) or aac (3)IV from pAC1H or pAC1A to create pRRH and pRRA respectively (only pRRH is shown). (D) Restriction digest analysis confirmed the function of all introduced sites. (E) The resistance markers from pRRK, pRRC, pRRH and pRRA were inserted into the C. jejuni 81–176 genome, and each resulting strain was analyzed for microaerobic growth and survival in shaken Mueller-Hinton (MH) broth by counting CFU over 48 hours at both 42°C (left panel) and 37°C (right panel). (F) To determine if the introduction of either marker contributed any fitness cost that could affect competitiveness against wild-type or the other marked strains, a competition assay was performed. Equal numbers of wild-type marked with hygromycin, apramycin, chloramphenicol and kanamycin resistance markers were co-cultured with unmarked wild-type in shaking MH broth at 37°C under microaerobic conditions. CFU were assessed by plating a dilution series on MH agar. (G) CFU were further assessed from the co-culture by plating on MH only (the total CFU, same data as in F) or MH supplemented with each antibiotic, representing the number of bacteria resistant to each antibiotic.

    Techniques Used: Expressing, Homologous Recombination, Inverse PCR, Amplification, Marker, Competitive Binding Assay, Cell Culture, Co-Culture Assay

    Mutagenesis of the arylsulfatase gene astA with aph (7″) or aac (3)IV non-polar markers and complementation of Δ astA via genomic insertion with pRRH or pRRA. (A) Loci arrangement of astA single-gene operon in C. jejuni 81–176. (B) Deletion of astA with either aph (7″) or aac (3)IV from pAC1H or pAC1H. (C) Introduction of promoterless astA into pRRH or pRRA in the same orientation as the cat promoter created pRRH+ astA or pRRA+ astA and resulted in polycistronic expression of astA with aph (7″) or aac (3)IV. (D) Promoterless astA inserted in the opposite orientation to the cat promoter (designated pRRH+ astA (reverse) or pRRA+ astA (reverse) (E) Insertion of the endogenous astA promoter and astA in the opposite orientation to the cat promoter in pRRH and pRRA created pRRH+(p) astA (reverse) and pRRA+(p) astA (reverse). Only Hyg R plasmids/strains are depicted in B–E, but both Hyg R and Apr R plasmids represented with Hyg R in C, D and E were integrated into the genome of the Δ astA strain, DRH461. (F) Arylsulfatase activity of the deletion and complementation strains was assessed by spotting 10 µL of OD-standardized cultures onto MH agar plates supplemented with the chromogen XS cleaved by arylsulfatase. A blue-green color indicates activity, and the spots correspond to labels on the bar graph below. (G) Quantification of arylsulfatase activity from broth cultures to assess transcription of astA .
    Figure Legend Snippet: Mutagenesis of the arylsulfatase gene astA with aph (7″) or aac (3)IV non-polar markers and complementation of Δ astA via genomic insertion with pRRH or pRRA. (A) Loci arrangement of astA single-gene operon in C. jejuni 81–176. (B) Deletion of astA with either aph (7″) or aac (3)IV from pAC1H or pAC1H. (C) Introduction of promoterless astA into pRRH or pRRA in the same orientation as the cat promoter created pRRH+ astA or pRRA+ astA and resulted in polycistronic expression of astA with aph (7″) or aac (3)IV. (D) Promoterless astA inserted in the opposite orientation to the cat promoter (designated pRRH+ astA (reverse) or pRRA+ astA (reverse) (E) Insertion of the endogenous astA promoter and astA in the opposite orientation to the cat promoter in pRRH and pRRA created pRRH+(p) astA (reverse) and pRRA+(p) astA (reverse). Only Hyg R plasmids/strains are depicted in B–E, but both Hyg R and Apr R plasmids represented with Hyg R in C, D and E were integrated into the genome of the Δ astA strain, DRH461. (F) Arylsulfatase activity of the deletion and complementation strains was assessed by spotting 10 µL of OD-standardized cultures onto MH agar plates supplemented with the chromogen XS cleaved by arylsulfatase. A blue-green color indicates activity, and the spots correspond to labels on the bar graph below. (G) Quantification of arylsulfatase activity from broth cultures to assess transcription of astA .

    Techniques Used: Mutagenesis, Expressing, Activity Assay

    6) Product Images from "Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection"

    Article Title: Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002781

    Identification of ORFs 75a/b/c as virion components. A. BHK-21 cells were incubated with wild-type MuHV-4 virions (5 p.f.u./cell, 6 h, 37°C), with or without cycloheximide (100 µg/ml) to block new protein synthesis or bafilomycin (500 nM) to block virion membrane fusion. The cells were then fixed in 4% paraformaldehyde, permeabilized with Triton X-100 and stained (green) for ORF75b, ORF75c, the ORF65 virion capsid component or glycoprotein N (gN). Nuclei were counterstained with DAPI (blue). B. BHK-21 cells were exposed to MuHV-4 virions as in A without drug treatment, then fixed, permeabilized and stained for ORF75a. At the exposure necessary to see strong positive staining, background staining of uninfected controls was also evident. C. 35 S-cysteine/methionine-labelled, MuHV-4-infected BHK-21 cultures were separated into infected cell and virion fractions, lysed in Triton X-100 and precipitated with mAbs specific for thymidine kinase (TK), ORF75c (75c) or ORF75a (75a), plus protein A/protein G-sepharose. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography. The predicted sizes of ORF75a (142 kDa), ORF75c (146 kDa) and TK (72 kDa) are marked.
    Figure Legend Snippet: Identification of ORFs 75a/b/c as virion components. A. BHK-21 cells were incubated with wild-type MuHV-4 virions (5 p.f.u./cell, 6 h, 37°C), with or without cycloheximide (100 µg/ml) to block new protein synthesis or bafilomycin (500 nM) to block virion membrane fusion. The cells were then fixed in 4% paraformaldehyde, permeabilized with Triton X-100 and stained (green) for ORF75b, ORF75c, the ORF65 virion capsid component or glycoprotein N (gN). Nuclei were counterstained with DAPI (blue). B. BHK-21 cells were exposed to MuHV-4 virions as in A without drug treatment, then fixed, permeabilized and stained for ORF75a. At the exposure necessary to see strong positive staining, background staining of uninfected controls was also evident. C. 35 S-cysteine/methionine-labelled, MuHV-4-infected BHK-21 cultures were separated into infected cell and virion fractions, lysed in Triton X-100 and precipitated with mAbs specific for thymidine kinase (TK), ORF75c (75c) or ORF75a (75a), plus protein A/protein G-sepharose. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography. The predicted sizes of ORF75a (142 kDa), ORF75c (146 kDa) and TK (72 kDa) are marked.

    Techniques Used: Incubation, Blocking Assay, Staining, Infection, Immunoprecipitation, SDS Page, Autoradiography

    No evidence for ORF75c deficiency compromising virion morphogenesis. A. Wild-type (WT), ORF75c − (75c − .4, 75c − .7, 75c − .9) and revertant (rev.1, rev.2) virus stocks were normalized by genome content, denatured in Laemmli's buffer and immunoblotted for the ORF17 capsid component with mAb 150-7D1, for gB (C-terminal furin cleavage product) with mAb MG-4D11, and for thymidine kinase (TK) with mAb CS-4A5. B. Virus stocks were denatured, resolved by SDS-PAGE and analyzed by Coomassie staining. The positions of bovine albumin (alb, 63 kDa), the ORF25 capsid component (160 kDa) and the ORFs 75a/b/c (150 kDa) are shown. C. BHK-21 cells were infected with wild-type (WT) MuHV-4 (1 p.f.u./cell, 18 h) or ORF75c − .4 (ORF75c − ) MuHV-4 to an equivalent infection level based on viral eGFP expression, then processed for transmission electron microscopy. The scale bars show 1 µM in the overview images. nuc = nucleus, cyt = cytoplasm. The boxed regions in the overview images correspond to the zoomed images 1–4. The arrows show examples of mature virions.
    Figure Legend Snippet: No evidence for ORF75c deficiency compromising virion morphogenesis. A. Wild-type (WT), ORF75c − (75c − .4, 75c − .7, 75c − .9) and revertant (rev.1, rev.2) virus stocks were normalized by genome content, denatured in Laemmli's buffer and immunoblotted for the ORF17 capsid component with mAb 150-7D1, for gB (C-terminal furin cleavage product) with mAb MG-4D11, and for thymidine kinase (TK) with mAb CS-4A5. B. Virus stocks were denatured, resolved by SDS-PAGE and analyzed by Coomassie staining. The positions of bovine albumin (alb, 63 kDa), the ORF25 capsid component (160 kDa) and the ORFs 75a/b/c (150 kDa) are shown. C. BHK-21 cells were infected with wild-type (WT) MuHV-4 (1 p.f.u./cell, 18 h) or ORF75c − .4 (ORF75c − ) MuHV-4 to an equivalent infection level based on viral eGFP expression, then processed for transmission electron microscopy. The scale bars show 1 µM in the overview images. nuc = nucleus, cyt = cytoplasm. The boxed regions in the overview images correspond to the zoomed images 1–4. The arrows show examples of mature virions.

    Techniques Used: SDS Page, Staining, Infection, Expressing, Transmission Assay, Electron Microscopy

    Immunostaining of ORFs 75a/b/c. A. 293T cells were transfected with a mammalian expression vector encoding ORF75a, ORF75b or ORF75c. 48 h later the cells were fixed in 4% paraformaldehyde, permeabilized with Triton-X100 and stained with mAbs derived from MuHV-4-infected mice. Positive staining is green. Nuclei were counter-stained with DAPI (blue). Each field shown is representative of at least 50 examined. B. FGARAT-deficient CHO-AdeB cells were transfected with ORF75a/b/c expression constructs as in A, then selected for 2 weeks in G418 and switched to purine-free medium with hypoxanthine for a further 3 weeks. This led to growth arrest. The cells were then switched back to normal medium and ORF75a/b/c expression tested by immunostaining as in A. C. BHK-21 cells were infected with wild-type MuHV-4 (2 p.f.u./cell, 18 h), then fixed in 4% paraformaldehyde, permeabilized with Triton-X100 and stained with ORF75a/b/c-specific mAbs. Nuclei were counterstained with DAPI. Only merged images are shown for the uninfected controls.
    Figure Legend Snippet: Immunostaining of ORFs 75a/b/c. A. 293T cells were transfected with a mammalian expression vector encoding ORF75a, ORF75b or ORF75c. 48 h later the cells were fixed in 4% paraformaldehyde, permeabilized with Triton-X100 and stained with mAbs derived from MuHV-4-infected mice. Positive staining is green. Nuclei were counter-stained with DAPI (blue). Each field shown is representative of at least 50 examined. B. FGARAT-deficient CHO-AdeB cells were transfected with ORF75a/b/c expression constructs as in A, then selected for 2 weeks in G418 and switched to purine-free medium with hypoxanthine for a further 3 weeks. This led to growth arrest. The cells were then switched back to normal medium and ORF75a/b/c expression tested by immunostaining as in A. C. BHK-21 cells were infected with wild-type MuHV-4 (2 p.f.u./cell, 18 h), then fixed in 4% paraformaldehyde, permeabilized with Triton-X100 and stained with ORF75a/b/c-specific mAbs. Nuclei were counterstained with DAPI. Only merged images are shown for the uninfected controls.

    Techniques Used: Immunostaining, Transfection, Expressing, Plasmid Preparation, Staining, Derivative Assay, Infection, Mouse Assay, Construct

    7) Product Images from "A Practical Approach to T-Cell Receptor Cloning and Expression"

    Article Title: A Practical Approach to T-Cell Receptor Cloning and Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027930

    Electroporation of mRNA encoding MART-1 specific TcRs. (a) SupT1 were electroporated with or without 20 µg mRNA encoding the indicated TcR_2A. Twelve hours later, the cells were stained with HLA-A2/MART-1 multimer-PE and anti-CD3 Alexa Fluor 647. (b) Human PBMC were electroporated with 20 µg mRNA (same constructs as in (a)) and cultured for 4.5 hours. Following the addition of T2 cells pre-loaded or not (grey) with MART-1 peptide (10 µM final concentration), the cells were co-incubated for an additional 5 hours in the presence of anti-CD107a/b Alexa Fluor 647 antibodies, monensin and brefeldin A. Prior to analysis, cells were stained with anti-CD8 PE. The percentage of CD107a/b positive cells from the CD8 positive population is plotted. PHA was used to control for similar maximal degranulation levels regardless of the mRNA used for electroporation. Each bar represents the mean values of duplicates.
    Figure Legend Snippet: Electroporation of mRNA encoding MART-1 specific TcRs. (a) SupT1 were electroporated with or without 20 µg mRNA encoding the indicated TcR_2A. Twelve hours later, the cells were stained with HLA-A2/MART-1 multimer-PE and anti-CD3 Alexa Fluor 647. (b) Human PBMC were electroporated with 20 µg mRNA (same constructs as in (a)) and cultured for 4.5 hours. Following the addition of T2 cells pre-loaded or not (grey) with MART-1 peptide (10 µM final concentration), the cells were co-incubated for an additional 5 hours in the presence of anti-CD107a/b Alexa Fluor 647 antibodies, monensin and brefeldin A. Prior to analysis, cells were stained with anti-CD8 PE. The percentage of CD107a/b positive cells from the CD8 positive population is plotted. PHA was used to control for similar maximal degranulation levels regardless of the mRNA used for electroporation. Each bar represents the mean values of duplicates.

