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  • 99
    New England Biolabs sfi i
    PFGE patterns of <t>Sfi</t> I-digested DNA of C. diphtheriae strains. Lanes 1 through 4 and 6, strains of the mitis biotype, including PFGE types F (lane 1), E (lane 2), H (lane 3), L (lane 4), and J (lane 6); lanes 5 and 7 through 9, strains of the gravis biotype, including PFGE types I (lane 5), A (lane 7), M (lane 8, ATCC 13812), and N (lane 9, NCTC 10648); lane 10, combination of λ ladder and low-range PFGE size markers (kilobases).
    Sfi I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore sfi
    <t>SFI</t> reverses the <t>cisplatin</t> resistance of A549/DDP cells. (a) Direct cytotoxic effect of SFI on A549/DDP cells. A549/DDP cells were treated with various concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 mg/mL) of SFI for 26 hours. The IC 5 , IC 10 , and IC 20 values were 2, 3.78, and 35.18 mg/mL, respectively. Each data point represents the mean ± standard deviation of results from four individual measurements ( ∗ : P
    Sfi, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sfii  (TaKaRa)
    94
    TaKaRa sfii
    PFGE analysis of environmental ctx + and reference (ATCC) strains of V. mimicus (VM) and of ctx + non-O1/non-O139 (VCE 233), O1 El Tor (VC N16961), and O1 classical (VC O395) strains of V. cholerae . (A) Gel image of PFGE profiles of undigested gDNA showing similar sizes of the two chromosomes of ctx + V. mimicus and ATCC V. mimicus strains. (B and C) Gel images showing PFGE patterns of <t>NotI-digested</t> (B) and <t>SfiI-digested</t> (C) gDNA of ctx + V. mimicus and the reference strains. The ctx + V. mimicus strains were clonal but differed in 1 to 2 bands, indicated by arrows. NotI- and SfiI-digested gDNA of ctx + V. mimicus strains generated two (a and b) and three (I, II, and III) PFGE profiles, respectively. “MW” represents the molecular weight standard of the Hansenula wingei chromosomes (Bio-Rad) for undigested gDNA (A) and lambda ladder (Bio-Rad) for digested gDNA (B and C).
    Sfii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher sfi i
    (a) Fingerprint patterns obtained from PFGE of <t>Sfi</t> I-digested DNA of clinical and environmental V. cholerae O1, O139, and non-O1, non-O139 isolates. Lanes M, 1-kb molecular weight ladder; lanes 1 to 3 and 12, O1 strains VO1, VO2, VO3, and VO13, respectively; lanes 4 to 6, O1 strains VO4, VO5, and VO7, respectively; lanes 7 to 11 and 13 to 15, O139 strains MO45 (= ATCC 51394), CO594, CO766, CO788, VO12, VO14, VO15, and VO16, respectively; lanes 16 to 19, O139 strains VO17, VO18, VO19, and VO20, respectively; lanes 20, and 21, non-O1, non-O139 strains VO22, and VO23, respectively; lanes 22 to 26, non-O1, non-O139 strains VO24, VO25, VO26, VO27, and VO28, respectively; (b) Digitized PFGE analysis of Sfi I-digested profiles obtained from genomic DNA of clinical and environmental V. cholerae O1, O139, and non-O1, non-O139 isolates. The Dendrogram was generated by using the average percentages of matched bands summarizing the degrees of similarity of the <t>Sfi</t> I restriction patterns of genomic DNA of V. cholerae O1, O139, and non-O1, non-O139 strains.
    Sfi I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Boehringer Mannheim sfi i
    Structures of the β-YAC globin loci of ΔHS3c β-YAC transgenic mouse lines. The upper part of the figure shows the 140-kb <t>Sfi</t> I fragment encompassing most of the β-globin locus from 5′ HS3 to the breakpoint of HPFH6 approximately 53 kb downstream of the β-globin gene. Arrows indicate HSs. Agarose plugs containing high-molecular-weight DNA from liver tissue were isolated from four ΔHS3c β-YAC transgenic lines. The plugs were digested with Sfi I, fractionated by PFGE, and subjected to Southern hybridization analyses. The location of each of the probes used in the structural analysis is identified on the map. Schematic representations of the structures of the Sfi I fragments are drawn below each autoradiogram. (A) Line A has an intact 140-kb fragment and an additional 120-kb fragment which is deleted from sequences 3′ of the δ-globin gene. (B) Line B has a single 140-kb fragment identified by each of the probes. (C) Line C has a 150-kb fragment containing the intact locus from 5′ HS3 to position H500 (placed between the breakpoints of HPFH3 and HPFH6). A second 160-kb fragment extends from 5′ HS3 to the β-globin gene. This fragment can be seen in the doublets apparent in the lighter exposure of the same autoradiogram in the upper portion of panel C. (D) Line D has a 140-kb fragment containing an intact β-globin locus and two additional fragments of 120 and 170 kb, from both of which most of the β-globin locus is deleted. The first lane in each of the autoradiograms contains control DNA from a mouse erythroleukemia cell line containing a single intact β-YAC, as determined by structural analysis and fluorescent in situ hybridization.
    Sfi I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 91/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs restriction endonuclease sfi i
    Construction of plasmid <t>pComb3XSS-Fab</t> for the production of Fab antibody fragments in E coli . Genes of light-chain and heavy-chain Fd fragments were f used by overlap-extension PCR, and cloned directionally by using two asymmetric sites of the rare cutter Sfi I. Fab was transcribed as a single transcript under the control of one LacZ promoter. The amber stop codon (cross) between the antibody genes and bacteriophage gene III enables the production of soluble Fab fragments in a non suppressor strain of E coli . (A) The genes for the variable and constant regions were amplified separately. (B) Heavy-chain Fd and light chain DNA were assembled by variable regions and their constant counterpart respectively by using overlap PCR. (C) Fd and light chain were fused to form Fab-encoding sequences by overlap PCR. Fab genes were directionally cloned into pComb3XSS phagemid by using the <t>Sfi</t> I site. (D) Both L chain fragment and Fd fragment were transported to the periplasm of E coli .
    Restriction Endonuclease Sfi I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GenScript sfi i restriction sites
    Construction of plasmid <t>pComb3XSS-Fab</t> for the production of Fab antibody fragments in E coli . Genes of light-chain and heavy-chain Fd fragments were f used by overlap-extension PCR, and cloned directionally by using two asymmetric sites of the rare cutter Sfi I. Fab was transcribed as a single transcript under the control of one LacZ promoter. The amber stop codon (cross) between the antibody genes and bacteriophage gene III enables the production of soluble Fab fragments in a non suppressor strain of E coli . (A) The genes for the variable and constant regions were amplified separately. (B) Heavy-chain Fd and light chain DNA were assembled by variable regions and their constant counterpart respectively by using overlap PCR. (C) Fd and light chain were fused to form Fab-encoding sequences by overlap PCR. Fab genes were directionally cloned into pComb3XSS phagemid by using the <t>Sfi</t> I site. (D) Both L chain fragment and Fd fragment were transported to the periplasm of E coli .
    Sfi I Restriction Sites, supplied by GenScript, used in various techniques. Bioz Stars score: 86/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega sfi i
    PFGE profiles of genomic DNAs West Lothian, Highland, and central Scotland outbreak isolates cleaved with <t>Xba</t> I (lanes 2 to 4), Xho I (lanes 5 to 7), Not I (lanes 8 to 10), and <t>Sfi</t> I (lanes 11 to 13). Lanes 1 and 14, bacteriophage lambda concatemer size markers. The unnumbered lanes correspond to lanes 1 to 14 from left to right, respectively.
    Sfi I, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Boehringer Mannheim restriction endonuclease sfi i
    PFGE profiles of genomic DNAs West Lothian, Highland, and central Scotland outbreak isolates cleaved with <t>Xba</t> I (lanes 2 to 4), Xho I (lanes 5 to 7), Not I (lanes 8 to 10), and <t>Sfi</t> I (lanes 11 to 13). Lanes 1 and 14, bacteriophage lambda concatemer size markers. The unnumbered lanes correspond to lanes 1 to 14 from left to right, respectively.
    Restriction Endonuclease Sfi I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher fastdigest sfi i
    PFGE profiles of genomic DNAs West Lothian, Highland, and central Scotland outbreak isolates cleaved with <t>Xba</t> I (lanes 2 to 4), Xho I (lanes 5 to 7), Not I (lanes 8 to 10), and <t>Sfi</t> I (lanes 11 to 13). Lanes 1 and 14, bacteriophage lambda concatemer size markers. The unnumbered lanes correspond to lanes 1 to 14 from left to right, respectively.
    Fastdigest Sfi I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Boehringer Mannheim restriction enzyme sfi i
