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  • 99
    New England Biolabs sspi
    Southern hybridization to detect lptA . After digestion of chromosomal <t>DNA</t> with <t>SspI,</t> the DNA was transferred to a charged nylon membrane and probed with a digoxigenin-labeled probe specific for lptA . The lanes represent DNA isolated from the following
    Sspi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sspi
    Analysis of the <t>PV94</t> cohesive ends. (A) AdeI, Bgll and <t>SspI</t> restriction patterns of PV94 phage DNA. Lane M, DNA size marker (λ DNA Eco130I), lanes (−), unheated phage DNA, lanes (+), phage DNA that had been heated before digestion. (B) Determination of protruding nucleotides. Chromatograms of run-off sequencing reactions using phage DNA as template and primers (PPNF and PPNR) binding close to the genomic ends. The 16 bp overhanging sequences are shaded and marked by arrows.
    Sspi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs sspi hf
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Sspi Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fastdigest sspi
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Fastdigest Sspi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Promega sspi restriction enzyme
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Sspi Restriction Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 81/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Promega sspi
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Sspi, supplied by Promega, used in various techniques. Bioz Stars score: 82/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sspi  (Roche)
    81
    Roche sspi
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Sspi, supplied by Roche, used in various techniques. Bioz Stars score: 81/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    TaKaRa sspi restriction enzyme
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Sspi Restriction Enzyme, supplied by TaKaRa, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    GE Healthcare sspi
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Sspi, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    SibEnzyme sspi
    Gel Electrophoresis of <t>PCR</t> Products of CYP3A5*3 after Digestion by <t>SspI</t> enzyme.
    Sspi, supplied by SibEnzyme, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Promega sspi endonucleases
    Gel Electrophoresis of <t>PCR</t> Products of CYP3A5*3 after Digestion by <t>SspI</t> enzyme.
    Sspi Endonucleases, supplied by Promega, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Roche restriction endonuclease sspi
    Gel Electrophoresis of <t>PCR</t> Products of CYP3A5*3 after Digestion by <t>SspI</t> enzyme.
    Restriction Endonuclease Sspi, supplied by Roche, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Medtronic percutaneous sspi
    Gel Electrophoresis of <t>PCR</t> Products of CYP3A5*3 after Digestion by <t>SspI</t> enzyme.
    Percutaneous Sspi, supplied by Medtronic, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher sspi scai sites
    Gel Electrophoresis of <t>PCR</t> Products of CYP3A5*3 after Digestion by <t>SspI</t> enzyme.
    Sspi Scai Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    TaKaRa mouse sspi genomic library
    Gel Electrophoresis of <t>PCR</t> Products of CYP3A5*3 after Digestion by <t>SspI</t> enzyme.
    Mouse Sspi Genomic Library, supplied by TaKaRa, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sspi  (TaKaRa)
    99
    TaKaRa sspi
    Gel Electrophoresis of <t>PCR</t> Products of CYP3A5*3 after Digestion by <t>SspI</t> enzyme.
    Sspi, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Southern hybridization to detect lptA . After digestion of chromosomal DNA with SspI, the DNA was transferred to a charged nylon membrane and probed with a digoxigenin-labeled probe specific for lptA . The lanes represent DNA isolated from the following

    Journal: Infection and Immunity

    Article Title: Lack of Lipid A Pyrophosphorylation and Functional lptA Reduces Inflammation by Neisseria Commensals

    doi: 10.1128/IAI.00506-12

    Figure Lengend Snippet: Southern hybridization to detect lptA . After digestion of chromosomal DNA with SspI, the DNA was transferred to a charged nylon membrane and probed with a digoxigenin-labeled probe specific for lptA . The lanes represent DNA isolated from the following

    Article Snippet: Chromosomal DNA was digested with SspI (New England BioLabs, Ipswich, MA) and electrophoresed on a 1.2% Tris-borate-EDTA (TBE) agarose gel , transferred onto a charged nylon membrane (DuPont, Wilmington, DE) using the alkaline transfer method of Reed and Mann , and fixed onto the membrane by UV exposure (1.5 J/cm2 ).

    Techniques: Hybridization, Labeling, Isolation

    Expression of HIV-1 P24 from pSA-HP24-6His vectors with or without rare tRNA genes array. To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E . coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E . coli co - transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A) , immuno-blotting (B) , and band densitometery (C) . The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.