    Techniques Used: Electroporation, Staining, Construct, Cell Culture, Concentration Assay, Incubation

    8) Product Images from "Towards a Rigorous Network of Protein-Protein Interactions of the Model Sulfate Reducer Desulfovibrio vulgaris Hildenborough"

    Article Title: Towards a Rigorous Network of Protein-Protein Interactions of the Model Sulfate Reducer Desulfovibrio vulgaris Hildenborough

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021470

    Gene expression correlations between interacting and non-interacting pairs in D. vulgaris Hildenborough. Shown are the D. vulgaris Hildenborough (JW801) gene co-expression distributions, measured as the centered Pearson correlation between vectors of gene expression values, for pairs of genes whose corresponding proteins were found to interact with high confidence (black) or not (red). The y-axis shows the fraction of all interacting or non-interacting protein pairs.
    Figure Legend Snippet: Gene expression correlations between interacting and non-interacting pairs in D. vulgaris Hildenborough. Shown are the D. vulgaris Hildenborough (JW801) gene co-expression distributions, measured as the centered Pearson correlation between vectors of gene expression values, for pairs of genes whose corresponding proteins were found to interact with high confidence (black) or not (red). The y-axis shows the fraction of all interacting or non-interacting protein pairs.

    Techniques Used: Expressing

    Partial D. vulgaris Hildenborough (JW801) high confidence protein-protein interaction network. Shown are the high confidence bait-prey protein interaction pairs from this study. Edges connecting nodes indicate a detected high confidence interaction between a bait and a pulled down prey protein. Nodes in the network are colored by TIGR functional role, as are edges where both nodes belong to the same TIGR role. Bait protein nodes are surrounded by a thicker black circle proportional to the normalized adjusted median-max emPAI value for the bait protein. The dotted node indicates the bait, which was not observed (Rub). Dotted edges indicate interactions with a median-max emPAI value equal to the control but where the bait was also observed in the control with a high emPAI value (see Methods ). Interconnected sets of nodes belonging to the same TIGR role are shaded with a lighter hue of the TIGR functional role color. Head-on arrows indicate reciprocally detected interactions and the width of the arrow corresponds to the normalized adjusted median-max emPAI value for the prey protein. Interactions corresponding to p
    Figure Legend Snippet: Partial D. vulgaris Hildenborough (JW801) high confidence protein-protein interaction network. Shown are the high confidence bait-prey protein interaction pairs from this study. Edges connecting nodes indicate a detected high confidence interaction between a bait and a pulled down prey protein. Nodes in the network are colored by TIGR functional role, as are edges where both nodes belong to the same TIGR role. Bait protein nodes are surrounded by a thicker black circle proportional to the normalized adjusted median-max emPAI value for the bait protein. The dotted node indicates the bait, which was not observed (Rub). Dotted edges indicate interactions with a median-max emPAI value equal to the control but where the bait was also observed in the control with a high emPAI value (see Methods ). Interconnected sets of nodes belonging to the same TIGR role are shaded with a lighter hue of the TIGR functional role color. Head-on arrows indicate reciprocally detected interactions and the width of the arrow corresponds to the normalized adjusted median-max emPAI value for the prey protein. Interactions corresponding to p

    Techniques Used: Functional Assay

    9) Product Images from "Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end"

    Article Title: Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end

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

    doi: 10.1073/pnas.1203118109

    Experiment setup and supercoil relaxation assay. ( A ) A supercoiled dsDNA is tethered between a surface and a paramagnetic bead. As plectonemic supercoils are introduced, DNA extension is reduced. ( B ) When supercoiling is relaxed, an increase in DNA extension
    Figure Legend Snippet: Experiment setup and supercoil relaxation assay. ( A ) A supercoiled dsDNA is tethered between a surface and a paramagnetic bead. As plectonemic supercoils are introduced, DNA extension is reduced. ( B ) When supercoiling is relaxed, an increase in DNA extension

    Techniques Used:

    10) Product Images from "The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice"

    Article Title: The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice

    Journal: eLife

    doi: 10.7554/eLife.18197

    Generation of TBC1D3 transgenic mouse. ( A ) Schematic structure of the construct used for generating TBC1D3 transgenic mice. The expression of TBC1D3 was under the control of promoter composed of the second intron enhancer of rat nestin gene and the minimum promoter of heat shock protein 68 (hsp68) ( Kawaguchi et al., 2001 ). P1 and P2, primers used for genotyping. ( B ) Relative copy numbers of TBC1D3 genes inserted in the genome of transgenic mouse founders (10#, 14#, 51#) were determined by real-time PCR using the tail genomic DNA as template. The number of inserted TBC1D3 gene was normalized to that of glcoe4 (n = 8 mice, mean = 3.15, SEM = 0.47 for 10#; n = 2 mice, mean = 0.54, SEM = 0.03 for 14#; n = 4, mean = 0.46, SEM = 0.03 for 51#). ( C ) Protein levels of TBC1D3 in brains of E12.5 TG mouse lines (L10, L51), from founders 10# or 51#, were measured by immunoblot (left panel) and quantified (right panel). GAPDH was used as the internal control (L10: n = 4 mice, mean = 2.92, SEM = 0.72; L51: n = 5 mice, mean = 1.00, SEM = 0.08). ( D ) Immunostaining for the expression of TBC1D3 in E12.5 TG (L10) mouse brain. Note the magnified area (D1) showing cytosol distribution of TBC1D3. Scale bars, 200 μm ( D ) and 20 μm (D1). ( E ) TBC1D3 protein levels in TG mice (L10) at indicated developmental stages. Shown in the histogram is mean of 2 mice in each stage with GAPDH as control. DOI: http://dx.doi.org/10.7554/eLife.18197.019
    Figure Legend Snippet: Generation of TBC1D3 transgenic mouse. ( A ) Schematic structure of the construct used for generating TBC1D3 transgenic mice. The expression of TBC1D3 was under the control of promoter composed of the second intron enhancer of rat nestin gene and the minimum promoter of heat shock protein 68 (hsp68) ( Kawaguchi et al., 2001 ). P1 and P2, primers used for genotyping. ( B ) Relative copy numbers of TBC1D3 genes inserted in the genome of transgenic mouse founders (10#, 14#, 51#) were determined by real-time PCR using the tail genomic DNA as template. The number of inserted TBC1D3 gene was normalized to that of glcoe4 (n = 8 mice, mean = 3.15, SEM = 0.47 for 10#; n = 2 mice, mean = 0.54, SEM = 0.03 for 14#; n = 4, mean = 0.46, SEM = 0.03 for 51#). ( C ) Protein levels of TBC1D3 in brains of E12.5 TG mouse lines (L10, L51), from founders 10# or 51#, were measured by immunoblot (left panel) and quantified (right panel). GAPDH was used as the internal control (L10: n = 4 mice, mean = 2.92, SEM = 0.72; L51: n = 5 mice, mean = 1.00, SEM = 0.08). ( D ) Immunostaining for the expression of TBC1D3 in E12.5 TG (L10) mouse brain. Note the magnified area (D1) showing cytosol distribution of TBC1D3. Scale bars, 200 μm ( D ) and 20 μm (D1). ( E ) TBC1D3 protein levels in TG mice (L10) at indicated developmental stages. Shown in the histogram is mean of 2 mice in each stage with GAPDH as control. DOI: http://dx.doi.org/10.7554/eLife.18197.019

    Techniques Used: Transgenic Assay, Construct, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Immunostaining

    11) Product Images from "Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]"

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.15.01663

    -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).
    Figure Legend Snippet: -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Techniques Used: Polymerase Chain Reaction, Amplification, Generated

    locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.
    Figure Legend Snippet: locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Techniques Used: Southern Blot, Mutagenesis, Hybridization

    12) Product Images from "Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]"

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.15.01663

    -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).
    Figure Legend Snippet: -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Techniques Used: Polymerase Chain Reaction, Amplification, Generated

    locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.
    Figure Legend Snippet: locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Techniques Used: Southern Blot, Mutagenesis, Hybridization

    13) Product Images from "Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni"

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095084

    Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.
    Figure Legend Snippet: Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Techniques Used: Binding Assay, Amplification, TA Cloning, Clone Assay, Plasmid Preparation, Subcloning, Marker

    14) Product Images from "Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species"

    Article Title: Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species

    Journal: Journal of Clinical Microbiology

    doi:

    AFLP fingerprint of M. genitalium G-37 T . Bgl II plus Mfe I AFLP templates were prepared on three occasions with the same batch of genomic DNA of M. genitalium G-37 T . Amplification products obtained from each experiment (blue, black, and red patterns) were detected on an ABI 373A sequencer by GeneScan 1.2.2.-1 software. The complete AFLP patterns are superimposed and divided into four parts (A to D). The fragment size scale (base pairs) is indicated above each panel. y axes indicate relative amounts of amplicons (in fluorescence units).
    Figure Legend Snippet: AFLP fingerprint of M. genitalium G-37 T . Bgl II plus Mfe I AFLP templates were prepared on three occasions with the same batch of genomic DNA of M. genitalium G-37 T . Amplification products obtained from each experiment (blue, black, and red patterns) were detected on an ABI 373A sequencer by GeneScan 1.2.2.-1 software. The complete AFLP patterns are superimposed and divided into four parts (A to D). The fragment size scale (base pairs) is indicated above each panel. y axes indicate relative amounts of amplicons (in fluorescence units).

    Techniques Used: Amplification, Software, Fluorescence

    Genescan-derived electropherogram traces of AFLP templates of different complexity. AFLP templates of a field isolate of Escherichia coli and M. genitalium G-37 T were prepared by the digestion of genomic DNAs with Bgl II and Mfe I and subsequent ligation of corresponding adapters. PCR products of individual samples and a mixture of the AFLP templates, containing adjusted DNA concentrations, were detected on an ABI 373A sequencer. The hybrid pattern (middle panel) contains all of the bands detected in individual AFLP templates. The fragment size scale (base pairs) is indicated above the top panel.
    Figure Legend Snippet: Genescan-derived electropherogram traces of AFLP templates of different complexity. AFLP templates of a field isolate of Escherichia coli and M. genitalium G-37 T were prepared by the digestion of genomic DNAs with Bgl II and Mfe I and subsequent ligation of corresponding adapters. PCR products of individual samples and a mixture of the AFLP templates, containing adjusted DNA concentrations, were detected on an ABI 373A sequencer. The hybrid pattern (middle panel) contains all of the bands detected in individual AFLP templates. The fragment size scale (base pairs) is indicated above the top panel.

    Techniques Used: Derivative Assay, Ligation, Polymerase Chain Reaction

    15) Product Images from "Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni"

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095084

    Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.
    Figure Legend Snippet: Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Techniques Used: Binding Assay, Amplification, TA Cloning, Clone Assay, Plasmid Preparation, Subcloning, Marker

    16) Product Images from "Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni"

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095084

    Adaptation of the pRRC gene delivery and expression system to harbor hygromycin B or apramycin resistance, and testing of genome-integrated markers for detrimental effects of resistance genes. (A) Schematic of pRRC, which inserts into any of 3 rRNA clusters in the genome by homologous recombination. (B) Inverse PCR amplification of pRRC with primers 5705 ( Kpn I) and 5706 deleted the chloramphenicol resistance gene but conserved the Campylobacter -optimized cat promoter. (C) The inverse PCR product was digested with Kpn I and Xba I, and ligated to similarly digested aph (7″) or aac (3)IV from pAC1H or pAC1A to create pRRH and pRRA respectively (only pRRH is shown). (D) Restriction digest analysis confirmed the function of all introduced sites. (E) The resistance markers from pRRK, pRRC, pRRH and pRRA were inserted into the C. jejuni 81–176 genome, and each resulting strain was analyzed for microaerobic growth and survival in shaken Mueller-Hinton (MH) broth by counting CFU over 48 hours at both 42°C (left panel) and 37°C (right panel). (F) To determine if the introduction of either marker contributed any fitness cost that could affect competitiveness against wild-type or the other marked strains, a competition assay was performed. Equal numbers of wild-type marked with hygromycin, apramycin, chloramphenicol and kanamycin resistance markers were co-cultured with unmarked wild-type in shaking MH broth at 37°C under microaerobic conditions. CFU were assessed by plating a dilution series on MH agar. (G) CFU were further assessed from the co-culture by plating on MH only (the total CFU, same data as in F) or MH supplemented with each antibiotic, representing the number of bacteria resistant to each antibiotic.
    Figure Legend Snippet: Adaptation of the pRRC gene delivery and expression system to harbor hygromycin B or apramycin resistance, and testing of genome-integrated markers for detrimental effects of resistance genes. (A) Schematic of pRRC, which inserts into any of 3 rRNA clusters in the genome by homologous recombination. (B) Inverse PCR amplification of pRRC with primers 5705 ( Kpn I) and 5706 deleted the chloramphenicol resistance gene but conserved the Campylobacter -optimized cat promoter. (C) The inverse PCR product was digested with Kpn I and Xba I, and ligated to similarly digested aph (7″) or aac (3)IV from pAC1H or pAC1A to create pRRH and pRRA respectively (only pRRH is shown). (D) Restriction digest analysis confirmed the function of all introduced sites. (E) The resistance markers from pRRK, pRRC, pRRH and pRRA were inserted into the C. jejuni 81–176 genome, and each resulting strain was analyzed for microaerobic growth and survival in shaken Mueller-Hinton (MH) broth by counting CFU over 48 hours at both 42°C (left panel) and 37°C (right panel). (F) To determine if the introduction of either marker contributed any fitness cost that could affect competitiveness against wild-type or the other marked strains, a competition assay was performed. Equal numbers of wild-type marked with hygromycin, apramycin, chloramphenicol and kanamycin resistance markers were co-cultured with unmarked wild-type in shaking MH broth at 37°C under microaerobic conditions. CFU were assessed by plating a dilution series on MH agar. (G) CFU were further assessed from the co-culture by plating on MH only (the total CFU, same data as in F) or MH supplemented with each antibiotic, representing the number of bacteria resistant to each antibiotic.