    PFGE profiles of genomic DNAs West Lothian, Highland, and central Scotland outbreak isolates cleaved with <t>Xba</t> I (lanes 2 to 4), Xho I (lanes 5 to 7), Not I (lanes 8 to 10), and <t>Sfi</t> I (lanes 11 to 13). Lanes 1 and 14, bacteriophage lambda concatemer size markers. The unnumbered lanes correspond to lanes 1 to 14 from left to right, respectively.
    Restriction Enzyme Sfi I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    I-stem sfi i stem loop sequences
    The <t>Sfi</t> I-site in the 5′ UTR near the AUG start codon increases the levels of PDR5 mRNA. Northern blot analysis was performed on late-logarithmically grown AD and AD/sec6-4 cells that expressed PDR5 with differently modified 5′ UTRs (WT = unmodified; strains labeled SP, S or P contained the SfiI/PacI, the SfiI or the PacI site, respectively). AD strains contained the PGK1 terminator and the URA3 marker at the 3′ end of PDR5 while AD/sec6-4 strains contained the 3′ UTR of PDR5 . Three control strains were included: two wild-type PDR5 expressing strains (AH22 and SY1) and AD/pABC3 as the negative (∆PDR5 ) control. A Upper panel - 10 μg total RNA extracts separated on a 1.2% denaturing agarose gel and stained with EtBr (top), lower panels - autoradiographs of blots probed with PDR5 and ACT1 . The band intensities for the top two PDR5 and ACT1 panels can be directly compared as they experienced the same treatment ( PDR5 and ACT1 probes were combined for the hybridization with the Northern blot) while the autoradiograph at the bottom was overexposed so that PDR5 bands of weaker intensities could be measured accurately. B , C , and D show the intensities of bands in panel A quantified with the ImageJ software program [ 30 ]. B shows the expression of PDR5 relative to the expression of the housekeeping gene ACT1 that was used as an internal standard (the intensities for ACT1 in AD/SP-PDR5 were ~10-times higher than in AD/pABC3 while ACT1 varied no more than +/− 50% in the remaining samples). C and D show the -fold differences in normalized PDR5 mRNA levels relative to AD/sec6-4/PDR5 (C) and AD/P-PDR5 (D) , respectively (the results for the Sfi I-site containing strains are shown with black bars, for wt-PDR5 with dark grey and Pac I-site containing strains with light grey bars). The numbers above individual bars in B , C , and D give the actual values represented by the bars.
    Sfi I Stem Loop Sequences, supplied by I-stem, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc sfi i digested psbbi pur
    The <t>Sfi</t> I-site in the 5′ UTR near the AUG start codon increases the levels of PDR5 mRNA. Northern blot analysis was performed on late-logarithmically grown AD and AD/sec6-4 cells that expressed PDR5 with differently modified 5′ UTRs (WT = unmodified; strains labeled SP, S or P contained the SfiI/PacI, the SfiI or the PacI site, respectively). AD strains contained the PGK1 terminator and the URA3 marker at the 3′ end of PDR5 while AD/sec6-4 strains contained the 3′ UTR of PDR5 . Three control strains were included: two wild-type PDR5 expressing strains (AH22 and SY1) and AD/pABC3 as the negative (∆PDR5 ) control. A Upper panel - 10 μg total RNA extracts separated on a 1.2% denaturing agarose gel and stained with EtBr (top), lower panels - autoradiographs of blots probed with PDR5 and ACT1 . The band intensities for the top two PDR5 and ACT1 panels can be directly compared as they experienced the same treatment ( PDR5 and ACT1 probes were combined for the hybridization with the Northern blot) while the autoradiograph at the bottom was overexposed so that PDR5 bands of weaker intensities could be measured accurately. B , C , and D show the intensities of bands in panel A quantified with the ImageJ software program [ 30 ]. B shows the expression of PDR5 relative to the expression of the housekeeping gene ACT1 that was used as an internal standard (the intensities for ACT1 in AD/SP-PDR5 were ~10-times higher than in AD/pABC3 while ACT1 varied no more than +/− 50% in the remaining samples). C and D show the -fold differences in normalized PDR5 mRNA levels relative to AD/sec6-4/PDR5 (C) and AD/P-PDR5 (D) , respectively (the results for the Sfi I-site containing strains are shown with black bars, for wt-PDR5 with dark grey and Pac I-site containing strains with light grey bars). The numbers above individual bars in B , C , and D give the actual values represented by the bars.
    Sfi I Digested Psbbi Pur, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher sfi i enzyme
    The <t>Sfi</t> I-site in the 5′ UTR near the AUG start codon increases the levels of PDR5 mRNA. Northern blot analysis was performed on late-logarithmically grown AD and AD/sec6-4 cells that expressed PDR5 with differently modified 5′ UTRs (WT = unmodified; strains labeled SP, S or P contained the SfiI/PacI, the SfiI or the PacI site, respectively). AD strains contained the PGK1 terminator and the URA3 marker at the 3′ end of PDR5 while AD/sec6-4 strains contained the 3′ UTR of PDR5 . Three control strains were included: two wild-type PDR5 expressing strains (AH22 and SY1) and AD/pABC3 as the negative (∆PDR5 ) control. A Upper panel - 10 μg total RNA extracts separated on a 1.2% denaturing agarose gel and stained with EtBr (top), lower panels - autoradiographs of blots probed with PDR5 and ACT1 . The band intensities for the top two PDR5 and ACT1 panels can be directly compared as they experienced the same treatment ( PDR5 and ACT1 probes were combined for the hybridization with the Northern blot) while the autoradiograph at the bottom was overexposed so that PDR5 bands of weaker intensities could be measured accurately. B , C , and D show the intensities of bands in panel A quantified with the ImageJ software program [ 30 ]. B shows the expression of PDR5 relative to the expression of the housekeeping gene ACT1 that was used as an internal standard (the intensities for ACT1 in AD/SP-PDR5 were ~10-times higher than in AD/pABC3 while ACT1 varied no more than +/− 50% in the remaining samples). C and D show the -fold differences in normalized PDR5 mRNA levels relative to AD/sec6-4/PDR5 (C) and AD/P-PDR5 (D) , respectively (the results for the Sfi I-site containing strains are shown with black bars, for wt-PDR5 with dark grey and Pac I-site containing strains with light grey bars). The numbers above individual bars in B , C , and D give the actual values represented by the bars.
    Sfi I Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare sfi i
    The <t>Sfi</t> I-site in the 5′ UTR near the AUG start codon increases the levels of PDR5 mRNA. Northern blot analysis was performed on late-logarithmically grown AD and AD/sec6-4 cells that expressed PDR5 with differently modified 5′ UTRs (WT = unmodified; strains labeled SP, S or P contained the SfiI/PacI, the SfiI or the PacI site, respectively). AD strains contained the PGK1 terminator and the URA3 marker at the 3′ end of PDR5 while AD/sec6-4 strains contained the 3′ UTR of PDR5 . Three control strains were included: two wild-type PDR5 expressing strains (AH22 and SY1) and AD/pABC3 as the negative (∆PDR5 ) control. A Upper panel - 10 μg total RNA extracts separated on a 1.2% denaturing agarose gel and stained with EtBr (top), lower panels - autoradiographs of blots probed with PDR5 and ACT1 . The band intensities for the top two PDR5 and ACT1 panels can be directly compared as they experienced the same treatment ( PDR5 and ACT1 probes were combined for the hybridization with the Northern blot) while the autoradiograph at the bottom was overexposed so that PDR5 bands of weaker intensities could be measured accurately. B , C , and D show the intensities of bands in panel A quantified with the ImageJ software program [ 30 ]. B shows the expression of PDR5 relative to the expression of the housekeeping gene ACT1 that was used as an internal standard (the intensities for ACT1 in AD/SP-PDR5 were ~10-times higher than in AD/pABC3 while ACT1 varied no more than +/− 50% in the remaining samples). C and D show the -fold differences in normalized PDR5 mRNA levels relative to AD/sec6-4/PDR5 (C) and AD/P-PDR5 (D) , respectively (the results for the Sfi I-site containing strains are shown with black bars, for wt-PDR5 with dark grey and Pac I-site containing strains with light grey bars). The numbers above individual bars in B , C , and D give the actual values represented by the bars.
    Sfi I, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PFGE patterns of Sfi I-digested DNA of C. diphtheriae strains. Lanes 1 through 4 and 6, strains of the mitis biotype, including PFGE types F (lane 1), E (lane 2), H (lane 3), L (lane 4), and J (lane 6); lanes 5 and 7 through 9, strains of the gravis biotype, including PFGE types I (lane 5), A (lane 7), M (lane 8, ATCC 13812), and N (lane 9, NCTC 10648); lane 10, combination of λ ladder and low-range PFGE size markers (kilobases).