    Journal: PLoS ONE

    Article Title: Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli

    doi: 10.1371/journal.pone.0130446

    Figure Lengend Snippet: Expression of HIV-1 P24 from pSA-HP24-6His vectors with or without rare tRNA genes array. To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E . coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E . coli co - transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A) , immuno-blotting (B) , and band densitometery (C) . The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.

    Article Snippet: Construction of pSA-HNef-6His-RIL The pACYC-RIL vector (5μg) was restricted with Ssp I (NEB, #R0132) and Fsp I (NEB, #R0135) for 4h at 37°C, and an 874bp DNA fragment that contained arg U, ile Y, leu W tRNA genes, was purified from 1.5% agarose gel.

    Techniques: Expressing, Plasmid Preparation, Transformation Assay, Chick Chorioallantoic Membrane Assay, Concentration Assay, SDS Page, Staining

    Expression of Nef from pSA-HNef-6His vectors with or without rare tRNA genes array. To evaluate the effect of the introduction of rare tRNA genes array in pSA-HNef-6His vector, the NiCo21 E . coli were individually transformed with pSA-HNef-6His, pSA-HNef-6His-RIL(CW), and pSA-HNef-6His-RIL(CCW) and Nef expression was compared with NiCo21 E . coli co - transformed with pSA-HNef-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HNef-6His or pSA-HNef-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HNef-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD 600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 15% SDS-PAGE gel and analyzed by ( A ) Coomassie staining, ( B ) immuno-blotting and ( C ) band densitometry. Nef expression in NiCo21 transformed with pSA-HNef-6His-RIL (CW/CCW) was comparable with NiCo21 co-transformed with pSA-HNef-6His and pACYC-RIL. A prominent band running at 25 kDa (red arrows) appears in NiCo21 harboring pSA-HNef-6His/ pACYC-RIL. This is most probably chloramphenicol acetyltransferase monomer. Higher baseline expression for un-induced cultures were noted in NiCo21 harboring pSA-HNef-6His-RIL(CCW) (see green arrows). This suggests that ileY tRNA gene most probably doesn’t contain a transcription terminator downstream and the baseline expression of Nef is a result of transcriptional read-through of mRNA synthesis from ile Y promoter.

    Journal: PLoS ONE

    Article Title: Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli

    doi: 10.1371/journal.pone.0130446

    Figure Lengend Snippet: Expression of Nef from pSA-HNef-6His vectors with or without rare tRNA genes array. To evaluate the effect of the introduction of rare tRNA genes array in pSA-HNef-6His vector, the NiCo21 E . coli were individually transformed with pSA-HNef-6His, pSA-HNef-6His-RIL(CW), and pSA-HNef-6His-RIL(CCW) and Nef expression was compared with NiCo21 E . coli co - transformed with pSA-HNef-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HNef-6His or pSA-HNef-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HNef-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD 600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 15% SDS-PAGE gel and analyzed by ( A ) Coomassie staining, ( B ) immuno-blotting and ( C ) band densitometry. Nef expression in NiCo21 transformed with pSA-HNef-6His-RIL (CW/CCW) was comparable with NiCo21 co-transformed with pSA-HNef-6His and pACYC-RIL. A prominent band running at 25 kDa (red arrows) appears in NiCo21 harboring pSA-HNef-6His/ pACYC-RIL. This is most probably chloramphenicol acetyltransferase monomer. Higher baseline expression for un-induced cultures were noted in NiCo21 harboring pSA-HNef-6His-RIL(CCW) (see green arrows). This suggests that ileY tRNA gene most probably doesn’t contain a transcription terminator downstream and the baseline expression of Nef is a result of transcriptional read-through of mRNA synthesis from ile Y promoter.

    Article Snippet: Construction of pSA-HNef-6His-RIL The pACYC-RIL vector (5μg) was restricted with Ssp I (NEB, #R0132) and Fsp I (NEB, #R0135) for 4h at 37°C, and an 874bp DNA fragment that contained arg U, ile Y, leu W tRNA genes, was purified from 1.5% agarose gel.