    Techniques Used: Expressing, Homologous Recombination, Inverse PCR, Amplification, Marker, Competitive Binding Assay, Cell Culture, Co-Culture Assay

    Mutagenesis of the arylsulfatase gene astA with aph (7″) or aac (3)IV non-polar markers and complementation of Δ astA via genomic insertion with pRRH or pRRA. (A) Loci arrangement of astA single-gene operon in C. jejuni 81–176. (B) Deletion of astA with either aph (7″) or aac (3)IV from pAC1H or pAC1H. (C) Introduction of promoterless astA into pRRH or pRRA in the same orientation as the cat promoter created pRRH+ astA or pRRA+ astA and resulted in polycistronic expression of astA with aph (7″) or aac (3)IV. (D) Promoterless astA inserted in the opposite orientation to the cat promoter (designated pRRH+ astA (reverse) or pRRA+ astA (reverse) (E) Insertion of the endogenous astA promoter and astA in the opposite orientation to the cat promoter in pRRH and pRRA created pRRH+(p) astA (reverse) and pRRA+(p) astA (reverse). Only Hyg R plasmids/strains are depicted in B–E, but both Hyg R and Apr R plasmids represented with Hyg R in C, D and E were integrated into the genome of the Δ astA strain, DRH461. (F) Arylsulfatase activity of the deletion and complementation strains was assessed by spotting 10 µL of OD-standardized cultures onto MH agar plates supplemented with the chromogen XS cleaved by arylsulfatase. A blue-green color indicates activity, and the spots correspond to labels on the bar graph below. (G) Quantification of arylsulfatase activity from broth cultures to assess transcription of astA .
    Figure Legend Snippet: Mutagenesis of the arylsulfatase gene astA with aph (7″) or aac (3)IV non-polar markers and complementation of Δ astA via genomic insertion with pRRH or pRRA. (A) Loci arrangement of astA single-gene operon in C. jejuni 81–176. (B) Deletion of astA with either aph (7″) or aac (3)IV from pAC1H or pAC1H. (C) Introduction of promoterless astA into pRRH or pRRA in the same orientation as the cat promoter created pRRH+ astA or pRRA+ astA and resulted in polycistronic expression of astA with aph (7″) or aac (3)IV. (D) Promoterless astA inserted in the opposite orientation to the cat promoter (designated pRRH+ astA (reverse) or pRRA+ astA (reverse) (E) Insertion of the endogenous astA promoter and astA in the opposite orientation to the cat promoter in pRRH and pRRA created pRRH+(p) astA (reverse) and pRRA+(p) astA (reverse). Only Hyg R plasmids/strains are depicted in B–E, but both Hyg R and Apr R plasmids represented with Hyg R in C, D and E were integrated into the genome of the Δ astA strain, DRH461. (F) Arylsulfatase activity of the deletion and complementation strains was assessed by spotting 10 µL of OD-standardized cultures onto MH agar plates supplemented with the chromogen XS cleaved by arylsulfatase. A blue-green color indicates activity, and the spots correspond to labels on the bar graph below. (G) Quantification of arylsulfatase activity from broth cultures to assess transcription of astA .

    Techniques Used: Mutagenesis, Expressing, Activity Assay

    17) Product Images from "Recombinant Respiratory Syncytial Virus with the G and F Genes Shifted to the Promoter-Proximal Positions"

    Article Title: Recombinant Respiratory Syncytial Virus with the G and F Genes Shifted to the Promoter-Proximal Positions

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.23.11931-11942.2002

    Shift of the G or F gene individually or together to a promoter-proximal position(s) in the RSV genome. The diagram at the top illustrates the wild-type RSV genome from which the SH gene had been deleted and in which a Blp I restriction site had been inserted immediately upstream of the NS1 ORF to create Blp/ΔSH. The box underneath and on the left illustrates the insertion of the G gene alone, the F gene alone, or the G and F genes together into the Blp I site of Blp/ΔSH to create G1/ΔSH, F1/ΔSH, and G1F2/ΔSH, respectively. The creation of the Blp I site involved two nucleotide substitutions, and the original assignments are shown in lowercase below the underlined site. To create the G or F single-gene insert, the complete G or F ORF (each illustrated by an open rectangle with the translational initiation codon indicated) with the downstream noncoding region and GE signal (illustrated by a shaded box) was engineered to be followed by a hexanucleotide intergenic sequence (CATATT or CACAAT, identical to the first 6 nt of the naturally occurring G-F or F-M2 IG sequence, respectively), followed by the NS1 GS signal. The G-F double-gene cDNA insert was constructed to contain (in upstream-to-downstream order) the complete G ORF, its downstream noncoding region and GE signal, the G-F IG sequence, the complete F gene, the above-mentioned hexanucleotide from the F-M2 IG, and the NS1 GS signal. All inserts are flanked by Blp I sites. The box on the right shows the details of the deletion of the gene(s) from the naturally occurring position(s), which was done leaving intact the indicated naturally occurring IG regions and without the addition of any heterologous sequence.
    Figure Legend Snippet: Shift of the G or F gene individually or together to a promoter-proximal position(s) in the RSV genome. The diagram at the top illustrates the wild-type RSV genome from which the SH gene had been deleted and in which a Blp I restriction site had been inserted immediately upstream of the NS1 ORF to create Blp/ΔSH. The box underneath and on the left illustrates the insertion of the G gene alone, the F gene alone, or the G and F genes together into the Blp I site of Blp/ΔSH to create G1/ΔSH, F1/ΔSH, and G1F2/ΔSH, respectively. The creation of the Blp I site involved two nucleotide substitutions, and the original assignments are shown in lowercase below the underlined site. To create the G or F single-gene insert, the complete G or F ORF (each illustrated by an open rectangle with the translational initiation codon indicated) with the downstream noncoding region and GE signal (illustrated by a shaded box) was engineered to be followed by a hexanucleotide intergenic sequence (CATATT or CACAAT, identical to the first 6 nt of the naturally occurring G-F or F-M2 IG sequence, respectively), followed by the NS1 GS signal. The G-F double-gene cDNA insert was constructed to contain (in upstream-to-downstream order) the complete G ORF, its downstream noncoding region and GE signal, the G-F IG sequence, the complete F gene, the above-mentioned hexanucleotide from the F-M2 IG, and the NS1 GS signal. All inserts are flanked by Blp I sites. The box on the right shows the details of the deletion of the gene(s) from the naturally occurring position(s), which was done leaving intact the indicated naturally occurring IG regions and without the addition of any heterologous sequence.

    Techniques Used: Sequencing, Construct

    18) Product Images from "Recombinant Respiratory Syncytial Virus with the G and F Genes Shifted to the Promoter-Proximal Positions"

    Article Title: Recombinant Respiratory Syncytial Virus with the G and F Genes Shifted to the Promoter-Proximal Positions

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.23.11931-11942.2002

    Shift of the G or F gene individually or together to a promoter-proximal position(s) in the RSV genome. The diagram at the top illustrates the wild-type RSV genome from which the SH gene had been deleted and in which a Blp I restriction site had been inserted immediately upstream of the NS1 ORF to create Blp/ΔSH. The box underneath and on the left illustrates the insertion of the G gene alone, the F gene alone, or the G and F genes together into the Blp I site of Blp/ΔSH to create G1/ΔSH, F1/ΔSH, and G1F2/ΔSH, respectively. The creation of the Blp I site involved two nucleotide substitutions, and the original assignments are shown in lowercase below the underlined site. To create the G or F single-gene insert, the complete G or F ORF (each illustrated by an open rectangle with the translational initiation codon indicated) with the downstream noncoding region and GE signal (illustrated by a shaded box) was engineered to be followed by a hexanucleotide intergenic sequence (CATATT or CACAAT, identical to the first 6 nt of the naturally occurring G-F or F-M2 IG sequence, respectively), followed by the NS1 GS signal. The G-F double-gene cDNA insert was constructed to contain (in upstream-to-downstream order) the complete G ORF, its downstream noncoding region and GE signal, the G-F IG sequence, the complete F gene, the above-mentioned hexanucleotide from the F-M2 IG, and the NS1 GS signal. All inserts are flanked by Blp I sites. The box on the right shows the details of the deletion of the gene(s) from the naturally occurring position(s), which was done leaving intact the indicated naturally occurring IG regions and without the addition of any heterologous sequence.
    Figure Legend Snippet: Shift of the G or F gene individually or together to a promoter-proximal position(s) in the RSV genome. The diagram at the top illustrates the wild-type RSV genome from which the SH gene had been deleted and in which a Blp I restriction site had been inserted immediately upstream of the NS1 ORF to create Blp/ΔSH. The box underneath and on the left illustrates the insertion of the G gene alone, the F gene alone, or the G and F genes together into the Blp I site of Blp/ΔSH to create G1/ΔSH, F1/ΔSH, and G1F2/ΔSH, respectively. The creation of the Blp I site involved two nucleotide substitutions, and the original assignments are shown in lowercase below the underlined site. To create the G or F single-gene insert, the complete G or F ORF (each illustrated by an open rectangle with the translational initiation codon indicated) with the downstream noncoding region and GE signal (illustrated by a shaded box) was engineered to be followed by a hexanucleotide intergenic sequence (CATATT or CACAAT, identical to the first 6 nt of the naturally occurring G-F or F-M2 IG sequence, respectively), followed by the NS1 GS signal. The G-F double-gene cDNA insert was constructed to contain (in upstream-to-downstream order) the complete G ORF, its downstream noncoding region and GE signal, the G-F IG sequence, the complete F gene, the above-mentioned hexanucleotide from the F-M2 IG, and the NS1 GS signal. All inserts are flanked by Blp I sites. The box on the right shows the details of the deletion of the gene(s) from the naturally occurring position(s), which was done leaving intact the indicated naturally occurring IG regions and without the addition of any heterologous sequence.

    Techniques Used: Sequencing, Construct

    19) Product Images from "Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end"

    Article Title: Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end

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

    doi: 10.1073/pnas.1203118109

    Experiment setup and supercoil relaxation assay. ( A ) A supercoiled dsDNA is tethered between a surface and a paramagnetic bead. As plectonemic supercoils are introduced, DNA extension is reduced. ( B ) When supercoiling is relaxed, an increase in DNA extension
    Figure Legend Snippet: Experiment setup and supercoil relaxation assay. ( A ) A supercoiled dsDNA is tethered between a surface and a paramagnetic bead. As plectonemic supercoils are introduced, DNA extension is reduced. ( B ) When supercoiling is relaxed, an increase in DNA extension

    Techniques Used:

    20) Product Images from "Identification of trichlormethiazide as a Mdr1a/b gene expression enhancer via a dual secretion-based promoter assay"

    Article Title: Identification of trichlormethiazide as a Mdr1a/b gene expression enhancer via a dual secretion-based promoter assay

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.109

    Characterization of Mdr1a and 1b reporter constructs. (A) Core promoter regions of Mdr1a and b according to literature (Hsu et al. 1990 ; Cohen et al. 1991 ). An in silico analysis revealed about 60–70 transcription factor binding sites in both sequences (MatInspector Analysis). Predicted binding sites for p53 (green) and HSF1 (blue) are indicated by arrows and given in detail in the table below. For comparison, we included HSF-binding sites of the human promoter region. As HSF1 and 2 are able to form heterocomplexes (Lecomte et al. 2013 ), we also included information for this transcription factor. (B) Basal activity of both promoters was measured using single Gaussia -based reporter plasmids. N2A cells were transfected and cell supernatant with secreted enzyme collected at the indicated time points. RLU (relative light units) are given as mean ± standard deviation from three independent experiments performed in triplicate. Cells transfected with the promoter-less control vector (mock) were used as a control and values obtained for those cells at 16 h post transfection were set to 100%. All data obtained for promoter-containing constructs were significantly higher than the respective control sample (one-way ANOVA, Bonferroni posttest, P
    Figure Legend Snippet: Characterization of Mdr1a and 1b reporter constructs. (A) Core promoter regions of Mdr1a and b according to literature (Hsu et al. 1990 ; Cohen et al. 1991 ). An in silico analysis revealed about 60–70 transcription factor binding sites in both sequences (MatInspector Analysis). Predicted binding sites for p53 (green) and HSF1 (blue) are indicated by arrows and given in detail in the table below. For comparison, we included HSF-binding sites of the human promoter region. As HSF1 and 2 are able to form heterocomplexes (Lecomte et al. 2013 ), we also included information for this transcription factor. (B) Basal activity of both promoters was measured using single Gaussia -based reporter plasmids. N2A cells were transfected and cell supernatant with secreted enzyme collected at the indicated time points. RLU (relative light units) are given as mean ± standard deviation from three independent experiments performed in triplicate. Cells transfected with the promoter-less control vector (mock) were used as a control and values obtained for those cells at 16 h post transfection were set to 100%. All data obtained for promoter-containing constructs were significantly higher than the respective control sample (one-way ANOVA, Bonferroni posttest, P

    Techniques Used: Construct, In Silico, Binding Assay, Activity Assay, Transfection, Standard Deviation, Plasmid Preparation

    Screening of FDA-approved drugs for Mdr1a and b expression modifying potential. (A) Mdr1a and b promoter activities upon 24 or 48 h of incubation with respective substances. N2A cells were transiently cotransfected with Mdr1a- and Mdr1b promoter reporter and after end of transfection period (7 h) drugs of the FDA-approved compound library were added in fresh culture medium. After 24 or 48 h of treatment, cell supernatant aliquots were collected and subjected to dual luciferase reporter gene assay. RLU were normalized to values of DMSO-treated control cells. TSA (15 nmol/L) served as an internal positive control (data not shown). All substances were tested in three independent experiments; drugs that resulted in experimental data with a standard deviation of > 30% were retested in two additional experiments. Values are given as means, standard deviations are not included in the graph for reasons of clarity (red: gemcitabine; green: trichlormethiazide; gray: oltipraz). (B) Outcome of screening. Drugs were defined as hits (inhibitors or activators of the respective promoter activity) when RLU were obtained > 130% or
    Figure Legend Snippet: Screening of FDA-approved drugs for Mdr1a and b expression modifying potential. (A) Mdr1a and b promoter activities upon 24 or 48 h of incubation with respective substances. N2A cells were transiently cotransfected with Mdr1a- and Mdr1b promoter reporter and after end of transfection period (7 h) drugs of the FDA-approved compound library were added in fresh culture medium. After 24 or 48 h of treatment, cell supernatant aliquots were collected and subjected to dual luciferase reporter gene assay. RLU were normalized to values of DMSO-treated control cells. TSA (15 nmol/L) served as an internal positive control (data not shown). All substances were tested in three independent experiments; drugs that resulted in experimental data with a standard deviation of > 30% were retested in two additional experiments. Values are given as means, standard deviations are not included in the graph for reasons of clarity (red: gemcitabine; green: trichlormethiazide; gray: oltipraz). (B) Outcome of screening. Drugs were defined as hits (inhibitors or activators of the respective promoter activity) when RLU were obtained > 130% or