    Journal: Journal of Clinical Microbiology

    Article Title: Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

    doi:

    Figure Lengend Snippet: PFGE patterns of Sfi I-digested DNA of C. diphtheriae strains. Lanes 1 through 4 and 6, strains of the mitis biotype, including PFGE types F (lane 1), E (lane 2), H (lane 3), L (lane 4), and J (lane 6); lanes 5 and 7 through 9, strains of the gravis biotype, including PFGE types I (lane 5), A (lane 7), M (lane 8, ATCC 13812), and N (lane 9, NCTC 10648); lane 10, combination of λ ladder and low-range PFGE size markers (kilobases).

    Article Snippet: The plugs were incubated (30 min, room temperature) with restriction enzyme buffer, the DNA in the plugs was digested by incubation (50°C, 5 h) of the plugs with Sfi I (New England Biolabs, Beverly, Mass.), and electrophoresis was performed with 1% SeaKem Gold agarose in 0.5× Tris-borate-EDTA buffer.

    Techniques:

    Dendrogram portraying the genetic diversity of the Georgian and Russian epidemic C. diphtheriae strains. Representative PFGE patterns of Sfi I-digested DNA of C. diphtheriae strains are shown. Data for strains are number of Georgian strains/number of Russian strains, unless otherwise indicated.

    Journal: Journal of Clinical Microbiology

    Article Title: Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

    doi:

    Figure Lengend Snippet: Dendrogram portraying the genetic diversity of the Georgian and Russian epidemic C. diphtheriae strains. Representative PFGE patterns of Sfi I-digested DNA of C. diphtheriae strains are shown. Data for strains are number of Georgian strains/number of Russian strains, unless otherwise indicated.

    Article Snippet: The plugs were incubated (30 min, room temperature) with restriction enzyme buffer, the DNA in the plugs was digested by incubation (50°C, 5 h) of the plugs with Sfi I (New England Biolabs, Beverly, Mass.), and electrophoresis was performed with 1% SeaKem Gold agarose in 0.5× Tris-borate-EDTA buffer.

    Techniques:

    SFI reverses the cisplatin resistance of A549/DDP cells. (a) Direct cytotoxic effect of SFI on A549/DDP cells. A549/DDP cells were treated with various concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 mg/mL) of SFI for 26 hours. The IC 5 , IC 10 , and IC 20 values were 2, 3.78, and 35.18 mg/mL, respectively. Each data point represents the mean ± standard deviation of results from four individual measurements ( ∗ : P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Shenqi Fuzheng Injection Reverses Cisplatin Resistance through Mitofusin-2-Mediated Cell Cycle Arrest and Apoptosis in A549/DDP Cells

    doi: 10.1155/2018/8258246

    Figure Lengend Snippet: SFI reverses the cisplatin resistance of A549/DDP cells. (a) Direct cytotoxic effect of SFI on A549/DDP cells. A549/DDP cells were treated with various concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 mg/mL) of SFI for 26 hours. The IC 5 , IC 10 , and IC 20 values were 2, 3.78, and 35.18 mg/mL, respectively. Each data point represents the mean ± standard deviation of results from four individual measurements ( ∗ : P

    Article Snippet: Hoechst Staining Following coincubation with cisplatin and SFI, A549/DDP cells were secured in 70% ethanol and then incubated along with 10 μ g/mL bisbenzimide trihydrochloride (Hoechst 33258) staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes.

    Techniques: Standard Deviation

    Cotreatment with cisplatin and SFI induces cell cycle arrest in A549/DDP cells. A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. (a) Cell cycle distribution by PI staining and DNA contents were determined by flow cytometry. (b) Cell lysates were prepared and subjected to immunoblotting with antibodies to p53, p21, and β -actin. Data are presented in the format of mean ± standard deviation of three independent experiments ( ∗ : P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Shenqi Fuzheng Injection Reverses Cisplatin Resistance through Mitofusin-2-Mediated Cell Cycle Arrest and Apoptosis in A549/DDP Cells

    doi: 10.1155/2018/8258246

    Figure Lengend Snippet: Cotreatment with cisplatin and SFI induces cell cycle arrest in A549/DDP cells. A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. (a) Cell cycle distribution by PI staining and DNA contents were determined by flow cytometry. (b) Cell lysates were prepared and subjected to immunoblotting with antibodies to p53, p21, and β -actin. Data are presented in the format of mean ± standard deviation of three independent experiments ( ∗ : P

    Article Snippet: Hoechst Staining Following coincubation with cisplatin and SFI, A549/DDP cells were secured in 70% ethanol and then incubated along with 10 μ g/mL bisbenzimide trihydrochloride (Hoechst 33258) staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes.

    Techniques: Staining, Flow Cytometry, Cytometry, Standard Deviation

    Mfn2 is involved in reversing cell cycle inhibition and cell apoptosis upon cotreatment with cisplatin and SFI. (a, b, and c) A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. After drug intervention, (a) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (b) cells were incubated with JC-1 and analyzed by flow cytometry; and (c) cells were labeled with DCFH-DA and the fluorescence intensity of the oxidized product DCF in individual cells was detected by flow cytometry and fluorescence microscopy. (d, e, and f) A549/DDP cells were pretreated with or without 2.5 mM of NAC, followed by cisplatin (40 μ g/mL) and SFI (35.18 mg/mL) cotreatment. After drug intervention, (d) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (e) cell cycle distribution by PI staining and DNA contents were determined by flow cytometry; and (f) apoptosis was determined by Annexin V-FITC/PI staining and analyzed by flow cytometry.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Shenqi Fuzheng Injection Reverses Cisplatin Resistance through Mitofusin-2-Mediated Cell Cycle Arrest and Apoptosis in A549/DDP Cells

    doi: 10.1155/2018/8258246

    Figure Lengend Snippet: Mfn2 is involved in reversing cell cycle inhibition and cell apoptosis upon cotreatment with cisplatin and SFI. (a, b, and c) A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. After drug intervention, (a) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (b) cells were incubated with JC-1 and analyzed by flow cytometry; and (c) cells were labeled with DCFH-DA and the fluorescence intensity of the oxidized product DCF in individual cells was detected by flow cytometry and fluorescence microscopy. (d, e, and f) A549/DDP cells were pretreated with or without 2.5 mM of NAC, followed by cisplatin (40 μ g/mL) and SFI (35.18 mg/mL) cotreatment. After drug intervention, (d) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (e) cell cycle distribution by PI staining and DNA contents were determined by flow cytometry; and (f) apoptosis was determined by Annexin V-FITC/PI staining and analyzed by flow cytometry.