    Techniques: Expressing, Plasmid Preparation, Transformation Assay, Chick Chorioallantoic Membrane Assay, Concentration Assay, SDS Page, Staining

    Expression of HIV-1 Vif from pSA-HVif-6His vectors with or without rare tRNA genes array. To evaluate the effect of the introduction of rare tRNA genes array in pSA-HVif-6His vector, the NiCo21(DE3) E . coli were individually transformed with pSA-HVif-6His, and pSA-HVif-6His-RIL and Vif expression was compared with NiCo21(DE3) E . coli co - transformed with pSA-HVif-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HVif-6His or pSA-HVif-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HVif-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A) , immuno-blotting (B) , and band densitometery (C) . The Vif expression in NiCo21(DE3) transformed with pSA-HVif-6His-RIL was comparable with NiCo21 co-transformed with pSA-HVif-6His and pACYC-RIL.

    Journal: PLoS ONE

    Article Title: Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli

    doi: 10.1371/journal.pone.0130446

    Figure Lengend Snippet: Expression of HIV-1 Vif from pSA-HVif-6His vectors with or without rare tRNA genes array. To evaluate the effect of the introduction of rare tRNA genes array in pSA-HVif-6His vector, the NiCo21(DE3) E . coli were individually transformed with pSA-HVif-6His, and pSA-HVif-6His-RIL and Vif expression was compared with NiCo21(DE3) E . coli co - transformed with pSA-HVif-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HVif-6His or pSA-HVif-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HVif-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A) , immuno-blotting (B) , and band densitometery (C) . The Vif expression in NiCo21(DE3) transformed with pSA-HVif-6His-RIL was comparable with NiCo21 co-transformed with pSA-HVif-6His and pACYC-RIL.

    Article Snippet: Construction of pSA-HNef-6His-RIL The pACYC-RIL vector (5μg) was restricted with Ssp I (NEB, #R0132) and Fsp I (NEB, #R0135) for 4h at 37°C, and an 874bp DNA fragment that contained arg U, ile Y, leu W tRNA genes, was purified from 1.5% agarose gel.

    Techniques: Expressing, Plasmid Preparation, Transformation Assay, Chick Chorioallantoic Membrane Assay, Concentration Assay, SDS Page, Staining

    Production and purification of HIV-1 Nef and P24. NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD 600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD 600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector.

    Journal: PLoS ONE

    Article Title: Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli

    doi: 10.1371/journal.pone.0130446

    Figure Lengend Snippet: Production and purification of HIV-1 Nef and P24. NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD 600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD 600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector.

    Article Snippet: Construction of pSA-HNef-6His-RIL The pACYC-RIL vector (5μg) was restricted with Ssp I (NEB, #R0132) and Fsp I (NEB, #R0135) for 4h at 37°C, and an 874bp DNA fragment that contained arg U, ile Y, leu W tRNA genes, was purified from 1.5% agarose gel.

    Techniques: Purification, Transformation Assay, Concentration Assay, Expressing, Plasmid Preparation

    Analysis of the PV94 cohesive ends. (A) AdeI, Bgll and SspI restriction patterns of PV94 phage DNA. Lane M, DNA size marker (λ DNA Eco130I), lanes (−), unheated phage DNA, lanes (+), phage DNA that had been heated before digestion. (B) Determination of protruding nucleotides. Chromatograms of run-off sequencing reactions using phage DNA as template and primers (PPNF and PPNR) binding close to the genomic ends. The 16 bp overhanging sequences are shaded and marked by arrows.

    Journal: PLoS ONE

    Article Title: Vibrio vulnificus Phage PV94 Is Closely Related to Temperate Phages of V. cholerae and Other Vibrio Species

    doi: 10.1371/journal.pone.0094707

    Figure Lengend Snippet: Analysis of the PV94 cohesive ends. (A) AdeI, Bgll and SspI restriction patterns of PV94 phage DNA. Lane M, DNA size marker (λ DNA Eco130I), lanes (−), unheated phage DNA, lanes (+), phage DNA that had been heated before digestion. (B) Determination of protruding nucleotides. Chromatograms of run-off sequencing reactions using phage DNA as template and primers (PPNF and PPNR) binding close to the genomic ends. The 16 bp overhanging sequences are shaded and marked by arrows.