    Techniques Used: Expressing, Incubation, Transfection, Luciferase, Reporter Gene Assay, Positive Control, Standard Deviation, Activity Assay

    Definition of experimental parameters and evaluation of the Mdr1 dual promoter assay. (A) Measurement of luciferase enzymatic activity in dependency of SDS supplementation. Supernatants from cells transfected with the respective reporter were supplemented with SDS as indicated (v/v). Water served as a solvent control (0% SDS) and values were normalized to this control (mean ± standard deviation from technical replicates). (B) Experimental setup for dual reporter gene assay. Cell supernatants were first supplemented with Gaussia p . substrate and light emission detected for 10 sec. The signal was subsequently quenched by addition of 0.01% SDS in Cypridina n . substrate mixture, followed by another 10-sec detection period. (C) Comparison of single- and dual reporter gene assay. To demonstrate reliability of the newly established dual reporter gene assay for Mdr1a and 1b, cells were cotransfected with both reporter gene constructs and transcription factor expression plasmids or empty vector (mock). Overexpression of transcription factors was proven by western blotting of respective cell lysates (see right part of the figure). Additionally, cells only transfected with reporter constructs were treated with TSA (15 nmol/L). Supernatants were collected and either luciferase activities were measured separately (mono) or in the dual setup. Values are given as mean ± standard deviation from three independently conducted experiments ( n = 9, one-way ANOVA, Dunnett’s multiple comparison test; all data have a P
    Figure Legend Snippet: Definition of experimental parameters and evaluation of the Mdr1 dual promoter assay. (A) Measurement of luciferase enzymatic activity in dependency of SDS supplementation. Supernatants from cells transfected with the respective reporter were supplemented with SDS as indicated (v/v). Water served as a solvent control (0% SDS) and values were normalized to this control (mean ± standard deviation from technical replicates). (B) Experimental setup for dual reporter gene assay. Cell supernatants were first supplemented with Gaussia p . substrate and light emission detected for 10 sec. The signal was subsequently quenched by addition of 0.01% SDS in Cypridina n . substrate mixture, followed by another 10-sec detection period. (C) Comparison of single- and dual reporter gene assay. To demonstrate reliability of the newly established dual reporter gene assay for Mdr1a and 1b, cells were cotransfected with both reporter gene constructs and transcription factor expression plasmids or empty vector (mock). Overexpression of transcription factors was proven by western blotting of respective cell lysates (see right part of the figure). Additionally, cells only transfected with reporter constructs were treated with TSA (15 nmol/L). Supernatants were collected and either luciferase activities were measured separately (mono) or in the dual setup. Values are given as mean ± standard deviation from three independently conducted experiments ( n = 9, one-way ANOVA, Dunnett’s multiple comparison test; all data have a P

    Techniques Used: Promoter Assay, Luciferase, Activity Assay, Transfection, Standard Deviation, Reporter Gene Assay, Size-exclusion Chromatography, Construct, Expressing, Plasmid Preparation, Over Expression, Western Blot

    Effect of selected hits in wild-type and PGP knockout mice on risperidone transport into the brain. (A) Distribution of risperidone and its metabolite 9-OH-risperidone in candidate-drug pretreated mice. FVB/N male mice (wt) were treated with the respective drug 48 h by a daily injection (oltipraz: 30 mg/kg; trichlormethiazide: 8 mg/kg; gemcitabine: 20 mg/kg). DMSO-injected mice served as a control. Mice were subsequently injected i.p. with risperidone (3 mg/kg) and brains and serum collected after 3 h. Analysis of risperidone and the active metabolite was performed by HPLC and concentrations were determined by standard curves. Values obtained for brain samples were divided by those of serum and ratios were compared to those of control animals. Data represent mean ± standard error of samples from n ≥ 3 animals per group. (B) Expression of Mdr1a/b in total brain of mice pretreated with drug candidates. Mice were treated as described in A. Subsequently, mRNA was prepared from brain tissue homogenate and analyzed by real-time RT-PCR for Mdr1a and b mRNA. Values were calculated using a standard curve and normalized to Gapdh mRNA. Values represent mean ± standard error of samples from n ≥ 3 animals per group, performed in technical duplicates. (C) Impact of trichlormethiazide on risperidone/9-OH-risperidone transport in PGP knockout mice. Male PGP knockout (ko) mice were treated as described in A ( n = 5 for control, n = 4 for ko). Samples from serum and brain homogenates were analyzed for risperidone and 9-OH-risperidone content by HPLC (unpaired student’s t -test versus control; all P > 0.05).
    Figure Legend Snippet: Effect of selected hits in wild-type and PGP knockout mice on risperidone transport into the brain. (A) Distribution of risperidone and its metabolite 9-OH-risperidone in candidate-drug pretreated mice. FVB/N male mice (wt) were treated with the respective drug 48 h by a daily injection (oltipraz: 30 mg/kg; trichlormethiazide: 8 mg/kg; gemcitabine: 20 mg/kg). DMSO-injected mice served as a control. Mice were subsequently injected i.p. with risperidone (3 mg/kg) and brains and serum collected after 3 h. Analysis of risperidone and the active metabolite was performed by HPLC and concentrations were determined by standard curves. Values obtained for brain samples were divided by those of serum and ratios were compared to those of control animals. Data represent mean ± standard error of samples from n ≥ 3 animals per group. (B) Expression of Mdr1a/b in total brain of mice pretreated with drug candidates. Mice were treated as described in A. Subsequently, mRNA was prepared from brain tissue homogenate and analyzed by real-time RT-PCR for Mdr1a and b mRNA. Values were calculated using a standard curve and normalized to Gapdh mRNA. Values represent mean ± standard error of samples from n ≥ 3 animals per group, performed in technical duplicates. (C) Impact of trichlormethiazide on risperidone/9-OH-risperidone transport in PGP knockout mice. Male PGP knockout (ko) mice were treated as described in A ( n = 5 for control, n = 4 for ko). Samples from serum and brain homogenates were analyzed for risperidone and 9-OH-risperidone content by HPLC (unpaired student’s t -test versus control; all P > 0.05).

    Techniques Used: Knock-Out, Mouse Assay, Injection, High Performance Liquid Chromatography, Expressing, Quantitative RT-PCR

    21) Product Images from "Identification of trichlormethiazide as a Mdr1a/b gene expression enhancer via a dual secretion-based promoter assay"

    Article Title: Identification of trichlormethiazide as a Mdr1a/b gene expression enhancer via a dual secretion-based promoter assay

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.109

    Definition of experimental parameters and evaluation of the Mdr1 dual promoter assay. (A) Measurement of luciferase enzymatic activity in dependency of SDS supplementation. Supernatants from cells transfected with the respective reporter were supplemented with SDS as indicated (v/v). Water served as a solvent control (0% SDS) and values were normalized to this control (mean ± standard deviation from technical replicates). (B) Experimental setup for dual reporter gene assay. Cell supernatants were first supplemented with Gaussia p . substrate and light emission detected for 10 sec. The signal was subsequently quenched by addition of 0.01% SDS in Cypridina n . substrate mixture, followed by another 10-sec detection period. (C) Comparison of single- and dual reporter gene assay. To demonstrate reliability of the newly established dual reporter gene assay for Mdr1a and 1b, cells were cotransfected with both reporter gene constructs and transcription factor expression plasmids or empty vector (mock). Overexpression of transcription factors was proven by western blotting of respective cell lysates (see right part of the figure). Additionally, cells only transfected with reporter constructs were treated with TSA (15 nmol/L). Supernatants were collected and either luciferase activities were measured separately (mono) or in the dual setup. Values are given as mean ± standard deviation from three independently conducted experiments ( n = 9, one-way ANOVA, Dunnett’s multiple comparison test; all data have a P
    Figure Legend Snippet: Definition of experimental parameters and evaluation of the Mdr1 dual promoter assay. (A) Measurement of luciferase enzymatic activity in dependency of SDS supplementation. Supernatants from cells transfected with the respective reporter were supplemented with SDS as indicated (v/v). Water served as a solvent control (0% SDS) and values were normalized to this control (mean ± standard deviation from technical replicates). (B) Experimental setup for dual reporter gene assay. Cell supernatants were first supplemented with Gaussia p . substrate and light emission detected for 10 sec. The signal was subsequently quenched by addition of 0.01% SDS in Cypridina n . substrate mixture, followed by another 10-sec detection period. (C) Comparison of single- and dual reporter gene assay. To demonstrate reliability of the newly established dual reporter gene assay for Mdr1a and 1b, cells were cotransfected with both reporter gene constructs and transcription factor expression plasmids or empty vector (mock). Overexpression of transcription factors was proven by western blotting of respective cell lysates (see right part of the figure). Additionally, cells only transfected with reporter constructs were treated with TSA (15 nmol/L). Supernatants were collected and either luciferase activities were measured separately (mono) or in the dual setup. Values are given as mean ± standard deviation from three independently conducted experiments ( n = 9, one-way ANOVA, Dunnett’s multiple comparison test; all data have a P

    Techniques Used: Promoter Assay, Luciferase, Activity Assay, Transfection, Standard Deviation, Reporter Gene Assay, Size-exclusion Chromatography, Construct, Expressing, Plasmid Preparation, Over Expression, Western Blot

    22) Product Images from "Murine cytomegalovirus degrades MHC class II to colonize the salivary glands"

    Article Title: Murine cytomegalovirus degrades MHC class II to colonize the salivary glands

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006905

    Significant M78 - MCMV rescue by CD4 + T cell loss. a . C57BL/6 mice were given WT or M78 - MCMV i.n. (3x10 4 p.f.u.). 56d later sera were assayed for MCMV-specific IgG and IgM by ELISA. Naive = age-matched, uninfected controls. Each point shows the mean of results for 7 mice. M78 - MCMV elicited significantly less IgG response than WT (p
    Figure Legend Snippet: Significant M78 - MCMV rescue by CD4 + T cell loss. a . C57BL/6 mice were given WT or M78 - MCMV i.n. (3x10 4 p.f.u.). 56d later sera were assayed for MCMV-specific IgG and IgM by ELISA. Naive = age-matched, uninfected controls. Each point shows the mean of results for 7 mice. M78 - MCMV elicited significantly less IgG response than WT (p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    MHC II degradation by MCMV requires M78. a . RAW-264 and RAW-C2TA cells were infected at low multiplicity (0.01 p.f.u. / cell, 1h) and washed x3 in PBS. RAW-C2TA cells were also infected at high multiplicity (3 p.f.u. / cell) by centrifuging virus onto the cells (400 x g , 30min), then washed x 3 in PBS. The cells were cultured in complete medium, and at each time point, triplicate cultures were plaque-assayed for infectious virus. Symbols show mean ± SEM. Time = 0 shows the calculated virus input. M78 - MCMV had similar growth defects in MHC II + and MHC II - cells. b . RAW-C2TA cells were infected with WT or M78 - βgal + MCMV (1 p.f.u. / cell, 72h), or left uninfected (UI), then stained for surface MHC II (non-permeabilized) before fixation, permeabilization and staining for βgal; or fixed, permeabilized then stained for MHC class II plus βgal (permeabilized). MHC II either just at the cell surface (non-permeabilized) or also in the cell (permeabilized) was assayed by flow cytometry. Numbers show % total cells in each gated region. This was significantly less for M78 - than for WT MCMV (p
    Figure Legend Snippet: MHC II degradation by MCMV requires M78. a . RAW-264 and RAW-C2TA cells were infected at low multiplicity (0.01 p.f.u. / cell, 1h) and washed x3 in PBS. RAW-C2TA cells were also infected at high multiplicity (3 p.f.u. / cell) by centrifuging virus onto the cells (400 x g , 30min), then washed x 3 in PBS. The cells were cultured in complete medium, and at each time point, triplicate cultures were plaque-assayed for infectious virus. Symbols show mean ± SEM. Time = 0 shows the calculated virus input. M78 - MCMV had similar growth defects in MHC II + and MHC II - cells. b . RAW-C2TA cells were infected with WT or M78 - βgal + MCMV (1 p.f.u. / cell, 72h), or left uninfected (UI), then stained for surface MHC II (non-permeabilized) before fixation, permeabilization and staining for βgal; or fixed, permeabilized then stained for MHC class II plus βgal (permeabilized). MHC II either just at the cell surface (non-permeabilized) or also in the cell (permeabilized) was assayed by flow cytometry. Numbers show % total cells in each gated region. This was significantly less for M78 - than for WT MCMV (p

    Techniques Used: Infection, Cell Culture, Staining, Flow Cytometry, Cytometry

    M78 relocalizes MHC II. a . Cloned RAW-C2TA cells were transduced with vector alone or with M78 (line 1 and line 2), or infected with MCMV-GFP (+ MCMV, 1 p.f.u. / cell, 72h), then stained for MHC II (dashed lines). Solid lines show uninfected, untransduced RAW-C2TA cells. The biphasic population with MCMV corresponds to GFP + (MHC II - ) and GFP - (MHC II + ) cells. M78 transfection had much less effect than MCMV infection on cell surface MHC II expression. b . MCMV-GFP-infected RAW-C2TA cells were stained for M78. GFP was visualized directly. Nuclei were stained with DAPI. Arrows show example infected cells with M78 in vesicles. c . Untransduced (control) and M78-transduced RAW-C2TA cells were stained for M78 and CD44. Arrows show M78 staining, which did not co-localize with CD44. d . M78-transduced RAW-C2TA cells were stained for M78 and MHC II. M78 occupied intracellular vesicles, as did MHC class II in M78 + cells. White arrows show examples of co-localization. Grey arrows show M78 - cells with MHC II on the plasma membrane. e . Macrophages grown from bone marrow were infected with WT MCMV (1 p.f.u. / cell, 24h), then fixed and stained for M78 and MHC II. The white arrow shows a typical uninfected MHC II + cell. The yellow arrow shows a typical infected cell with MHC II in vesicles. > 80% of vesicles were M78 + MHC II + ,
    Figure Legend Snippet: M78 relocalizes MHC II. a . Cloned RAW-C2TA cells were transduced with vector alone or with M78 (line 1 and line 2), or infected with MCMV-GFP (+ MCMV, 1 p.f.u. / cell, 72h), then stained for MHC II (dashed lines). Solid lines show uninfected, untransduced RAW-C2TA cells. The biphasic population with MCMV corresponds to GFP + (MHC II - ) and GFP - (MHC II + ) cells. M78 transfection had much less effect than MCMV infection on cell surface MHC II expression. b . MCMV-GFP-infected RAW-C2TA cells were stained for M78. GFP was visualized directly. Nuclei were stained with DAPI. Arrows show example infected cells with M78 in vesicles. c . Untransduced (control) and M78-transduced RAW-C2TA cells were stained for M78 and CD44. Arrows show M78 staining, which did not co-localize with CD44. d . M78-transduced RAW-C2TA cells were stained for M78 and MHC II. M78 occupied intracellular vesicles, as did MHC class II in M78 + cells. White arrows show examples of co-localization. Grey arrows show M78 - cells with MHC II on the plasma membrane. e . Macrophages grown from bone marrow were infected with WT MCMV (1 p.f.u. / cell, 24h), then fixed and stained for M78 and MHC II. The white arrow shows a typical uninfected MHC II + cell. The yellow arrow shows a typical infected cell with MHC II in vesicles. > 80% of vesicles were M78 + MHC II + ,

    Techniques Used: Clone Assay, Transduction, Plasmid Preparation, Infection, Staining, Transfection, Expressing

    M78 - MCMV replication in vivo . a . BALB/c mice were given i.n. WT or M78 - MCMV (3x10 4 p.f.u.). 3d later lung sections were stained for MCMV IE1 and MHC II. Nuclei were stained with DAPI. Arrows show infected cells. b . Quantitation of staining as in a , for sections from 3 mice. Few WT infected cells were MHC II + . At d1 and d3, significantly more M78 - -infected cells were MHC II + . c . Mice were infected as in a . Lungs and salivary glands were plaque-assayed for infectious virus, and QPCR-assayed for viral genomes relative to cellular (βactin) genomes. Bars show means, other symbols show individual mice. Dashed lines show assay sensitivity limits. Significant differences are indicated.
    Figure Legend Snippet: M78 - MCMV replication in vivo . a . BALB/c mice were given i.n. WT or M78 - MCMV (3x10 4 p.f.u.). 3d later lung sections were stained for MCMV IE1 and MHC II. Nuclei were stained with DAPI. Arrows show infected cells. b . Quantitation of staining as in a , for sections from 3 mice. Few WT infected cells were MHC II + . At d1 and d3, significantly more M78 - -infected cells were MHC II + . c . Mice were infected as in a . Lungs and salivary glands were plaque-assayed for infectious virus, and QPCR-assayed for viral genomes relative to cellular (βactin) genomes. Bars show means, other symbols show individual mice. Dashed lines show assay sensitivity limits. Significant differences are indicated.

    Techniques Used: In Vivo, Mouse Assay, Staining, Infection, Quantitation Assay, Real-time Polymerase Chain Reaction

    23) Product Images from "Murine cytomegalovirus degrades MHC class II to colonize the salivary glands"

    Article Title: Murine cytomegalovirus degrades MHC class II to colonize the salivary glands

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006905

    Significant M78 - MCMV rescue by CD4 + T cell loss. a . C57BL/6 mice were given WT or M78 - MCMV i.n. (3x10 4 p.f.u.). 56d later sera were assayed for MCMV-specific IgG and IgM by ELISA. Naive = age-matched, uninfected controls. Each point shows the mean of results for 7 mice. M78 - MCMV elicited significantly less IgG response than WT (p
    Figure Legend Snippet: Significant M78 - MCMV rescue by CD4 + T cell loss. a . C57BL/6 mice were given WT or M78 - MCMV i.n. (3x10 4 p.f.u.). 56d later sera were assayed for MCMV-specific IgG and IgM by ELISA. Naive = age-matched, uninfected controls. Each point shows the mean of results for 7 mice. M78 - MCMV elicited significantly less IgG response than WT (p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    MHC II degradation by MCMV requires M78. a . RAW-264 and RAW-C2TA cells were infected at low multiplicity (0.01 p.f.u. / cell, 1h) and washed x3 in PBS. RAW-C2TA cells were also infected at high multiplicity (3 p.f.u. / cell) by centrifuging virus onto the cells (400 x g , 30min), then washed x 3 in PBS. The cells were cultured in complete medium, and at each time point, triplicate cultures were plaque-assayed for infectious virus. Symbols show mean ± SEM. Time = 0 shows the calculated virus input. M78 - MCMV had similar growth defects in MHC II + and MHC II - cells. b . RAW-C2TA cells were infected with WT or M78 - βgal + MCMV (1 p.f.u. / cell, 72h), or left uninfected (UI), then stained for surface MHC II (non-permeabilized) before fixation, permeabilization and staining for βgal; or fixed, permeabilized then stained for MHC class II plus βgal (permeabilized). MHC II either just at the cell surface (non-permeabilized) or also in the cell (permeabilized) was assayed by flow cytometry. Numbers show % total cells in each gated region. This was significantly less for M78 - than for WT MCMV (p
    Figure Legend Snippet: MHC II degradation by MCMV requires M78. a . RAW-264 and RAW-C2TA cells were infected at low multiplicity (0.01 p.f.u. / cell, 1h) and washed x3 in PBS. RAW-C2TA cells were also infected at high multiplicity (3 p.f.u. / cell) by centrifuging virus onto the cells (400 x g , 30min), then washed x 3 in PBS. The cells were cultured in complete medium, and at each time point, triplicate cultures were plaque-assayed for infectious virus. Symbols show mean ± SEM. Time = 0 shows the calculated virus input. M78 - MCMV had similar growth defects in MHC II + and MHC II - cells. b . RAW-C2TA cells were infected with WT or M78 - βgal + MCMV (1 p.f.u. / cell, 72h), or left uninfected (UI), then stained for surface MHC II (non-permeabilized) before fixation, permeabilization and staining for βgal; or fixed, permeabilized then stained for MHC class II plus βgal (permeabilized). MHC II either just at the cell surface (non-permeabilized) or also in the cell (permeabilized) was assayed by flow cytometry. Numbers show % total cells in each gated region. This was significantly less for M78 - than for WT MCMV (p

    Techniques Used: Infection, Cell Culture, Staining, Flow Cytometry, Cytometry

    M78 relocalizes MHC II. a . Cloned RAW-C2TA cells were transduced with vector alone or with M78 (line 1 and line 2), or infected with MCMV-GFP (+ MCMV, 1 p.f.u. / cell, 72h), then stained for MHC II (dashed lines). Solid lines show uninfected, untransduced RAW-C2TA cells. The biphasic population with MCMV corresponds to GFP + (MHC II - ) and GFP - (MHC II + ) cells. M78 transfection had much less effect than MCMV infection on cell surface MHC II expression. b . MCMV-GFP-infected RAW-C2TA cells were stained for M78. GFP was visualized directly. Nuclei were stained with DAPI. Arrows show example infected cells with M78 in vesicles. c . Untransduced (control) and M78-transduced RAW-C2TA cells were stained for M78 and CD44. Arrows show M78 staining, which did not co-localize with CD44. d . M78-transduced RAW-C2TA cells were stained for M78 and MHC II. M78 occupied intracellular vesicles, as did MHC class II in M78 + cells. White arrows show examples of co-localization. Grey arrows show M78 - cells with MHC II on the plasma membrane. e . Macrophages grown from bone marrow were infected with WT MCMV (1 p.f.u. / cell, 24h), then fixed and stained for M78 and MHC II. The white arrow shows a typical uninfected MHC II + cell. The yellow arrow shows a typical infected cell with MHC II in vesicles. > 80% of vesicles were M78 + MHC II + ,
    Figure Legend Snippet: M78 relocalizes MHC II. a . Cloned RAW-C2TA cells were transduced with vector alone or with M78 (line 1 and line 2), or infected with MCMV-GFP (+ MCMV, 1 p.f.u. / cell, 72h), then stained for MHC II (dashed lines). Solid lines show uninfected, untransduced RAW-C2TA cells. The biphasic population with MCMV corresponds to GFP + (MHC II - ) and GFP - (MHC II + ) cells. M78 transfection had much less effect than MCMV infection on cell surface MHC II expression. b . MCMV-GFP-infected RAW-C2TA cells were stained for M78. GFP was visualized directly. Nuclei were stained with DAPI. Arrows show example infected cells with M78 in vesicles. c . Untransduced (control) and M78-transduced RAW-C2TA cells were stained for M78 and CD44. Arrows show M78 staining, which did not co-localize with CD44. d . M78-transduced RAW-C2TA cells were stained for M78 and MHC II. M78 occupied intracellular vesicles, as did MHC class II in M78 + cells. White arrows show examples of co-localization. Grey arrows show M78 - cells with MHC II on the plasma membrane. e . Macrophages grown from bone marrow were infected with WT MCMV (1 p.f.u. / cell, 24h), then fixed and stained for M78 and MHC II. The white arrow shows a typical uninfected MHC II + cell. The yellow arrow shows a typical infected cell with MHC II in vesicles. > 80% of vesicles were M78 + MHC II + ,

    Techniques Used: Clone Assay, Transduction, Plasmid Preparation, Infection, Staining, Transfection, Expressing

    M78 - MCMV replication in vivo . a . BALB/c mice were given i.n. WT or M78 - MCMV (3x10 4 p.f.u.). 3d later lung sections were stained for MCMV IE1 and MHC II. Nuclei were stained with DAPI. Arrows show infected cells. b . Quantitation of staining as in a , for sections from 3 mice. Few WT infected cells were MHC II + . At d1 and d3, significantly more M78 - -infected cells were MHC II + . c . Mice were infected as in a . Lungs and salivary glands were plaque-assayed for infectious virus, and QPCR-assayed for viral genomes relative to cellular (βactin) genomes. Bars show means, other symbols show individual mice. Dashed lines show assay sensitivity limits. Significant differences are indicated.
    Figure Legend Snippet: M78 - MCMV replication in vivo . a . BALB/c mice were given i.n. WT or M78 - MCMV (3x10 4 p.f.u.). 3d later lung sections were stained for MCMV IE1 and MHC II. Nuclei were stained with DAPI. Arrows show infected cells. b . Quantitation of staining as in a , for sections from 3 mice. Few WT infected cells were MHC II + . At d1 and d3, significantly more M78 - -infected cells were MHC II + . c . Mice were infected as in a . Lungs and salivary glands were plaque-assayed for infectious virus, and QPCR-assayed for viral genomes relative to cellular (βactin) genomes. Bars show means, other symbols show individual mice. Dashed lines show assay sensitivity limits. Significant differences are indicated.

    Techniques Used: In Vivo, Mouse Assay, Staining, Infection, Quantitation Assay, Real-time Polymerase Chain Reaction

    24) Product Images from "Novel malaria antigen Plasmodium yoelii E140 induces antibody-mediated sterile protection in mice against malaria challenge"

    Article Title: Novel malaria antigen Plasmodium yoelii E140 induces antibody-mediated sterile protection in mice against malaria challenge

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0232234

    PyE140 immunization does not reduce liver stage parasite burden. Eleven week old CD1 mice were immunized with DNA and HuAd5 vectors expressing either PyE140 or PyCSP at weeks 0 and 6, respectively. The HuAd5-PyE140 vector encoded the codon-optimized PyE140co gene. Groups of null immunized and naïve mice were also included. Two weeks after the boost, mice were challenged with 20,000 P . yoelii 17XNL sporozoites by subcutaneous injection. Livers were harvested 42 hours after challenge for evaluation of Py18S RNA copies/murine GAPDH RNA copies by RT-qPCR. Filled symbols represent detectable Py18S RNA copies, and open symbols represent Py18S RNA copies below the limit of detection (10 copies of Py18S RNA) and are plotted at ½ the limit of detection (5 copies of Py18S RNA). ** indicates p
    Figure Legend Snippet: PyE140 immunization does not reduce liver stage parasite burden. Eleven week old CD1 mice were immunized with DNA and HuAd5 vectors expressing either PyE140 or PyCSP at weeks 0 and 6, respectively. The HuAd5-PyE140 vector encoded the codon-optimized PyE140co gene. Groups of null immunized and naïve mice were also included. Two weeks after the boost, mice were challenged with 20,000 P . yoelii 17XNL sporozoites by subcutaneous injection. Livers were harvested 42 hours after challenge for evaluation of Py18S RNA copies/murine GAPDH RNA copies by RT-qPCR. Filled symbols represent detectable Py18S RNA copies, and open symbols represent Py18S RNA copies below the limit of detection (10 copies of Py18S RNA) and are plotted at ½ the limit of detection (5 copies of Py18S RNA). ** indicates p

    Techniques Used: Mouse Assay, Expressing, Plasmid Preparation, Injection, Quantitative RT-PCR

    25) Product Images from "Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species"

    Article Title: Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species

    Journal: Journal of Clinical Microbiology

    doi:

    AFLP fingerprint of M. genitalium G-37 T . Bgl II plus Mfe I AFLP templates were prepared on three occasions with the same batch of genomic DNA of M. genitalium G-37 T . Amplification products obtained from each experiment (blue, black, and red patterns) were detected on an ABI 373A sequencer by GeneScan 1.2.2.-1 software. The complete AFLP patterns are superimposed and divided into four parts (A to D). The fragment size scale (base pairs) is indicated above each panel. y axes indicate relative amounts of amplicons (in fluorescence units).
    Figure Legend Snippet: AFLP fingerprint of M. genitalium G-37 T . Bgl II plus Mfe I AFLP templates were prepared on three occasions with the same batch of genomic DNA of M. genitalium G-37 T . Amplification products obtained from each experiment (blue, black, and red patterns) were detected on an ABI 373A sequencer by GeneScan 1.2.2.-1 software. The complete AFLP patterns are superimposed and divided into four parts (A to D). The fragment size scale (base pairs) is indicated above each panel. y axes indicate relative amounts of amplicons (in fluorescence units).

    Techniques Used: Amplification, Software, Fluorescence

    Genescan-derived electropherogram traces of AFLP templates of different complexity. AFLP templates of a field isolate of Escherichia coli and M. genitalium G-37 T were prepared by the digestion of genomic DNAs with Bgl II and Mfe I and subsequent ligation of corresponding adapters. PCR products of individual samples and a mixture of the AFLP templates, containing adjusted DNA concentrations, were detected on an ABI 373A sequencer. The hybrid pattern (middle panel) contains all of the bands detected in individual AFLP templates. The fragment size scale (base pairs) is indicated above the top panel.
    Figure Legend Snippet: Genescan-derived electropherogram traces of AFLP templates of different complexity. AFLP templates of a field isolate of Escherichia coli and M. genitalium G-37 T were prepared by the digestion of genomic DNAs with Bgl II and Mfe I and subsequent ligation of corresponding adapters. PCR products of individual samples and a mixture of the AFLP templates, containing adjusted DNA concentrations, were detected on an ABI 373A sequencer. The hybrid pattern (middle panel) contains all of the bands detected in individual AFLP templates. The fragment size scale (base pairs) is indicated above the top panel.

    Techniques Used: Derivative Assay, Ligation, Polymerase Chain Reaction

    26) Product Images from "Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]"

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.15.01663

    -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).
    Figure Legend Snippet: -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Techniques Used: Polymerase Chain Reaction, Amplification, Generated

    27) Product Images from "Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression"

    Article Title: Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression

    Journal: Nature protocols

    doi: 10.1038/nprot.2009.145

    Design of shRNA oligonucleotides for knocking down gene expression and silent mutations for knocking-in gene expression at internal gene regions. In A), annealed shRNA oligonucleotides form dsDNA fragment with BamH I/ Hind III sites, respectively (shown
    Figure Legend Snippet: Design of shRNA oligonucleotides for knocking down gene expression and silent mutations for knocking-in gene expression at internal gene regions. In A), annealed shRNA oligonucleotides form dsDNA fragment with BamH I/ Hind III sites, respectively (shown

    Techniques Used: shRNA, Expressing

    28) Product Images from "Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni"

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095084

    Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.
    Figure Legend Snippet: Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Techniques Used: Binding Assay, Amplification, TA Cloning, Clone Assay, Plasmid Preparation, Subcloning, Marker

    Adaptation of the pRRC gene delivery and expression system to harbor hygromycin B or apramycin resistance, and testing of genome-integrated markers for detrimental effects of resistance genes. (A) Schematic of pRRC, which inserts into any of 3 rRNA clusters in the genome by homologous recombination. (B) Inverse PCR amplification of pRRC with primers 5705 ( Kpn I) and 5706 deleted the chloramphenicol resistance gene but conserved the Campylobacter -optimized cat promoter. (C) The inverse PCR product was digested with Kpn I and Xba I, and ligated to similarly digested aph (7″) or aac (3)IV from pAC1H or pAC1A to create pRRH and pRRA respectively (only pRRH is shown). (D) Restriction digest analysis confirmed the function of all introduced sites. (E) The resistance markers from pRRK, pRRC, pRRH and pRRA were inserted into the C. jejuni 81–176 genome, and each resulting strain was analyzed for microaerobic growth and survival in shaken Mueller-Hinton (MH) broth by counting CFU over 48 hours at both 42°C (left panel) and 37°C (right panel). (F) To determine if the introduction of either marker contributed any fitness cost that could affect competitiveness against wild-type or the other marked strains, a competition assay was performed. Equal numbers of wild-type marked with hygromycin, apramycin, chloramphenicol and kanamycin resistance markers were co-cultured with unmarked wild-type in shaking MH broth at 37°C under microaerobic conditions. CFU were assessed by plating a dilution series on MH agar. (G) CFU were further assessed from the co-culture by plating on MH only (the total CFU, same data as in F) or MH supplemented with each antibiotic, representing the number of bacteria resistant to each antibiotic.
    Figure Legend Snippet: Adaptation of the pRRC gene delivery and expression system to harbor hygromycin B or apramycin resistance, and testing of genome-integrated markers for detrimental effects of resistance genes. (A) Schematic of pRRC, which inserts into any of 3 rRNA clusters in the genome by homologous recombination. (B) Inverse PCR amplification of pRRC with primers 5705 ( Kpn I) and 5706 deleted the chloramphenicol resistance gene but conserved the Campylobacter -optimized cat promoter. (C) The inverse PCR product was digested with Kpn I and Xba I, and ligated to similarly digested aph (7″) or aac (3)IV from pAC1H or pAC1A to create pRRH and pRRA respectively (only pRRH is shown). (D) Restriction digest analysis confirmed the function of all introduced sites. (E) The resistance markers from pRRK, pRRC, pRRH and pRRA were inserted into the C. jejuni 81–176 genome, and each resulting strain was analyzed for microaerobic growth and survival in shaken Mueller-Hinton (MH) broth by counting CFU over 48 hours at both 42°C (left panel) and 37°C (right panel). (F) To determine if the introduction of either marker contributed any fitness cost that could affect competitiveness against wild-type or the other marked strains, a competition assay was performed. Equal numbers of wild-type marked with hygromycin, apramycin, chloramphenicol and kanamycin resistance markers were co-cultured with unmarked wild-type in shaking MH broth at 37°C under microaerobic conditions. CFU were assessed by plating a dilution series on MH agar. (G) CFU were further assessed from the co-culture by plating on MH only (the total CFU, same data as in F) or MH supplemented with each antibiotic, representing the number of bacteria resistant to each antibiotic.

    Techniques Used: Expressing, Homologous Recombination, Inverse PCR, Amplification, Marker, Competitive Binding Assay, Cell Culture, Co-Culture Assay

    29) Product Images from "The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus"

    Article Title: The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus

    Journal: Journal of Bacteriology

    doi:

    Structures of plasmids pGB2, pAY1099, and pAY1105. The aadA gene is flanked by a pair of Bsp HI sites in each vector, and its translation initiates at an ATG codon within one of these sites. Also shown are unique sites within the polylinker of pGB2 derived
    Figure Legend Snippet: Structures of plasmids pGB2, pAY1099, and pAY1105. The aadA gene is flanked by a pair of Bsp HI sites in each vector, and its translation initiates at an ATG codon within one of these sites. Also shown are unique sites within the polylinker of pGB2 derived

    Techniques Used: Plasmid Preparation, Derivative Assay

    30) Product Images from "Photorespiratory glycolate oxidase is essential for the survival of the red alga Cyanidioschyzon merolae under ambient CO2 conditions"

    Article Title: Photorespiratory glycolate oxidase is essential for the survival of the red alga Cyanidioschyzon merolae under ambient CO2 conditions

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erw118

    Generation of a Δ gox knockout mutant in C. merolae . ( A ) Schematic presentation of the strategy to generate a knockout mutant for CmGOX . For detailed information see ‘Materials and methods’. ( B ) Verification of Δ gox mutant lines #43, #45, and #46. PCR was performed on genomic DNA of the M4 background mutant and the Δ gox mutant lines #43, #45, and #46 with primers flanking the CmGOX upstream and downstream regions (F1/R1) and the CmGOX coding region (F2/R2), respectively. Expected fragment sizes were F1/R1: 4.5kb (M4), 6kb (Δ gox ); F2/R2: 0.66kb (M4), – (Δ gox ). ( C ) Verification of absence of CmGOX transcripts in the Δ gox knockout lines #43 and #46. RT-PCR analysis was performed on cDNA isolated from WT, M4 background mutant, and the Δ gox mutant lines #43 and #46 with primers flanking the CmGOX coding region (F2/R2). Transcripts from the CMQ432C locus adjacent to the CmGOX locus were used as a control. Expected fragment sizes are CmGOX : 698bp; CMQ432C : 520bp.
    Figure Legend Snippet: Generation of a Δ gox knockout mutant in C. merolae . ( A ) Schematic presentation of the strategy to generate a knockout mutant for CmGOX . For detailed information see ‘Materials and methods’. ( B ) Verification of Δ gox mutant lines #43, #45, and #46. PCR was performed on genomic DNA of the M4 background mutant and the Δ gox mutant lines #43, #45, and #46 with primers flanking the CmGOX upstream and downstream regions (F1/R1) and the CmGOX coding region (F2/R2), respectively. Expected fragment sizes were F1/R1: 4.5kb (M4), 6kb (Δ gox ); F2/R2: 0.66kb (M4), – (Δ gox ). ( C ) Verification of absence of CmGOX transcripts in the Δ gox knockout lines #43 and #46. RT-PCR analysis was performed on cDNA isolated from WT, M4 background mutant, and the Δ gox mutant lines #43 and #46 with primers flanking the CmGOX coding region (F2/R2). Transcripts from the CMQ432C locus adjacent to the CmGOX locus were used as a control. Expected fragment sizes are CmGOX : 698bp; CMQ432C : 520bp.

    Techniques Used: Knock-Out, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Isolation

    31) Product Images from "Soluble expression, purification and characterization of the full length IS2 Transposase"

    Article Title: Soluble expression, purification and characterization of the full length IS2 Transposase

    Journal: Mobile DNA

    doi: 10.1186/1759-8753-2-14

    Electrophoretic mobility shift assays using purified and partially purified preparations of the IS 2 OrfAB-GFP fusion protein . (A) Purified OrfAB-GFP fusion protein preparations shown in Figure 4c and the purified native protein from refolding experiments were used in gel retardation reactions. 0.46 μM of the fusion protein and 6.02 μg of the refolded protein were reacted for 30 minutes at room temperature (20°C) with 2 nM of 32 P-labeled annealed 87-mer oligonucleotides containing the 41 bp inverted right repeat sequence. The reactions were run at 4°C at 120 mA for 2400 Vhr in a 5% native polyacrylamide gel. The arrow shows complexes formed with low efficiency. Lanes: 1. Protein-free control. 2. Refolded native OrfAB. 3. OrfAB-GFP. (B) Partially purified preparations of the OrfAB-GFP fusion protein shown in Figure 4a and crude extracts from overexpression of the pTW2 OrfAB-GFP construct used in binding reactions. Approximately 80 nM of the protein from the partially purified preparations shown in Figure 4a and from the crude extracts were reacted with 2 nM of the 32 P-labeled annealed 87-mer oligonucleotides as described in part A. The reactions were run for 1400 Vhrs at 4°C. Lanes: 1. Protein-free control. 2. Partially purified preparation of OrfAB-GFP. 3. Crude extract from the overexpressed pTW2 OrfAB-GFP plasmid. Bp: base pairs; GFP: green fluorescent protein; orf: open reading frame; Vhr: volt hour.
    Figure Legend Snippet: Electrophoretic mobility shift assays using purified and partially purified preparations of the IS 2 OrfAB-GFP fusion protein . (A) Purified OrfAB-GFP fusion protein preparations shown in Figure 4c and the purified native protein from refolding experiments were used in gel retardation reactions. 0.46 μM of the fusion protein and 6.02 μg of the refolded protein were reacted for 30 minutes at room temperature (20°C) with 2 nM of 32 P-labeled annealed 87-mer oligonucleotides containing the 41 bp inverted right repeat sequence. The reactions were run at 4°C at 120 mA for 2400 Vhr in a 5% native polyacrylamide gel. The arrow shows complexes formed with low efficiency. Lanes: 1. Protein-free control. 2. Refolded native OrfAB. 3. OrfAB-GFP. (B) Partially purified preparations of the OrfAB-GFP fusion protein shown in Figure 4a and crude extracts from overexpression of the pTW2 OrfAB-GFP construct used in binding reactions. Approximately 80 nM of the protein from the partially purified preparations shown in Figure 4a and from the crude extracts were reacted with 2 nM of the 32 P-labeled annealed 87-mer oligonucleotides as described in part A. The reactions were run for 1400 Vhrs at 4°C. Lanes: 1. Protein-free control. 2. Partially purified preparation of OrfAB-GFP. 3. Crude extract from the overexpressed pTW2 OrfAB-GFP plasmid. Bp: base pairs; GFP: green fluorescent protein; orf: open reading frame; Vhr: volt hour.

    Techniques Used: Electrophoretic Mobility Shift Assay, Purification, Labeling, Sequencing, Over Expression, Construct, Binding Assay, Plasmid Preparation

    Analysis of the coiled coil domain in IS 2 OrfAB aligned with similar domains in the IS 3 family . (A) The coiled coil sequence in IS 2 identified by the PCOILS analysis of coiled coils [ 57 , 58 ] annotated to show the four putative heptad repeats of a leucine zipper-like motif. Italicized letters a to g represent the repeated positions within each heptad. The critical d positions which favor hydrophobic leucines are highlighted in green (or in red for a non-canonical amino acid). The a -located buried asparagine (N94) is shown in red while green lettering identifies the three canonical a -located hydrophobics. The five randomly induced mutations are indicated by arrows. The corresponding GMF mutant strain is listed beneath each mutation. (B) Alignment of the coiled coil domains of seven members from the five principal subgroups of the IS 3 family showing their relationships to the putative heptads of a leucine-zipper motif. Annotation is as described in part A but for the IS 2 sequence the a positions are highlighted in aqua. (C) Analysis of the potential of the coiled coil sequence in IS 2 to function as a leucine zipper and the effect of mutations recovered within the motif on that function. The data suggest that the sequence which fails the 2ZIP test for a leucine zipper [ 59 ] may indeed have that function. Stabilization by the two d -located leucines is indicated by vertical bold green lines, by the a -located hydrophobics by narrow green lines and by the buried asparagine by a vertical broken red line. Weak salt bridges between glutamines in the g and e locations in heptads 1 and 2 are indicated by a large narrow-lined red × and the canonical ionic salt bridges between the g and e -located E and K residues in heptads 3 and 4, are indicated by a large bold red X. Binding efficiencies (see Figure 6) and transposition frequencies (see Table 2) are listed below the schematic. Additional annotation is as described in part A. GFP: green fluorescent protein; IS: insertion sequence.
    Figure Legend Snippet: Analysis of the coiled coil domain in IS 2 OrfAB aligned with similar domains in the IS 3 family . (A) The coiled coil sequence in IS 2 identified by the PCOILS analysis of coiled coils [ 57 , 58 ] annotated to show the four putative heptad repeats of a leucine zipper-like motif. Italicized letters a to g represent the repeated positions within each heptad. The critical d positions which favor hydrophobic leucines are highlighted in green (or in red for a non-canonical amino acid). The a -located buried asparagine (N94) is shown in red while green lettering identifies the three canonical a -located hydrophobics. The five randomly induced mutations are indicated by arrows. The corresponding GMF mutant strain is listed beneath each mutation. (B) Alignment of the coiled coil domains of seven members from the five principal subgroups of the IS 3 family showing their relationships to the putative heptads of a leucine-zipper motif. Annotation is as described in part A but for the IS 2 sequence the a positions are highlighted in aqua. (C) Analysis of the potential of the coiled coil sequence in IS 2 to function as a leucine zipper and the effect of mutations recovered within the motif on that function. The data suggest that the sequence which fails the 2ZIP test for a leucine zipper [ 59 ] may indeed have that function. Stabilization by the two d -located leucines is indicated by vertical bold green lines, by the a -located hydrophobics by narrow green lines and by the buried asparagine by a vertical broken red line. Weak salt bridges between glutamines in the g and e locations in heptads 1 and 2 are indicated by a large narrow-lined red × and the canonical ionic salt bridges between the g and e -located E and K residues in heptads 3 and 4, are indicated by a large bold red X. Binding efficiencies (see Figure 6) and transposition frequencies (see Table 2) are listed below the schematic. Additional annotation is as described in part A. GFP: green fluorescent protein; IS: insertion sequence.

    Techniques Used: Sequencing, Mutagenesis, Binding Assay

    Structure of plasmids used to create the IS 2OrfAB::GFP fusion construct . Modifications and alternations are indicated in red. (a) pGLO-ATG2, a derivative of the commercially available pGLO plasmid (Biotechnology Explorer GFP Chromatography kit, Bio-Rad Inc., Hercules, CA, USA) containing the GFPuv gene under the control of the P BAD promoter. An Eco RI- Nhe I cassetting site was created in the 5' multiple cloning site (MCS), to facilitate the cloning of the IS 2orfAB fused frame gene. A unique Eco RI site was deleted from its position adjacent to the GFP stop codon and transferred to a position downstream of the P BAD promoter and 9 bp from an existing Nhe I site which encodes the first two amino acids of GFP. The mutagenizing primer for this last step also deleted the GFP start codon to create pGLO-ATG2. (b) pLL18, a pUC19 derivative with IS 2 carrying the Km r reporter gene [ 6 ]. IS 2 in this construct contains the engineered orfAB gene described in Figure 1a ( ii ). (c) pLL2509A was created by removing the left inverted repeats and repositioning the existing Eco RI site to a location downstream of the P IRL promoter, effectively excluding this IS 2 endogenous promoter from subsequent cloning of the cassetted orfAB gene. (d) pLL2521HK was created by the successive steps of adding (i) the 3'-located cassetting Nhe I site which included the removal of the orfAB stop codon and (ii) the 6XHIS-Tag, downstream of the Eco RI cassetting site. (e) pLL2522 was formed when the Nhe I- Eco RI cassetted orfAB (part d) was cloned into the corresponding 5' cloning site of pGLO-ATG2 (part a). bp: basepair; GFP: green fluorescent protein; IS: insertion sequences.
    Figure Legend Snippet: Structure of plasmids used to create the IS 2OrfAB::GFP fusion construct . Modifications and alternations are indicated in red. (a) pGLO-ATG2, a derivative of the commercially available pGLO plasmid (Biotechnology Explorer GFP Chromatography kit, Bio-Rad Inc., Hercules, CA, USA) containing the GFPuv gene under the control of the P BAD promoter. An Eco RI- Nhe I cassetting site was created in the 5' multiple cloning site (MCS), to facilitate the cloning of the IS 2orfAB fused frame gene. A unique Eco RI site was deleted from its position adjacent to the GFP stop codon and transferred to a position downstream of the P BAD promoter and 9 bp from an existing Nhe I site which encodes the first two amino acids of GFP. The mutagenizing primer for this last step also deleted the GFP start codon to create pGLO-ATG2. (b) pLL18, a pUC19 derivative with IS 2 carrying the Km r reporter gene [ 6 ]. IS 2 in this construct contains the engineered orfAB gene described in Figure 1a ( ii ). (c) pLL2509A was created by removing the left inverted repeats and repositioning the existing Eco RI site to a location downstream of the P IRL promoter, effectively excluding this IS 2 endogenous promoter from subsequent cloning of the cassetted orfAB gene. (d) pLL2521HK was created by the successive steps of adding (i) the 3'-located cassetting Nhe I site which included the removal of the orfAB stop codon and (ii) the 6XHIS-Tag, downstream of the Eco RI cassetting site. (e) pLL2522 was formed when the Nhe I- Eco RI cassetted orfAB (part d) was cloned into the corresponding 5' cloning site of pGLO-ATG2 (part a). bp: basepair; GFP: green fluorescent protein; IS: insertion sequences.

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

    Comparative growth and fluorescence of colonies with the pGLO, pLL2522 and pLL 2524-XXX plasmids . (A) Contrasting growth patterns of colonies of XL1 Blue cells of E. coli (Stratagene Inc.) transformed with (a) the pGLO plasmid and (b) the pLL2522 (IS 2orfAB :: GFP ) plasmid. Cells were plated on lysogeny broth (LB) plus carbenicillin and arabinose, incubated at 37°C for 48 hours and irradiated with UV light. (B) XL1 Blue cells transformed with the ligation products generated by cloning PCR products recovered from the Genemorph II Random mutagenesis of IS 2orfAB DNA, into the Eco RI/ Nhe I sites of pGLO-ATG2. Colonies were generated as described above and viewed after 72 hours at 37°C. Arrows identify the faster growing more brightly fluorescing colonies, the vast majority of which contained plasmids pLL2524-XXX (IS 2orfAB :: GFP -GMF) with loss-of-function mutations in the orfAB gene. Isolated colonies at the periphery of the Petri dish (see white asterisk) occasionally produced false positives without mutations or with silent mutations, for example, A42T. PCR: polymerase chain reaction.
    Figure Legend Snippet: Comparative growth and fluorescence of colonies with the pGLO, pLL2522 and pLL 2524-XXX plasmids . (A) Contrasting growth patterns of colonies of XL1 Blue cells of E. coli (Stratagene Inc.) transformed with (a) the pGLO plasmid and (b) the pLL2522 (IS 2orfAB :: GFP ) plasmid. Cells were plated on lysogeny broth (LB) plus carbenicillin and arabinose, incubated at 37°C for 48 hours and irradiated with UV light. (B) XL1 Blue cells transformed with the ligation products generated by cloning PCR products recovered from the Genemorph II Random mutagenesis of IS 2orfAB DNA, into the Eco RI/ Nhe I sites of pGLO-ATG2. Colonies were generated as described above and viewed after 72 hours at 37°C. Arrows identify the faster growing more brightly fluorescing colonies, the vast majority of which contained plasmids pLL2524-XXX (IS 2orfAB :: GFP -GMF) with loss-of-function mutations in the orfAB gene. Isolated colonies at the periphery of the Petri dish (see white asterisk) occasionally produced false positives without mutations or with silent mutations, for example, A42T. PCR: polymerase chain reaction.

    Techniques Used: Fluorescence, Transformation Assay, Plasmid Preparation, Incubation, Irradiation, Ligation, Generated, Clone Assay, Polymerase Chain Reaction, Mutagenesis, Isolation, Produced

    12% SDS-PAGE analysis of proteins prepared under native conditions . (A) Analysis of fluorometrically determined peak fractions from Ni-NTA gravity flow affinity chromatography purification of the 6xHis-tagged OrfAB-GFP. Lanes: 1. Prestained Protein Molecular Weight markers (New England Biolabs). 2-4. Partial purification of the 74 kDa His-tagged OrfAB-GFP fusion protein (upper arrow) from cells with the pLL2522 plasmid. The lower arrow identifies the 17.5 kDa OrfA protein generated by programmed -1 translational frameshifting. These lanes represent peak fractions (determined fluorometrically) which were run out prior to pooling. (B) Analysis of the pooled fractions in part (A) following concentration and dialysis (see Methods). Lanes: 1. Hydrophobic interaction chromatography purification of the 27 kDa GFP from cells with the pGLO plasmid. 2. Pooled fractions from the purification protocol. 3. Protein preparation from the pGLO-ATG2 control plasmid. 4. Prestained protein molecular weight markers. (C) . Purification of the 74 kDa OrfAB-GFP fusion protein to near homogeneity with the IMPACT system (New England Biolabs) from overexpression of the fused orfAB::GFP genes cloned into the pTWIN2 vector. The eluted protein was subjected to a polishing step on an ion exchange Hi Trap Q sepharose column (GE Healthcare Biosciences). GFP: green fluorescent protein; kDA: kiloDaltons; orf: open reading frame.
    Figure Legend Snippet: 12% SDS-PAGE analysis of proteins prepared under native conditions . (A) Analysis of fluorometrically determined peak fractions from Ni-NTA gravity flow affinity chromatography purification of the 6xHis-tagged OrfAB-GFP. Lanes: 1. Prestained Protein Molecular Weight markers (New England Biolabs). 2-4. Partial purification of the 74 kDa His-tagged OrfAB-GFP fusion protein (upper arrow) from cells with the pLL2522 plasmid. The lower arrow identifies the 17.5 kDa OrfA protein generated by programmed -1 translational frameshifting. These lanes represent peak fractions (determined fluorometrically) which were run out prior to pooling. (B) Analysis of the pooled fractions in part (A) following concentration and dialysis (see Methods). Lanes: 1. Hydrophobic interaction chromatography purification of the 27 kDa GFP from cells with the pGLO plasmid. 2. Pooled fractions from the purification protocol. 3. Protein preparation from the pGLO-ATG2 control plasmid. 4. Prestained protein molecular weight markers. (C) . Purification of the 74 kDa OrfAB-GFP fusion protein to near homogeneity with the IMPACT system (New England Biolabs) from overexpression of the fused orfAB::GFP genes cloned into the pTWIN2 vector. The eluted protein was subjected to a polishing step on an ion exchange Hi Trap Q sepharose column (GE Healthcare Biosciences). GFP: green fluorescent protein; kDA: kiloDaltons; orf: open reading frame.

    Techniques Used: SDS Page, Flow Cytometry, Affinity Chromatography, Purification, Molecular Weight, Plasmid Preparation, Generated, Concentration Assay, Hydrophobic Interaction Chromatography, Over Expression, Clone Assay

    Electrophoretic mobility shift assays . Binding efficiencies of the IS 2 OrfAB Transposase derivatives from 22 randomly induced mutants. Reactions were carried out for 30 minutes at 20°C, with 10 nM of 32 P-labeled annealed 50-mer oligonucleotides (except where stated in part (f) below) containing the inverted right repeat sequence and 0.11 μM of the partially purified mutant or wild type IS 2 OrfAB-GFP protein derivatives (see Methods). Domain locations of the substitutions are color-coded and identified by a single letter code, that is, the binding domain (B) yellow, the leucine zipper-like (L) blue, the catalytic active site (C) green, and the middle interval (M) orange. Reactions were separated on 5% native polyacrylamide gels at 4°C at 120 mA as follows: (a) 450 Vhrs. (b) 420 Vhrs (c) 300 Vhrs (d) 450 Vhrs (e) 450 Vhrs (f) 12% native PAGE for 300 Vhrs using 87-mer annealed oligonucleotides. Binding efficiencies are identified as follows: 5 = Identical to that of the wild type, that is, absence of any dissociation of the complex. 4.5 = a slight loss of compactness of the undissociated complex seen in the wild type control. 4.0 = as in 4.5 but with a faster migrating tail of dissociated complexes. 3.5 = as in 4.0 but with a more prominent faster migrating tail of dissociated complexes. 3.0 = significant loss of compactness of the complex with a small amount of uncomplexed DNA. 2.5 = as in 3.0 but with significantly more uncomplexed DNA. 2.0 = as in 3.0 but mostly composed of uncomplexed DNA. 0.5 = mostly composed of uncomplexed DNA with a small tail of dissociated complex. 0 = no complex formation, identical to that of the protein-free controls (lane 1 in each panel) or the GFP control (part a lane 10). Double mutations are indicated within rectangular boxes. For GMF 18 the operative mutation, L97H, is shown in red (gel c, lane 4). GFP: green fluorescent protein; orf: open reading frame; Vhr: volt hour
    Figure Legend Snippet: Electrophoretic mobility shift assays . Binding efficiencies of the IS 2 OrfAB Transposase derivatives from 22 randomly induced mutants. Reactions were carried out for 30 minutes at 20°C, with 10 nM of 32 P-labeled annealed 50-mer oligonucleotides (except where stated in part (f) below) containing the inverted right repeat sequence and 0.11 μM of the partially purified mutant or wild type IS 2 OrfAB-GFP protein derivatives (see Methods). Domain locations of the substitutions are color-coded and identified by a single letter code, that is, the binding domain (B) yellow, the leucine zipper-like (L) blue, the catalytic active site (C) green, and the middle interval (M) orange. Reactions were separated on 5% native polyacrylamide gels at 4°C at 120 mA as follows: (a) 450 Vhrs. (b) 420 Vhrs (c) 300 Vhrs (d) 450 Vhrs (e) 450 Vhrs (f) 12% native PAGE for 300 Vhrs using 87-mer annealed oligonucleotides. Binding efficiencies are identified as follows: 5 = Identical to that of the wild type, that is, absence of any dissociation of the complex. 4.5 = a slight loss of compactness of the undissociated complex seen in the wild type control. 4.0 = as in 4.5 but with a faster migrating tail of dissociated complexes. 3.5 = as in 4.0 but with a more prominent faster migrating tail of dissociated complexes. 3.0 = significant loss of compactness of the complex with a small amount of uncomplexed DNA. 2.5 = as in 3.0 but with significantly more uncomplexed DNA. 2.0 = as in 3.0 but mostly composed of uncomplexed DNA. 0.5 = mostly composed of uncomplexed DNA with a small tail of dissociated complex. 0 = no complex formation, identical to that of the protein-free controls (lane 1 in each panel) or the GFP control (part a lane 10). Double mutations are indicated within rectangular boxes. For GMF 18 the operative mutation, L97H, is shown in red (gel c, lane 4). GFP: green fluorescent protein; orf: open reading frame; Vhr: volt hour

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Sequencing, Purification, Mutagenesis, Clear Native PAGE

    32) Product Images from "Soluble expression, purification and characterization of the full length IS2 Transposase"

    Article Title: Soluble expression, purification and characterization of the full length IS2 Transposase

    Journal: Mobile DNA

    doi: 10.1186/1759-8753-2-14

    12% SDS-PAGE analysis of proteins prepared under native conditions . (A) Analysis of fluorometrically determined peak fractions from Ni-NTA gravity flow affinity chromatography purification of the 6xHis-tagged OrfAB-GFP. Lanes: 1. Prestained Protein Molecular Weight markers (New England Biolabs). 2-4. Partial purification of the 74 kDa His-tagged OrfAB-GFP fusion protein (upper arrow) from cells with the pLL2522 plasmid. The lower arrow identifies the 17.5 kDa OrfA protein generated by programmed -1 translational frameshifting. These lanes represent peak fractions (determined fluorometrically) which were run out prior to pooling. (B) Analysis of the pooled fractions in part (A) following concentration and dialysis (see Methods). Lanes: 1. Hydrophobic interaction chromatography purification of the 27 kDa GFP from cells with the pGLO plasmid. 2. Pooled fractions from the purification protocol. 3. Protein preparation from the pGLO-ATG2 control plasmid. 4. Prestained protein molecular weight markers. (C) . Purification of the 74 kDa OrfAB-GFP fusion protein to near homogeneity with the IMPACT system (New England Biolabs) from overexpression of the fused orfAB::GFP genes cloned into the pTWIN2 vector. The eluted protein was subjected to a polishing step on an ion exchange Hi Trap Q sepharose column (GE Healthcare Biosciences). GFP: green fluorescent protein; kDA: kiloDaltons; orf: open reading frame.
    Figure Legend Snippet: 12% SDS-PAGE analysis of proteins prepared under native conditions . (A) Analysis of fluorometrically determined peak fractions from Ni-NTA gravity flow affinity chromatography purification of the 6xHis-tagged OrfAB-GFP. Lanes: 1. Prestained Protein Molecular Weight markers (New England Biolabs). 2-4. Partial purification of the 74 kDa His-tagged OrfAB-GFP fusion protein (upper arrow) from cells with the pLL2522 plasmid. The lower arrow identifies the 17.5 kDa OrfA protein generated by programmed -1 translational frameshifting. These lanes represent peak fractions (determined fluorometrically) which were run out prior to pooling. (B) Analysis of the pooled fractions in part (A) following concentration and dialysis (see Methods). Lanes: 1. Hydrophobic interaction chromatography purification of the 27 kDa GFP from cells with the pGLO plasmid. 2. Pooled fractions from the purification protocol. 3. Protein preparation from the pGLO-ATG2 control plasmid. 4. Prestained protein molecular weight markers. (C) . Purification of the 74 kDa OrfAB-GFP fusion protein to near homogeneity with the IMPACT system (New England Biolabs) from overexpression of the fused orfAB::GFP genes cloned into the pTWIN2 vector. The eluted protein was subjected to a polishing step on an ion exchange Hi Trap Q sepharose column (GE Healthcare Biosciences). GFP: green fluorescent protein; kDA: kiloDaltons; orf: open reading frame.

    Techniques Used: SDS Page, Flow Cytometry, Affinity Chromatography, Purification, Molecular Weight, Plasmid Preparation, Generated, Concentration Assay, Hydrophobic Interaction Chromatography, Over Expression, Clone Assay

    33) Product Images from "The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus"

    Article Title: The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus

    Journal: Journal of Bacteriology

    doi:

    Structures of plasmids pGB2, pAY1099, and pAY1105. The aadA gene is flanked by a pair of Bsp HI sites in each vector, and its translation initiates at an ATG codon within one of these sites. Also shown are unique sites within the polylinker of pGB2 derived
    Figure Legend Snippet: Structures of plasmids pGB2, pAY1099, and pAY1105. The aadA gene is flanked by a pair of Bsp HI sites in each vector, and its translation initiates at an ATG codon within one of these sites. Also shown are unique sites within the polylinker of pGB2 derived

    Techniques Used: Plasmid Preparation, Derivative Assay

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    Southern Blot:

    Article Title: Nuclear pores as versatile reference standards for quantitative superresolution microscopy
    Article Snippet: .. Southern blotting was performed in accordance Koch et al. Genomic DNA was prepared using the Wizard Genomic DNA Purification kit (Promega) and digested with SspI-HF and MfeI-HF (NEB). ..

    Multiple Displacement Amplification:

    Article Title: Chromatin remodeling by the CHD7 protein is impaired by mutations that cause human developmental disorders
    Article Snippet: .. Here, we measured the ability of remodeling factors to expose an MfeI restriction site at +28-bp inside the nucleosomes and used 1.5 U/μL of MfeI-HF (New England Biolabs). hSWI/SNF complex concentrations are given assuming a Molecular Mass of ∼1.2 MDa). .. Data were quantified using the ImageQuantTL (GE Healthcare Life Sciences) and cut DNA intensity was normalized to cytosine content before calculating cut/uncut ratios and plotting the data using GraphPad Prism.

    Purification:

    Article Title: Rapid identification and expression of human TCRs in retrogenic mice
    Article Snippet: .. Purified PCR products from the beta nested reaction and the TCR-pMIA vector were digested with Bst bI (NEB #R0519S) and Mfe I (NEB #R3589L). .. The digested PCR products were purified using a DNA purification kit (Zymo Research #11-305C).

    Generated:

    Article Title: Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern
    Article Snippet: .. For the double digestion, EcoRI-HF and MfeI-HF (NEB) generated cohesive ends that were compatible, yet recognizing slightly different sequences (GAATTC and CAATTG respectively). .. They were selected due to their extremely low star activity, extended enzymatic half-life suitable for overnight digestion and stronger activity in samples of lower purity, making them appropriate for digestion of crosslinked chromatin.

    DNA Purification:

    Article Title: Nuclear pores as versatile reference standards for quantitative superresolution microscopy
    Article Snippet: .. Southern blotting was performed in accordance Koch et al. Genomic DNA was prepared using the Wizard Genomic DNA Purification kit (Promega) and digested with SspI-HF and MfeI-HF (NEB). ..

    Polymerase Chain Reaction:

    Article Title: Rapid identification and expression of human TCRs in retrogenic mice
    Article Snippet: .. Purified PCR products from the beta nested reaction and the TCR-pMIA vector were digested with Bst bI (NEB #R0519S) and Mfe I (NEB #R3589L). .. The digested PCR products were purified using a DNA purification kit (Zymo Research #11-305C).

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]
    Article Snippet: .. To confirm the occurrence of in -tolerant rice plants, we performed PCR analysis coupled with Mfe I digestion using Mfe I-HF; a high fidelity version of Mfe I was supplied by New England Biolabs (Ipswich, MA). .. PCR products were subjected to direct sequence analysis or cloned into pCR-Blunt II-TOPO (Invitrogen, San Diego, CA) and then subjected to sequencing analysis using an ABI3130 sequencer (Applied Biosystems, Foster City, CA).

    Plasmid Preparation:

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    Article Snippet: .. Purified PCR products from the beta nested reaction and the TCR-pMIA vector were digested with Bst bI (NEB #R0519S) and Mfe I (NEB #R3589L). .. The digested PCR products were purified using a DNA purification kit (Zymo Research #11-305C).

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  • 94
    New England Biolabs mfe i hf
    -tolerant calli. A, Schematic representation of <t>PCR-</t> Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).
    Mfe I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mfe i digestion
    -tolerant calli. A, Schematic representation of <t>PCR-</t> Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).
    Mfe I Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mfe i
    Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include <t>Mfe</t> I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The <t>Mfe</t> I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.
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    -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Journal: Plant Physiology

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    doi: 10.1104/pp.15.01663

    Figure Lengend Snippet: -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Article Snippet: To confirm the occurrence of in -tolerant rice plants, we performed PCR analysis coupled with Mfe I digestion using Mfe I-HF; a high fidelity version of Mfe I was supplied by New England Biolabs (Ipswich, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Generated

    locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Journal: Plant Physiology

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    doi: 10.1104/pp.15.01663

    Figure Lengend Snippet: locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Article Snippet: To confirm the occurrence of in -tolerant rice plants, we performed PCR analysis coupled with Mfe I digestion using Mfe I-HF; a high fidelity version of Mfe I was supplied by New England Biolabs (Ipswich, MA).

    Techniques: Southern Blot, Mutagenesis, Hybridization

    -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Journal: Plant Physiology

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    doi: 10.1104/pp.15.01663

    Figure Lengend Snippet: -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Article Snippet: To confirm the occurrence of in -tolerant rice plants, we performed PCR analysis coupled with Mfe I digestion using Mfe I-HF; a high fidelity version of Mfe I was supplied by New England Biolabs (Ipswich, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Generated

    locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Journal: Plant Physiology

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    doi: 10.1104/pp.15.01663

    Figure Lengend Snippet: locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Article Snippet: To confirm the occurrence of in -tolerant rice plants, we performed PCR analysis coupled with Mfe I digestion using Mfe I-HF; a high fidelity version of Mfe I was supplied by New England Biolabs (Ipswich, MA).

    Techniques: Southern Blot, Mutagenesis, Hybridization

    Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Journal: PLoS ONE

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    doi: 10.1371/journal.pone.0095084

    Figure Lengend Snippet: Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Article Snippet: The initial aph (7″) PCR product was instead digested with Mfe I and Sph I (NEB), purified, and ligated to low-copy pBAD24 digested with Eco RI and Sph I.

    Techniques: Binding Assay, Amplification, TA Cloning, Clone Assay, Plasmid Preparation, Subcloning, Marker

    AFLP fingerprint of M. genitalium G-37 T . Bgl II plus Mfe I AFLP templates were prepared on three occasions with the same batch of genomic DNA of M. genitalium G-37 T . Amplification products obtained from each experiment (blue, black, and red patterns) were detected on an ABI 373A sequencer by GeneScan 1.2.2.-1 software. The complete AFLP patterns are superimposed and divided into four parts (A to D). The fragment size scale (base pairs) is indicated above each panel. y axes indicate relative amounts of amplicons (in fluorescence units).

    Journal: Journal of Clinical Microbiology

    Article Title: Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species

    doi:

    Figure Lengend Snippet: AFLP fingerprint of M. genitalium G-37 T . Bgl II plus Mfe I AFLP templates were prepared on three occasions with the same batch of genomic DNA of M. genitalium G-37 T . Amplification products obtained from each experiment (blue, black, and red patterns) were detected on an ABI 373A sequencer by GeneScan 1.2.2.-1 software. The complete AFLP patterns are superimposed and divided into four parts (A to D). The fragment size scale (base pairs) is indicated above each panel. y axes indicate relative amounts of amplicons (in fluorescence units).

    Article Snippet: Five microliters of the DNA samples containing approximately 200 to 600 ng of genomic DNA was simultaneously digested with 10 U of Bgl II and 10 U of Mfe I (New England Biolabs, Beverly, Mass.) at 37°C for 2 h in a restriction buffer containing 10 mM Tris-acetate (pH 7.5), 10 mM Mg acetate, 50 mM K acetate, 5 mM dithiothreitol, and 50 ng of bovine serum albumin per μl ( ).

    Techniques: Amplification, Software, Fluorescence

    Genescan-derived electropherogram traces of AFLP templates of different complexity. AFLP templates of a field isolate of Escherichia coli and M. genitalium G-37 T were prepared by the digestion of genomic DNAs with Bgl II and Mfe I and subsequent ligation of corresponding adapters. PCR products of individual samples and a mixture of the AFLP templates, containing adjusted DNA concentrations, were detected on an ABI 373A sequencer. The hybrid pattern (middle panel) contains all of the bands detected in individual AFLP templates. The fragment size scale (base pairs) is indicated above the top panel.

    Journal: Journal of Clinical Microbiology

    Article Title: Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species

    doi:

    Figure Lengend Snippet: Genescan-derived electropherogram traces of AFLP templates of different complexity. AFLP templates of a field isolate of Escherichia coli and M. genitalium G-37 T were prepared by the digestion of genomic DNAs with Bgl II and Mfe I and subsequent ligation of corresponding adapters. PCR products of individual samples and a mixture of the AFLP templates, containing adjusted DNA concentrations, were detected on an ABI 373A sequencer. The hybrid pattern (middle panel) contains all of the bands detected in individual AFLP templates. The fragment size scale (base pairs) is indicated above the top panel.

    Article Snippet: Five microliters of the DNA samples containing approximately 200 to 600 ng of genomic DNA was simultaneously digested with 10 U of Bgl II and 10 U of Mfe I (New England Biolabs, Beverly, Mass.) at 37°C for 2 h in a restriction buffer containing 10 mM Tris-acetate (pH 7.5), 10 mM Mg acetate, 50 mM K acetate, 5 mM dithiothreitol, and 50 ng of bovine serum albumin per μl ( ).

    Techniques: Derivative Assay, Ligation, Polymerase Chain Reaction