    Article Snippet: Hoechst Staining Following coincubation with cisplatin and SFI, A549/DDP cells were secured in 70% ethanol and then incubated along with 10 μ g/mL bisbenzimide trihydrochloride (Hoechst 33258) staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes.

    Techniques: Inhibition, Incubation, Flow Cytometry, Cytometry, Labeling, Fluorescence, Microscopy, Staining

    Cotreatment with cisplatin and SFI induces cell apoptosis in A549/DDP cells. A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. (a) Apoptosis determined by Hoechst staining. “→” shows apoptosis cells with nuclear condensation. (b) Apoptosis determined by Annexin V-FITC/PI staining. Each cytogram consists of data showing live cells (PI- and FITC-negative) in Q3; early apoptotic population (FITC-positive) in Q4; mid- to late-stage apoptosis (PI- and FITC-positive) in Q2; and necrotic/end-stage apoptotic cells (PI-positive and FITC-negative) in Q1. (c) Cell lysates were prepared and subjected to immunoblotting with antibodies to cleaved caspase 3, cleaved-PARP, Bcl-2, Bax, and β -actin. Data are presented in the format of mean ± standard deviation of three independent experiments ( ∗ : P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Shenqi Fuzheng Injection Reverses Cisplatin Resistance through Mitofusin-2-Mediated Cell Cycle Arrest and Apoptosis in A549/DDP Cells

    doi: 10.1155/2018/8258246

    Figure Lengend Snippet: Cotreatment with cisplatin and SFI induces cell apoptosis in A549/DDP cells. A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. (a) Apoptosis determined by Hoechst staining. “→” shows apoptosis cells with nuclear condensation. (b) Apoptosis determined by Annexin V-FITC/PI staining. Each cytogram consists of data showing live cells (PI- and FITC-negative) in Q3; early apoptotic population (FITC-positive) in Q4; mid- to late-stage apoptosis (PI- and FITC-positive) in Q2; and necrotic/end-stage apoptotic cells (PI-positive and FITC-negative) in Q1. (c) Cell lysates were prepared and subjected to immunoblotting with antibodies to cleaved caspase 3, cleaved-PARP, Bcl-2, Bax, and β -actin. Data are presented in the format of mean ± standard deviation of three independent experiments ( ∗ : P

    Article Snippet: Hoechst Staining Following coincubation with cisplatin and SFI, A549/DDP cells were secured in 70% ethanol and then incubated along with 10 μ g/mL bisbenzimide trihydrochloride (Hoechst 33258) staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes.

    Techniques: Staining, Standard Deviation

    DNA cleavage by SfiI. ( A ) Successive time-lapse images of a SfiI-DNA reaction obtained at 2.0 fps in the presence of Mg 2+ . The elapsed time is indicated in each image. The image is cropped from the original scan size of 600 nm × 450 nm.

    Journal: Biophysical Journal

    Article Title: Visual Analysis of Concerted Cleavage by Type IIF Restriction Enzyme SfiI in Subsecond Time Region

    doi: 10.1016/j.bpj.2011.09.064

    Figure Lengend Snippet: DNA cleavage by SfiI. ( A ) Successive time-lapse images of a SfiI-DNA reaction obtained at 2.0 fps in the presence of Mg 2+ . The elapsed time is indicated in each image. The image is cropped from the original scan size of 600 nm × 450 nm.

    Article Snippet: Reaction mixtures contained 0.004 units of SfiI, 5 ng/ μ l DNA fragment (905 bp), 10 mM HEPES (pH 7.5), 5 mM CaCl2 , and 50 mM NaCl in a total volume of 10 μ l. After incubating at an ambient temperature for 15 min, the complexes were filtered through a Montage PCR column (Millipore, Bedford, MA) to remove bovine serum albumin, and unbound protein.

    Techniques:

    Looping and translocation by SfiI. Successive time-lapse images of a SfiI-DNA complex obtained at 1.0 fps in the presence of Ca 2+ . The elapsed time is shown in each image. The image is cropped from the original scan size of 600 nm × 450 nm.

    Journal: Biophysical Journal

    Article Title: Visual Analysis of Concerted Cleavage by Type IIF Restriction Enzyme SfiI in Subsecond Time Region

    doi: 10.1016/j.bpj.2011.09.064

    Figure Lengend Snippet: Looping and translocation by SfiI. Successive time-lapse images of a SfiI-DNA complex obtained at 1.0 fps in the presence of Ca 2+ . The elapsed time is shown in each image. The image is cropped from the original scan size of 600 nm × 450 nm.

    Article Snippet: Reaction mixtures contained 0.004 units of SfiI, 5 ng/ μ l DNA fragment (905 bp), 10 mM HEPES (pH 7.5), 5 mM CaCl2 , and 50 mM NaCl in a total volume of 10 μ l. After incubating at an ambient temperature for 15 min, the complexes were filtered through a Montage PCR column (Millipore, Bedford, MA) to remove bovine serum albumin, and unbound protein.

    Techniques: Translocation Assay

    DNA and protein movement on a mica surface. ( A . SfiI tetramers were indicated by yellow arrowheads. AFM imaging was performed in the presence of Ca 2+ . ( B ) Movement of DNA strands on a mica. The contours of four individual

    Journal: Biophysical Journal

    Article Title: Visual Analysis of Concerted Cleavage by Type IIF Restriction Enzyme SfiI in Subsecond Time Region

    doi: 10.1016/j.bpj.2011.09.064

    Figure Lengend Snippet: DNA and protein movement on a mica surface. ( A . SfiI tetramers were indicated by yellow arrowheads. AFM imaging was performed in the presence of Ca 2+ . ( B ) Movement of DNA strands on a mica. The contours of four individual

    Article Snippet: Reaction mixtures contained 0.004 units of SfiI, 5 ng/ μ l DNA fragment (905 bp), 10 mM HEPES (pH 7.5), 5 mM CaCl2 , and 50 mM NaCl in a total volume of 10 μ l. After incubating at an ambient temperature for 15 min, the complexes were filtered through a Montage PCR column (Millipore, Bedford, MA) to remove bovine serum albumin, and unbound protein.

    Techniques: Imaging

    Visualization of SfiI-DNA complex. ( A ) The locations of the two SfiI recognition sites along a 905 bp fragment are shown. ( B ) AFM image of SfiI-DNA complex obtained under a Ca 2+ -containing buffer. Loop and short arms are indicated by yellow and

    Journal: Biophysical Journal

    Article Title: Visual Analysis of Concerted Cleavage by Type IIF Restriction Enzyme SfiI in Subsecond Time Region

    doi: 10.1016/j.bpj.2011.09.064

    Figure Lengend Snippet: Visualization of SfiI-DNA complex. ( A ) The locations of the two SfiI recognition sites along a 905 bp fragment are shown. ( B ) AFM image of SfiI-DNA complex obtained under a Ca 2+ -containing buffer. Loop and short arms are indicated by yellow and

    Article Snippet: Reaction mixtures contained 0.004 units of SfiI, 5 ng/ μ l DNA fragment (905 bp), 10 mM HEPES (pH 7.5), 5 mM CaCl2 , and 50 mM NaCl in a total volume of 10 μ l. After incubating at an ambient temperature for 15 min, the complexes were filtered through a Montage PCR column (Millipore, Bedford, MA) to remove bovine serum albumin, and unbound protein.

    Techniques:

    PFGE analysis of environmental ctx + and reference (ATCC) strains of V. mimicus (VM) and of ctx + non-O1/non-O139 (VCE 233), O1 El Tor (VC N16961), and O1 classical (VC O395) strains of V. cholerae . (A) Gel image of PFGE profiles of undigested gDNA showing similar sizes of the two chromosomes of ctx + V. mimicus and ATCC V. mimicus strains. (B and C) Gel images showing PFGE patterns of NotI-digested (B) and SfiI-digested (C) gDNA of ctx + V. mimicus and the reference strains. The ctx + V. mimicus strains were clonal but differed in 1 to 2 bands, indicated by arrows. NotI- and SfiI-digested gDNA of ctx + V. mimicus strains generated two (a and b) and three (I, II, and III) PFGE profiles, respectively. “MW” represents the molecular weight standard of the Hansenula wingei chromosomes (Bio-Rad) for undigested gDNA (A) and lambda ladder (Bio-Rad) for digested gDNA (B and C).

    Journal: Applied and Environmental Microbiology

    Article Title: Novel Cholera Toxin Variant and ToxT Regulon in Environmental Vibrio mimicus Isolates: Potential Resources for the Evolution of Vibrio cholerae Hybrid Strains

    doi: 10.1128/AEM.01977-18

    Figure Lengend Snippet: PFGE analysis of environmental ctx + and reference (ATCC) strains of V. mimicus (VM) and of ctx + non-O1/non-O139 (VCE 233), O1 El Tor (VC N16961), and O1 classical (VC O395) strains of V. cholerae . (A) Gel image of PFGE profiles of undigested gDNA showing similar sizes of the two chromosomes of ctx + V. mimicus and ATCC V. mimicus strains. (B and C) Gel images showing PFGE patterns of NotI-digested (B) and SfiI-digested (C) gDNA of ctx + V. mimicus and the reference strains. The ctx + V. mimicus strains were clonal but differed in 1 to 2 bands, indicated by arrows. NotI- and SfiI-digested gDNA of ctx + V. mimicus strains generated two (a and b) and three (I, II, and III) PFGE profiles, respectively. “MW” represents the molecular weight standard of the Hansenula wingei chromosomes (Bio-Rad) for undigested gDNA (A) and lambda ladder (Bio-Rad) for digested gDNA (B and C).

    Article Snippet: Briefly, freshly grown V. mimicus strains were embedded into 1% Seakem Gold agarose (Sigma-Aldrich, MO, USA) followed by lysis of the cells with 0.5 mg ml−1 proteinase K (P8044-5G; Sigma) and 1% sarcosine (Sigma) at 54°C for 1 h. Agarose blocks containing genomic DNA were digested with NotI and SfiI (TaKaRa Bio Inc., Otsu, Japan) (30 and 40 U, respectively) using appropriate buffer at 37°C for 3 h. DNA fragments were electrophoresed in 1% pulsed-field-certified agarose gel (Bio-Rad Laboratories, CA, USA) using a Chef Mapper (Bio-Rad).

    Techniques: Generated, Molecular Weight

    Generation of Dnmt3a2 and Dnmt3L conditional Tg mice. ( A ) Schematic representation of the conditional Tg allele. 2lox vector (upper) is designed to express only EGFP but not the downstream sequences from the chicken beta-actin promoter (pCAG). The floxed-EGFP cassette is removed in the presence of Cre recombinase, concomitantly with the induction of mCherry , Dnmt3a2 and Dnmt3L , all linked by T2A peptides, in the 1lox allele (lower). Tg mice were generated by microinjection of linearized 2lox vector (8.7 kb) by double digestion of Psp 1406I (P) and Sfi I (S). Vsp I (V) digestion was used for Southern hybridization. ( B ) Expression of the immunofluorescence reporters mCherry and EGFP in 1lox and 2lox ng oocytes, respectively. mCherry expression was not observed in 2lox ng oocytes. ( C ) qRT-PCR of Dnmt3a , detecting Dnmt3a and Dnmt3a2 , and Dnmt3L in 1lox (orange bar) and 2lox (green bar) ng oocytes, various sizes of WT oocytes (brown bars) and WT male germ cells derived from 16.5 dpc embryos (blue bar). Error bars represent standard deviation. Relative expression in 1lox ng oocytes was equal to or greater than in oocytes and male germ cells during imprinting establishment. A different letter over the bar represents a significant difference ( P

    Journal: Human Molecular Genetics

    Article Title: Forced expression of DNA methyltransferases during oocyte growth accelerates the establishment of methylation imprints but not functional genomic imprinting

    doi: 10.1093/hmg/ddu100

    Figure Lengend Snippet: Generation of Dnmt3a2 and Dnmt3L conditional Tg mice. ( A ) Schematic representation of the conditional Tg allele. 2lox vector (upper) is designed to express only EGFP but not the downstream sequences from the chicken beta-actin promoter (pCAG). The floxed-EGFP cassette is removed in the presence of Cre recombinase, concomitantly with the induction of mCherry , Dnmt3a2 and Dnmt3L , all linked by T2A peptides, in the 1lox allele (lower). Tg mice were generated by microinjection of linearized 2lox vector (8.7 kb) by double digestion of Psp 1406I (P) and Sfi I (S). Vsp I (V) digestion was used for Southern hybridization. ( B ) Expression of the immunofluorescence reporters mCherry and EGFP in 1lox and 2lox ng oocytes, respectively. mCherry expression was not observed in 2lox ng oocytes. ( C ) qRT-PCR of Dnmt3a , detecting Dnmt3a and Dnmt3a2 , and Dnmt3L in 1lox (orange bar) and 2lox (green bar) ng oocytes, various sizes of WT oocytes (brown bars) and WT male germ cells derived from 16.5 dpc embryos (blue bar). Error bars represent standard deviation. Relative expression in 1lox ng oocytes was equal to or greater than in oocytes and male germ cells during imprinting establishment. A different letter over the bar represents a significant difference ( P

    Article Snippet: Generation of Tg mice Tg mice harboring floxed-EGFP allele (2lox mice) were generated by microinjection of vectors linearized using Psp 1406I and Sfi I (Takara Bio, Tokyo, Japan) into the paternal pronucleus of C57BL/6N-background zygotes (Fig. A) ( ).

    Techniques: Mouse Assay, Plasmid Preparation, Generated, Hybridization, Expressing, Immunofluorescence, Quantitative RT-PCR, Derivative Assay, Standard Deviation

    The construction of expressible full-length cDNA library. A adapter with Sfi l A and Sfi l B restrictive sites was introduced into pXCS-HAStrep to generate binary vector pXCS-lib, which was fully compatible with Clontech Creator SMART cDNA Library Construction Kit. Then the full-length cDNA library was constructed in this vector

    Journal: SpringerPlus

    Article Title: Gain-of-function in Arabidopsis (GAINA) for identifying functional genes in Hevea brasiliensis

    doi: 10.1186/s40064-016-3523-4

    Figure Lengend Snippet: The construction of expressible full-length cDNA library. A adapter with Sfi l A and Sfi l B restrictive sites was introduced into pXCS-HAStrep to generate binary vector pXCS-lib, which was fully compatible with Clontech Creator SMART cDNA Library Construction Kit. Then the full-length cDNA library was constructed in this vector

    Article Snippet: The pXCS-LIB was digested by Sfi I, and then dephosphorylated by calf intestinal alkaline phosphatase (Takara).

    Techniques: cDNA Library Assay, Plasmid Preparation, Construct

    (a) Fingerprint patterns obtained from PFGE of Sfi I-digested DNA of clinical and environmental V. cholerae O1, O139, and non-O1, non-O139 isolates. Lanes M, 1-kb molecular weight ladder; lanes 1 to 3 and 12, O1 strains VO1, VO2, VO3, and VO13, respectively; lanes 4 to 6, O1 strains VO4, VO5, and VO7, respectively; lanes 7 to 11 and 13 to 15, O139 strains MO45 (= ATCC 51394), CO594, CO766, CO788, VO12, VO14, VO15, and VO16, respectively; lanes 16 to 19, O139 strains VO17, VO18, VO19, and VO20, respectively; lanes 20, and 21, non-O1, non-O139 strains VO22, and VO23, respectively; lanes 22 to 26, non-O1, non-O139 strains VO24, VO25, VO26, VO27, and VO28, respectively; (b) Digitized PFGE analysis of Sfi I-digested profiles obtained from genomic DNA of clinical and environmental V. cholerae O1, O139, and non-O1, non-O139 isolates. The Dendrogram was generated by using the average percentages of matched bands summarizing the degrees of similarity of the Sfi I restriction patterns of genomic DNA of V. cholerae O1, O139, and non-O1, non-O139 strains.

    Journal: Applied and Environmental Microbiology

    Article Title: Molecular Analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 Strains: Clonal Relationships between Clinical and Environmental Isolates

    doi: 10.1128/AEM.67.2.910-921.2001

    Figure Lengend Snippet: (a) Fingerprint patterns obtained from PFGE of Sfi I-digested DNA of clinical and environmental V. cholerae O1, O139, and non-O1, non-O139 isolates. Lanes M, 1-kb molecular weight ladder; lanes 1 to 3 and 12, O1 strains VO1, VO2, VO3, and VO13, respectively; lanes 4 to 6, O1 strains VO4, VO5, and VO7, respectively; lanes 7 to 11 and 13 to 15, O139 strains MO45 (= ATCC 51394), CO594, CO766, CO788, VO12, VO14, VO15, and VO16, respectively; lanes 16 to 19, O139 strains VO17, VO18, VO19, and VO20, respectively; lanes 20, and 21, non-O1, non-O139 strains VO22, and VO23, respectively; lanes 22 to 26, non-O1, non-O139 strains VO24, VO25, VO26, VO27, and VO28, respectively; (b) Digitized PFGE analysis of Sfi I-digested profiles obtained from genomic DNA of clinical and environmental V. cholerae O1, O139, and non-O1, non-O139 isolates. The Dendrogram was generated by using the average percentages of matched bands summarizing the degrees of similarity of the Sfi I restriction patterns of genomic DNA of V. cholerae O1, O139, and non-O1, non-O139 strains.

    Article Snippet: Digestion with Sfi I (GIBCO-BRL) was carried out at 50°C for 16 h in a solution containing 100 μg of bovine serum albumin per ml by using 40 U of enzyme per plug.

    Techniques: Molecular Weight, Generated

    Structures of the β-YAC globin loci of ΔHS3c β-YAC transgenic mouse lines. The upper part of the figure shows the 140-kb Sfi I fragment encompassing most of the β-globin locus from 5′ HS3 to the breakpoint of HPFH6 approximately 53 kb downstream of the β-globin gene. Arrows indicate HSs. Agarose plugs containing high-molecular-weight DNA from liver tissue were isolated from four ΔHS3c β-YAC transgenic lines. The plugs were digested with Sfi I, fractionated by PFGE, and subjected to Southern hybridization analyses. The location of each of the probes used in the structural analysis is identified on the map. Schematic representations of the structures of the Sfi I fragments are drawn below each autoradiogram. (A) Line A has an intact 140-kb fragment and an additional 120-kb fragment which is deleted from sequences 3′ of the δ-globin gene. (B) Line B has a single 140-kb fragment identified by each of the probes. (C) Line C has a 150-kb fragment containing the intact locus from 5′ HS3 to position H500 (placed between the breakpoints of HPFH3 and HPFH6). A second 160-kb fragment extends from 5′ HS3 to the β-globin gene. This fragment can be seen in the doublets apparent in the lighter exposure of the same autoradiogram in the upper portion of panel C. (D) Line D has a 140-kb fragment containing an intact β-globin locus and two additional fragments of 120 and 170 kb, from both of which most of the β-globin locus is deleted. The first lane in each of the autoradiograms contains control DNA from a mouse erythroleukemia cell line containing a single intact β-YAC, as determined by structural analysis and fluorescent in situ hybridization.

    Journal: Molecular and Cellular Biology

    Article Title: Developmental Specificity of the Interaction between the Locus Control Region and Embryonic or Fetal Globin Genes in Transgenic Mice with an HS3 Core Deletion

    doi:

    Figure Lengend Snippet: Structures of the β-YAC globin loci of ΔHS3c β-YAC transgenic mouse lines. The upper part of the figure shows the 140-kb Sfi I fragment encompassing most of the β-globin locus from 5′ HS3 to the breakpoint of HPFH6 approximately 53 kb downstream of the β-globin gene. Arrows indicate HSs. Agarose plugs containing high-molecular-weight DNA from liver tissue were isolated from four ΔHS3c β-YAC transgenic lines. The plugs were digested with Sfi I, fractionated by PFGE, and subjected to Southern hybridization analyses. The location of each of the probes used in the structural analysis is identified on the map. Schematic representations of the structures of the Sfi I fragments are drawn below each autoradiogram. (A) Line A has an intact 140-kb fragment and an additional 120-kb fragment which is deleted from sequences 3′ of the δ-globin gene. (B) Line B has a single 140-kb fragment identified by each of the probes. (C) Line C has a 150-kb fragment containing the intact locus from 5′ HS3 to position H500 (placed between the breakpoints of HPFH3 and HPFH6). A second 160-kb fragment extends from 5′ HS3 to the β-globin gene. This fragment can be seen in the doublets apparent in the lighter exposure of the same autoradiogram in the upper portion of panel C. (D) Line D has a 140-kb fragment containing an intact β-globin locus and two additional fragments of 120 and 170 kb, from both of which most of the β-globin locus is deleted. The first lane in each of the autoradiograms contains control DNA from a mouse erythroleukemia cell line containing a single intact β-YAC, as determined by structural analysis and fluorescent in situ hybridization.

    Article Snippet: Twelve agarose plugs were digested overnight at 50°C with 20 U of Sfi I (Boehringer Mannheim) in a total volume of 200 μl after preequilibration in 200 μl of 1× Sfi I buffer.

    Techniques: Transgenic Assay, Molecular Weight, Isolation, Hybridization, In Situ Hybridization

    Construction of plasmid pComb3XSS-Fab for the production of Fab antibody fragments in E coli . Genes of light-chain and heavy-chain Fd fragments were f used by overlap-extension PCR, and cloned directionally by using two asymmetric sites of the rare cutter Sfi I. Fab was transcribed as a single transcript under the control of one LacZ promoter. The amber stop codon (cross) between the antibody genes and bacteriophage gene III enables the production of soluble Fab fragments in a non suppressor strain of E coli . (A) The genes for the variable and constant regions were amplified separately. (B) Heavy-chain Fd and light chain DNA were assembled by variable regions and their constant counterpart respectively by using overlap PCR. (C) Fd and light chain were fused to form Fab-encoding sequences by overlap PCR. Fab genes were directionally cloned into pComb3XSS phagemid by using the Sfi I site. (D) Both L chain fragment and Fd fragment were transported to the periplasm of E coli .

    Journal: Acta Pharmacologica Sinica

    Article Title: Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    doi: 10.1038/aps.2010.209

    Figure Lengend Snippet: Construction of plasmid pComb3XSS-Fab for the production of Fab antibody fragments in E coli . Genes of light-chain and heavy-chain Fd fragments were f used by overlap-extension PCR, and cloned directionally by using two asymmetric sites of the rare cutter Sfi I. Fab was transcribed as a single transcript under the control of one LacZ promoter. The amber stop codon (cross) between the antibody genes and bacteriophage gene III enables the production of soluble Fab fragments in a non suppressor strain of E coli . (A) The genes for the variable and constant regions were amplified separately. (B) Heavy-chain Fd and light chain DNA were assembled by variable regions and their constant counterpart respectively by using overlap PCR. (C) Fd and light chain were fused to form Fab-encoding sequences by overlap PCR. Fab genes were directionally cloned into pComb3XSS phagemid by using the Sfi I site. (D) Both L chain fragment and Fd fragment were transported to the periplasm of E coli .

    Article Snippet: The vector pComb3XSS and the Fab fragments were digested by the restriction endonuclease Sfi I (New England Biolabs, Ipswich, MA, USA) , and ligated to create recombinants.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Amplification

    PFGE profiles of genomic DNAs West Lothian, Highland, and central Scotland outbreak isolates cleaved with Xba I (lanes 2 to 4), Xho I (lanes 5 to 7), Not I (lanes 8 to 10), and Sfi I (lanes 11 to 13). Lanes 1 and 14, bacteriophage lambda concatemer size markers. The unnumbered lanes correspond to lanes 1 to 14 from left to right, respectively.

    Journal: Journal of Clinical Microbiology

    Article Title: Characterization of a Recurrent Clonal Type of Escherichia coli O157:H7 Causing Major Outbreaks of Infection in Scotland

    doi:

    Figure Lengend Snippet: PFGE profiles of genomic DNAs West Lothian, Highland, and central Scotland outbreak isolates cleaved with Xba I (lanes 2 to 4), Xho I (lanes 5 to 7), Not I (lanes 8 to 10), and Sfi I (lanes 11 to 13). Lanes 1 and 14, bacteriophage lambda concatemer size markers. The unnumbered lanes correspond to lanes 1 to 14 from left to right, respectively.

    Article Snippet: Cleavage of the agarose-embedded DNA was achieved with Xba I (Promega, Southampton, United Kingdom), Not I (Promega), Sfi I (Promega), Xho I (Boehringer Mannheim, Lewes, United Kingdom), and Avr II (Boehringer Mannheim) according to the manufacturer's instructions.

    Techniques:

    The Sfi I-site in the 5′ UTR near the AUG start codon increases the levels of PDR5 mRNA. Northern blot analysis was performed on late-logarithmically grown AD and AD/sec6-4 cells that expressed PDR5 with differently modified 5′ UTRs (WT = unmodified; strains labeled SP, S or P contained the SfiI/PacI, the SfiI or the PacI site, respectively). AD strains contained the PGK1 terminator and the URA3 marker at the 3′ end of PDR5 while AD/sec6-4 strains contained the 3′ UTR of PDR5 . Three control strains were included: two wild-type PDR5 expressing strains (AH22 and SY1) and AD/pABC3 as the negative (∆PDR5 ) control. A Upper panel - 10 μg total RNA extracts separated on a 1.2% denaturing agarose gel and stained with EtBr (top), lower panels - autoradiographs of blots probed with PDR5 and ACT1 . The band intensities for the top two PDR5 and ACT1 panels can be directly compared as they experienced the same treatment ( PDR5 and ACT1 probes were combined for the hybridization with the Northern blot) while the autoradiograph at the bottom was overexposed so that PDR5 bands of weaker intensities could be measured accurately. B , C , and D show the intensities of bands in panel A quantified with the ImageJ software program [ 30 ]. B shows the expression of PDR5 relative to the expression of the housekeeping gene ACT1 that was used as an internal standard (the intensities for ACT1 in AD/SP-PDR5 were ~10-times higher than in AD/pABC3 while ACT1 varied no more than +/− 50% in the remaining samples). C and D show the -fold differences in normalized PDR5 mRNA levels relative to AD/sec6-4/PDR5 (C) and AD/P-PDR5 (D) , respectively (the results for the Sfi I-site containing strains are shown with black bars, for wt-PDR5 with dark grey and Pac I-site containing strains with light grey bars). The numbers above individual bars in B , C , and D give the actual values represented by the bars.

    Journal: Microbial Cell Factories

    Article Title: Small, synthetic, GC-rich mRNA stem-loop modules 5? proximal to the AUG start-codon predictably tune gene expression in yeast

    doi: 10.1186/1475-2859-12-74

    Figure Lengend Snippet: The Sfi I-site in the 5′ UTR near the AUG start codon increases the levels of PDR5 mRNA. Northern blot analysis was performed on late-logarithmically grown AD and AD/sec6-4 cells that expressed PDR5 with differently modified 5′ UTRs (WT = unmodified; strains labeled SP, S or P contained the SfiI/PacI, the SfiI or the PacI site, respectively). AD strains contained the PGK1 terminator and the URA3 marker at the 3′ end of PDR5 while AD/sec6-4 strains contained the 3′ UTR of PDR5 . Three control strains were included: two wild-type PDR5 expressing strains (AH22 and SY1) and AD/pABC3 as the negative (∆PDR5 ) control. A Upper panel - 10 μg total RNA extracts separated on a 1.2% denaturing agarose gel and stained with EtBr (top), lower panels - autoradiographs of blots probed with PDR5 and ACT1 . The band intensities for the top two PDR5 and ACT1 panels can be directly compared as they experienced the same treatment ( PDR5 and ACT1 probes were combined for the hybridization with the Northern blot) while the autoradiograph at the bottom was overexposed so that PDR5 bands of weaker intensities could be measured accurately. B , C , and D show the intensities of bands in panel A quantified with the ImageJ software program [ 30 ]. B shows the expression of PDR5 relative to the expression of the housekeeping gene ACT1 that was used as an internal standard (the intensities for ACT1 in AD/SP-PDR5 were ~10-times higher than in AD/pABC3 while ACT1 varied no more than +/− 50% in the remaining samples). C and D show the -fold differences in normalized PDR5 mRNA levels relative to AD/sec6-4/PDR5 (C) and AD/P-PDR5 (D) , respectively (the results for the Sfi I-site containing strains are shown with black bars, for wt-PDR5 with dark grey and Pac I-site containing strains with light grey bars). The numbers above individual bars in B , C , and D give the actual values represented by the bars.

    Article Snippet: Equimolar amounts of each pair of Sfi I-digested PCR fragments were ligated and aliquots (grey box in the middle) were then used to PCR amplify the fused fragments with primers pd5f/Rev-3 (Sfi I stem-loop sequences are highlighted in light blue).

    Techniques: Northern Blot, Modification, Labeling, Marker, Expressing, Agarose Gel Electrophoresis, Staining, Hybridization, Autoradiography, Software

    Drug resistance levels (MIC FLC ) of Cdr1p-expressing strains are directly proportional to the amount of Cdr1p expressed. A. SDS-PAGE of plasma membrane proteins (30 μg) isolated from AD∆ strains containing different Sfi I stem-loop constructs. The black arrowhead indicates Cdr1p and the white arrowhead indicates the prominent plasma membrane proton pump protein Pma1p. wt = AD∆/P-CDR1-URA3; SfiI = AD∆/construct9-CDR1; lanes labeled 8, 2 and 0 represent Cdr1p expressing strains with decreasing loop-size of 8 nucleotides (AD∆/construct6-CDR1), 2 nucleotides (AD∆/construct7-CDR1) or no loop at all (AD∆/construct8-CDR1); lanes labeled 5, 6 and 7 represent strains with increasing stem-size of 5 GC-pairs (AD∆/construct11-CDR1), 6 GC-pairs (AD∆/construct14-CDR1) and 7 GC-pairs (AD∆/construct15-CDR1). B. The MIC FLC values for each construct correlated well with the amounts of Cdr1p expressed (measured as pixels using the ImageJ software [ 30 ]). %CDR1 (Y-axis) and %MIC FLC (X-axis) are the expression levels and MIC FLC relative to wt Cdr1p. To the right is a graphical illustration of this correlation (constructs #14 and #15 were excluded from the graph because their Cdr1p expression was below the detection limit but the MIC FLC = 0.5 of the negative control strain AD (no Cdr1p) was included), and the dashed grey line shows the theoretical trend line expected for a direct linear correlation between MIC FLC values and the amounts of Cdr1p expressed.

    Journal: Microbial Cell Factories

    Article Title: Small, synthetic, GC-rich mRNA stem-loop modules 5? proximal to the AUG start-codon predictably tune gene expression in yeast

    doi: 10.1186/1475-2859-12-74

    Figure Lengend Snippet: Drug resistance levels (MIC FLC ) of Cdr1p-expressing strains are directly proportional to the amount of Cdr1p expressed. A. SDS-PAGE of plasma membrane proteins (30 μg) isolated from AD∆ strains containing different Sfi I stem-loop constructs. The black arrowhead indicates Cdr1p and the white arrowhead indicates the prominent plasma membrane proton pump protein Pma1p. wt = AD∆/P-CDR1-URA3; SfiI = AD∆/construct9-CDR1; lanes labeled 8, 2 and 0 represent Cdr1p expressing strains with decreasing loop-size of 8 nucleotides (AD∆/construct6-CDR1), 2 nucleotides (AD∆/construct7-CDR1) or no loop at all (AD∆/construct8-CDR1); lanes labeled 5, 6 and 7 represent strains with increasing stem-size of 5 GC-pairs (AD∆/construct11-CDR1), 6 GC-pairs (AD∆/construct14-CDR1) and 7 GC-pairs (AD∆/construct15-CDR1). B. The MIC FLC values for each construct correlated well with the amounts of Cdr1p expressed (measured as pixels using the ImageJ software [ 30 ]). %CDR1 (Y-axis) and %MIC FLC (X-axis) are the expression levels and MIC FLC relative to wt Cdr1p. To the right is a graphical illustration of this correlation (constructs #14 and #15 were excluded from the graph because their Cdr1p expression was below the detection limit but the MIC FLC = 0.5 of the negative control strain AD (no Cdr1p) was included), and the dashed grey line shows the theoretical trend line expected for a direct linear correlation between MIC FLC values and the amounts of Cdr1p expressed.

    Article Snippet: Equimolar amounts of each pair of Sfi I-digested PCR fragments were ligated and aliquots (grey box in the middle) were then used to PCR amplify the fused fragments with primers pd5f/Rev-3 (Sfi I stem-loop sequences are highlighted in light blue).

    Techniques: Expressing, SDS Page, Isolation, Construct, Labeling, Software, Negative Control

    mRNA secondary structures predicted for the 5′ UTRs of wild-type PDR5 and PDR5 containing the originally created Sfi I stem-loop at −4 (construct #1). The most representative mRNA secondary structures predicted for the entire 5′ UTRs of wild-type PDR5 (three transcription start sites were determined at −171, -174 and −175 [ 35 ]) and wild-type PDR5 that has been modified to contain the Sfi I site at −4 (construct #1) are shown in A and B , respectively. Arrows with numbers (kcal/mol) indicate secondary structures of significant stability. 5′ ends are highlighted with black circles and the AUG start-codons are highlighted as grey ovals. The calculated thermodynamic stabilities for the entire 5′ UTR of each predicted secondary structure is shown at the bottom of each structure.

    Journal: Microbial Cell Factories

    Article Title: Small, synthetic, GC-rich mRNA stem-loop modules 5? proximal to the AUG start-codon predictably tune gene expression in yeast

    doi: 10.1186/1475-2859-12-74

    Figure Lengend Snippet: mRNA secondary structures predicted for the 5′ UTRs of wild-type PDR5 and PDR5 containing the originally created Sfi I stem-loop at −4 (construct #1). The most representative mRNA secondary structures predicted for the entire 5′ UTRs of wild-type PDR5 (three transcription start sites were determined at −171, -174 and −175 [ 35 ]) and wild-type PDR5 that has been modified to contain the Sfi I site at −4 (construct #1) are shown in A and B , respectively. Arrows with numbers (kcal/mol) indicate secondary structures of significant stability. 5′ ends are highlighted with black circles and the AUG start-codons are highlighted as grey ovals. The calculated thermodynamic stabilities for the entire 5′ UTR of each predicted secondary structure is shown at the bottom of each structure.

    Article Snippet: Equimolar amounts of each pair of Sfi I-digested PCR fragments were ligated and aliquots (grey box in the middle) were then used to PCR amplify the fused fragments with primers pd5f/Rev-3 (Sfi I stem-loop sequences are highlighted in light blue).

    Techniques: Construct, Modification

    The Sfi I restriction enzyme cloning site severely inhibits gene expression in yeast. A. A schematic diagram of the cloning strategy used to over-express heterologous ORFs in S. cerevisiae . PDR5 (orange) was cloned as Sfi I/ Not I or Pac I/ Not I fragments into pABC1 (the pBluescriptIISK(+) vector backbone is light green and the multiple cloning site of the transformation cassette light blue). pABC1-PDR5 was digested with Fse I and Asc I to release the 7.4 kb transformation cassette [ PDR5 promoter (green)-ORF (orange)- PGK1 terminator (blue)- URA3 marker (purple)- PDR5 downstream region (green)]. The transformation cassettes were gel purified and used to transform S. cerevisiae AD to Ura + . B. Effect of 5′ UTR on Pdr5p activity. Strains expressing Pdr5p were created either using pABC1 (SfiI-PDR5, SfiI-PacI-PDR5) or pABC3 (PacI-PDR5), as previously described [ 14 ], and ∆PDR5 (AD) and wt-PDR5 (AD124567u - ) were used as negative (0% Pdr5p expression) and positive (100% Pdr5p expression) controls, respectively. The 32 nucleotides upstream of the ATG start-codon for each construct are shown. Sfi I and Pac I restriction sites are underlined and the nucleotides that differ from the wild-type PDR5 5′ UTR are shown in bold type. The right hand column lists the MIC FLC values for the strains. The MIC FLC values for three independent transformants were measured and did not vary by more than one dilution. C. SDS-PAGE of plasma membrane proteins (30 μg) isolated from the strains listed in B including AD/PDR5 (this strain is AD with its wild-type PDR5 locus restored). The black arrowhead indicates Pdr5p and the white arrowhead indicates the prominent plasma membrane proton pump protein Pma1p. SP = AD/SP-PDR5-URA3; S = AD/S-PDR5-URA3; P = AD/P-PDR5-URA3.

    Journal: Microbial Cell Factories

    Article Title: Small, synthetic, GC-rich mRNA stem-loop modules 5? proximal to the AUG start-codon predictably tune gene expression in yeast

    doi: 10.1186/1475-2859-12-74

    Figure Lengend Snippet: The Sfi I restriction enzyme cloning site severely inhibits gene expression in yeast. A. A schematic diagram of the cloning strategy used to over-express heterologous ORFs in S. cerevisiae . PDR5 (orange) was cloned as Sfi I/ Not I or Pac I/ Not I fragments into pABC1 (the pBluescriptIISK(+) vector backbone is light green and the multiple cloning site of the transformation cassette light blue). pABC1-PDR5 was digested with Fse I and Asc I to release the 7.4 kb transformation cassette [ PDR5 promoter (green)-ORF (orange)- PGK1 terminator (blue)- URA3 marker (purple)- PDR5 downstream region (green)]. The transformation cassettes were gel purified and used to transform S. cerevisiae AD to Ura + . B. Effect of 5′ UTR on Pdr5p activity. Strains expressing Pdr5p were created either using pABC1 (SfiI-PDR5, SfiI-PacI-PDR5) or pABC3 (PacI-PDR5), as previously described [ 14 ], and ∆PDR5 (AD) and wt-PDR5 (AD124567u - ) were used as negative (0% Pdr5p expression) and positive (100% Pdr5p expression) controls, respectively. The 32 nucleotides upstream of the ATG start-codon for each construct are shown. Sfi I and Pac I restriction sites are underlined and the nucleotides that differ from the wild-type PDR5 5′ UTR are shown in bold type. The right hand column lists the MIC FLC values for the strains. The MIC FLC values for three independent transformants were measured and did not vary by more than one dilution. C. SDS-PAGE of plasma membrane proteins (30 μg) isolated from the strains listed in B including AD/PDR5 (this strain is AD with its wild-type PDR5 locus restored). The black arrowhead indicates Pdr5p and the white arrowhead indicates the prominent plasma membrane proton pump protein Pma1p. SP = AD/SP-PDR5-URA3; S = AD/S-PDR5-URA3; P = AD/P-PDR5-URA3.

    Article Snippet: Equimolar amounts of each pair of Sfi I-digested PCR fragments were ligated and aliquots (grey box in the middle) were then used to PCR amplify the fused fragments with primers pd5f/Rev-3 (Sfi I stem-loop sequences are highlighted in light blue).

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Transformation Assay, Marker, Purification, Activity Assay, Construct, SDS Page, Isolation