    Article Snippet: Analysis of the cohesive ends Cohesive ends of PV94 were identified by digestion of phage DNA with AdeI, BglII and SspI (Thermo Scientific, Hudson, NA) followed by an incubation of the digests at 70°C for 10 min.

    Techniques: Marker, Sequencing, Binding Assay

    Identification of cagA copy number using Southern blot analysis. (A) Southern blot (upper panel) and real-time PCR (lower panel) analysis of PMSS1 and of single-colony derivatives 1-89, 1-107, 1-14, 1-77, and 1-100 at first passage from PMSS1. Control DNA used in the Southern blot was a mixture of a 3.5-kbp fragment that was linearized by SphI digestion of a pGEM clone of the hybridization probe and a 0.5-kbp fragment liberated by digestion of the same clone with EcoRI. Strain cagA -S F -1 was used for normalization of real-time PCR. The bar graphs indicate average cagA copy numbers, and error bars represent standard deviations, derived from results of 3 independent experiments. (B) Southern blot (upper panel) and real-time PCR (lower panel) analysis of the 1-107 and 1-100 colonies at first passage and of the single-colony derivatives 2-107-69 and 2-107-51 and single-colony derivatives 2-100-18 and 2-100-188 at second passage. (C) Schematic representation of the cagA repeats of PMSS1 derivatives cagA -M4, cagA -M3, cagA -M2, cagA -S, and Δ cagA . Three CHAs (CHA-ud, CHA-u, and CHA-d), SspI cleavage sites, and probe binding sites are indicated.

    Journal: mBio

    Article Title: Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease

    doi: 10.1128/mBio.01779-16

    Figure Lengend Snippet: Identification of cagA copy number using Southern blot analysis. (A) Southern blot (upper panel) and real-time PCR (lower panel) analysis of PMSS1 and of single-colony derivatives 1-89, 1-107, 1-14, 1-77, and 1-100 at first passage from PMSS1. Control DNA used in the Southern blot was a mixture of a 3.5-kbp fragment that was linearized by SphI digestion of a pGEM clone of the hybridization probe and a 0.5-kbp fragment liberated by digestion of the same clone with EcoRI. Strain cagA -S F -1 was used for normalization of real-time PCR. The bar graphs indicate average cagA copy numbers, and error bars represent standard deviations, derived from results of 3 independent experiments. (B) Southern blot (upper panel) and real-time PCR (lower panel) analysis of the 1-107 and 1-100 colonies at first passage and of the single-colony derivatives 2-107-69 and 2-107-51 and single-colony derivatives 2-100-18 and 2-100-188 at second passage. (C) Schematic representation of the cagA repeats of PMSS1 derivatives cagA -M4, cagA -M3, cagA -M2, cagA -S, and Δ cagA . Three CHAs (CHA-ud, CHA-u, and CHA-d), SspI cleavage sites, and probe binding sites are indicated.

    Article Snippet: A total of 0.5 μg of chromosomal DNA from each sample was digested with restriction enzyme SspI (Thermo Fisher Scientific) and resolved on a 0.8% agarose gel for 12 h at 25 V. SspI was used because the SspI site is not found within the cagA repeat region ( ).

    Techniques: Southern Blot, Real-time Polymerase Chain Reaction, Hybridization, Derivative Assay, Binding Assay

    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The SspI fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic DNA from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.

    Journal: mBio

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains

    doi: 10.1128/mBio.02321-16

    Figure Lengend Snippet: H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The SspI fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic DNA from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.

    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane.

    Techniques: Southern Blot, Polymerase Chain Reaction, Amplification, Clone Assay, Negative Control, Western Blot, Expressing, Software

    Construction and quantitation of rKSHV.294. (A) Schematic diagram of rKSHV.294 showing the insertion site in the KSHV genome; the BamHI sites flanking the 4.8-kb segment of the KSHV genome used are indicated. The relative positions of the SeAP, the GFP, and the Neo elements are shown with their respective promoters but not to scale. Beneath the 294 construct are shown the expected PCR products (a to e) with the primers used for analysis of viral DNA. (B) Hybridization analysis of rKSHV.294 and JSC-1 viral DNA. (Left) KSHV probe using the 4.8-kb BamHI fragment used to construct the virus. (Right) Neo probe. Lane 1, rKSHV.294 × AflII, predicted fragments of 15.9, 5.7, and 4.8 kb. The 4.8-kb fragment contains the SeAP/GFP/Neo insert with only 170 bp of KSHV DNA, which accounts for the weak band. Lane 2, JSC-1 × AflII, predicted fragments of 15.9 and 5.8 kb. Lane 3, rKSHV.294 × SspI, predicted fragments of 8 and 6.2 kb. Lane 4, JSC-1 × SspI, predicted fragments of 6.2 and 3.3 kb. Lane 5, rKSHV.294 × AflII, predicted fragment of 4.8 kb. Lane 6, JSC-1 × AflII. Lane 7, rKSHV.294 DNA × SspI, predicted fragment of 8 kb. Lane 8, JSC-1 × SspI. DNA markers in kb are on the left side. (C) Ethidium bromide-stained agarose gel following electrophoresis of the PCR products resulting with rKSHV.294 viral DNA with the indicated primers described in Materials and Methods. Lane a, ORF56f (outside BamHI site) and SeAPr; lane b, ORF57u and SeAPr; lane c, SeAPf and GFPf; lane d, GFPr and Neor; lane e, Neof and K9. (D) rKSHV.294 was tested for tTA control of SeAP expression by the infection of 293 cells or of 293 cells expressing tTA at an MOI of

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿

    doi: 10.1128/AAC.01295-10

    Figure Lengend Snippet: Construction and quantitation of rKSHV.294. (A) Schematic diagram of rKSHV.294 showing the insertion site in the KSHV genome; the BamHI sites flanking the 4.8-kb segment of the KSHV genome used are indicated. The relative positions of the SeAP, the GFP, and the Neo elements are shown with their respective promoters but not to scale. Beneath the 294 construct are shown the expected PCR products (a to e) with the primers used for analysis of viral DNA. (B) Hybridization analysis of rKSHV.294 and JSC-1 viral DNA. (Left) KSHV probe using the 4.8-kb BamHI fragment used to construct the virus. (Right) Neo probe. Lane 1, rKSHV.294 × AflII, predicted fragments of 15.9, 5.7, and 4.8 kb. The 4.8-kb fragment contains the SeAP/GFP/Neo insert with only 170 bp of KSHV DNA, which accounts for the weak band. Lane 2, JSC-1 × AflII, predicted fragments of 15.9 and 5.8 kb. Lane 3, rKSHV.294 × SspI, predicted fragments of 8 and 6.2 kb. Lane 4, JSC-1 × SspI, predicted fragments of 6.2 and 3.3 kb. Lane 5, rKSHV.294 × AflII, predicted fragment of 4.8 kb. Lane 6, JSC-1 × AflII. Lane 7, rKSHV.294 DNA × SspI, predicted fragment of 8 kb. Lane 8, JSC-1 × SspI. DNA markers in kb are on the left side. (C) Ethidium bromide-stained agarose gel following electrophoresis of the PCR products resulting with rKSHV.294 viral DNA with the indicated primers described in Materials and Methods. Lane a, ORF56f (outside BamHI site) and SeAPr; lane b, ORF57u and SeAPr; lane c, SeAPf and GFPf; lane d, GFPr and Neor; lane e, Neof and K9. (D) rKSHV.294 was tested for tTA control of SeAP expression by the infection of 293 cells or of 293 cells expressing tTA at an MOI of

    Article Snippet: Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

    Techniques: Quantitation Assay, Construct, Polymerase Chain Reaction, Hybridization, Staining, Agarose Gel Electrophoresis, Electrophoresis, Expressing, Infection

    Gel Electrophoresis of PCR Products of CYP3A5*3 after Digestion by SspI enzyme.

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    Article Title: Association of CYP3A5*3 and CYP1A1*2C Polymorphism with Development of Acute Myeloid Leukemia in Egyptian Patients

    doi: 10.22034/APJCP.2017.18.3.747

    Figure Lengend Snippet: Gel Electrophoresis of PCR Products of CYP3A5*3 after Digestion by SspI enzyme.

    Article Snippet: Ten ul of PCR products were digested at 37°C overnight with 10 units of SspI (SibEnzyme) (New England Biolabs, USA) in 1X buffer supplied with the enzyme and supplemented with 100 ng/ul Bovine Serum Albumin (BSA).

    Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction