120 bp fragment Promega Search Results


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  • 91
    Promega bst eii
    PRA patterns of the M. tuberculosis complex organisms upon digestion of the 441-bp PCR product with <t>Bst</t> <t>EII</t> (A), Hae III (B), Aci I (C), or Hha I (D) and the sequence of the hsp65 fragment from “ M. canettii ” (E). Lanes 1, M. tuberculosis ; lanes 2, M. bovis ; lanes 3, M. bovis BCG; lanes 4, M. africanum ; lanes 5, “ M. canettii ”; lanes C, negative control; lanes M, 100-bp ladder. The underlined portion in panel E shows the single mutation at position 235 (C-to-T transition) that is linked to the disappearance of a Hha I site and that corresponds to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv.
    Bst Eii, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher 25bp dna step ladder
    PRA patterns of the M. tuberculosis complex organisms upon digestion of the 441-bp PCR product with <t>Bst</t> <t>EII</t> (A), Hae III (B), Aci I (C), or Hha I (D) and the sequence of the hsp65 fragment from “ M. canettii ” (E). Lanes 1, M. tuberculosis ; lanes 2, M. bovis ; lanes 3, M. bovis BCG; lanes 4, M. africanum ; lanes 5, “ M. canettii ”; lanes C, negative control; lanes M, 100-bp ladder. The underlined portion in panel E shows the single mutation at position 235 (C-to-T transition) that is linked to the disappearance of a Hha I site and that corresponds to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv.
    25bp Dna Step Ladder, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pgem t easy
    PRA patterns of the M. tuberculosis complex organisms upon digestion of the 441-bp PCR product with <t>Bst</t> <t>EII</t> (A), Hae III (B), Aci I (C), or Hha I (D) and the sequence of the hsp65 fragment from “ M. canettii ” (E). Lanes 1, M. tuberculosis ; lanes 2, M. bovis ; lanes 3, M. bovis BCG; lanes 4, M. africanum ; lanes 5, “ M. canettii ”; lanes C, negative control; lanes M, 100-bp ladder. The underlined portion in panel E shows the single mutation at position 235 (C-to-T transition) that is linked to the disappearance of a Hha I site and that corresponds to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv.
    Pgem T Easy, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 33392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega bcii restriction enzymes
    PRA patterns of the M. tuberculosis complex organisms upon digestion of the 441-bp PCR product with <t>Bst</t> <t>EII</t> (A), Hae III (B), Aci I (C), or Hha I (D) and the sequence of the hsp65 fragment from “ M. canettii ” (E). Lanes 1, M. tuberculosis ; lanes 2, M. bovis ; lanes 3, M. bovis BCG; lanes 4, M. africanum ; lanes 5, “ M. canettii ”; lanes C, negative control; lanes M, 100-bp ladder. The underlined portion in panel E shows the single mutation at position 235 (C-to-T transition) that is linked to the disappearance of a Hha I site and that corresponds to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv.
    Bcii Restriction Enzymes, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pgl3 vector
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Pgl3 Vector, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 9304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega wizard plus minipreps system
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Wizard Plus Minipreps System, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pgl3 basic
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Pgl3 Basic, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 12565 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega klenow enzyme
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Klenow Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega pdnas encoding photinus pyralis luciferase
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Pdnas Encoding Photinus Pyralis Luciferase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega psp73 vector
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Psp73 Vector, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega nhei restriction sites
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Nhei Restriction Sites, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega riboprobe transcription system
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Riboprobe Transcription System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega taq 1 restriction endonuclease
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Taq 1 Restriction Endonuclease, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr vector pgem t
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Pcr Vector Pgem T, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega colorless gotaq flexi buffer
    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the <t>pGL3-Nab2</t> or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =
    Colorless Gotaq Flexi Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega pgl3 basic luciferase vector
    rs1175550 and rs143702418 independently increase luciferase activity. Four DNA fragments of SMIM1 intron 2 (full sequences in Supplementary Table S2 ) corresponding to all four possible alleles of the two biallelic variants were inserted upstream of the luciferase gene in the <t>pGL3-Basic</t> vector and assayed for luciferase activity in K562 ( b , left panel) and HEL cells ( b , right panel). Values are fireflly/renilla ratios normalised to pGL3-Basic without insert (ctr), plotted as mean ± SEM and based on two different experiments performed in triplicate (n = 6) for each cell line. Pairwise, two-tailed unpaired t-tests were performed on all groups; selected p -values less than 0.05 are indicated. All groups were significantly different from the pGL3-Basic control vector.
    Pgl3 Basic Luciferase Vector, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 2072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega pgem t plasmid
    rs1175550 and rs143702418 independently increase luciferase activity. Four DNA fragments of SMIM1 intron 2 (full sequences in Supplementary Table S2 ) corresponding to all four possible alleles of the two biallelic variants were inserted upstream of the luciferase gene in the <t>pGL3-Basic</t> vector and assayed for luciferase activity in K562 ( b , left panel) and HEL cells ( b , right panel). Values are fireflly/renilla ratios normalised to pGL3-Basic without insert (ctr), plotted as mean ± SEM and based on two different experiments performed in triplicate (n = 6) for each cell line. Pairwise, two-tailed unpaired t-tests were performed on all groups; selected p -values less than 0.05 are indicated. All groups were significantly different from the pGL3-Basic control vector.
    Pgem T Plasmid, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 1244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega promoterless luciferase reporter plasmid pgl3 basic
    rs1175550 and rs143702418 independently increase luciferase activity. Four DNA fragments of SMIM1 intron 2 (full sequences in Supplementary Table S2 ) corresponding to all four possible alleles of the two biallelic variants were inserted upstream of the luciferase gene in the <t>pGL3-Basic</t> vector and assayed for luciferase activity in K562 ( b , left panel) and HEL cells ( b , right panel). Values are fireflly/renilla ratios normalised to pGL3-Basic without insert (ctr), plotted as mean ± SEM and based on two different experiments performed in triplicate (n = 6) for each cell line. Pairwise, two-tailed unpaired t-tests were performed on all groups; selected p -values less than 0.05 are indicated. All groups were significantly different from the pGL3-Basic control vector.
    Promoterless Luciferase Reporter Plasmid Pgl3 Basic, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega pgl2 basic
    Mutant analysis of IGFBP-3 promoter for the induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of <t>pGL2-210</t> and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.
    Pgl2 Basic, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1911 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega transcriptase kit
    Mutant analysis of IGFBP-3 promoter for the induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of <t>pGL2-210</t> and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.
    Transcriptase Kit, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcat basic vector
    Mutant analysis of IGFBP-3 promoter for the induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of <t>pGL2-210</t> and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.
    Pcat Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega psicheck2
    Mutant analysis of IGFBP-3 promoter for the induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of <t>pGL2-210</t> and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.
    Psicheck2, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 1316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PRA patterns of the M. tuberculosis complex organisms upon digestion of the 441-bp PCR product with Bst EII (A), Hae III (B), Aci I (C), or Hha I (D) and the sequence of the hsp65 fragment from “ M. canettii ” (E). Lanes 1, M. tuberculosis ; lanes 2, M. bovis ; lanes 3, M. bovis BCG; lanes 4, M. africanum ; lanes 5, “ M. canettii ”; lanes C, negative control; lanes M, 100-bp ladder. The underlined portion in panel E shows the single mutation at position 235 (C-to-T transition) that is linked to the disappearance of a Hha I site and that corresponds to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv.

    Journal: Journal of Clinical Microbiology

    Article Title: Rapid Differentiation of "Mycobacterium canettii" from Other Mycobacterium tuberculosis Complex Organisms by PCR-Restriction Analysis of the hsp65 Gene

    doi: 10.1128/JCM.39.10.3705-3708.2001

    Figure Lengend Snippet: PRA patterns of the M. tuberculosis complex organisms upon digestion of the 441-bp PCR product with Bst EII (A), Hae III (B), Aci I (C), or Hha I (D) and the sequence of the hsp65 fragment from “ M. canettii ” (E). Lanes 1, M. tuberculosis ; lanes 2, M. bovis ; lanes 3, M. bovis BCG; lanes 4, M. africanum ; lanes 5, “ M. canettii ”; lanes C, negative control; lanes M, 100-bp ladder. The underlined portion in panel E shows the single mutation at position 235 (C-to-T transition) that is linked to the disappearance of a Hha I site and that corresponds to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv.

    Article Snippet: Although the results of PRA after Bst EII, Hae III, and Aci I enzyme digestions did not discriminate among the various members of the M. tuberculosis complex, we found an excellent correlation between the theoretical sizes of the fragments based on hsp65 sequencing data and PRA results.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Mutagenesis

    ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the pGL3-Nab2 or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =

    Journal: BMC Molecular Biology

    Article Title: Differential regulation of NAB corepressor genes in Schwann cells

    doi: 10.1186/1471-2199-8-117

    Figure Lengend Snippet: ETS activation of NAB promoters . A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the pGL3-Nab2 or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =

    Article Snippet: Plasmids A 370 bp NotI/PvuII fragment of the mouse Nab1 promoter (-250 to +120 relative to the transcription start site) and a 1000 bp KpnI/SmaI fragment of the mouse Nab2 promoter (-850 to +150) were fused to the luciferase gene in the pGL3 vector (Promega).

    Techniques: Activation Assay, Transfection, Expressing, Activity Assay

    Diagrammatic representation of plasmid constructs and analysis of RNA transcripts. (A) Structure of all the constructs that were used for making RNAs and also for transfection into mouse cell lines. The T7-U6 1-28 -18 nt-IRES was PCR amplified and cloned into the pGL3 basic vector using Sac I and Bgl II sites. The Hpa ) was inserted into the Eco RI site to increase the distance between the IRES and the U6 capping signal. The constructs are not drawn to scale. T7, T7 promoter; m, mutant; U6 1-28 , U6 snRNA capping signal; Luc, luciferase. (B) In vitro transcription was carried out in the presence of CH3-pppG to get γ-monomethylphosphate-capped mRNAs and in the presence of m 7 GpppG to get m 7 G-capped RNAs. The RNAs were run on an agarose gel and stained with ethidium bromide to estimate quality and quantity of RNAs.

    Journal: Gene Expression

    Article Title: Inhibition of Translation of mRNAs Containing γ-Monomethylphosphate Cap Structure in Frog Oocytes and in Mammalian Cells

    doi:

    Figure Lengend Snippet: Diagrammatic representation of plasmid constructs and analysis of RNA transcripts. (A) Structure of all the constructs that were used for making RNAs and also for transfection into mouse cell lines. The T7-U6 1-28 -18 nt-IRES was PCR amplified and cloned into the pGL3 basic vector using Sac I and Bgl II sites. The Hpa ) was inserted into the Eco RI site to increase the distance between the IRES and the U6 capping signal. The constructs are not drawn to scale. T7, T7 promoter; m, mutant; U6 1-28 , U6 snRNA capping signal; Luc, luciferase. (B) In vitro transcription was carried out in the presence of CH3-pppG to get γ-monomethylphosphate-capped mRNAs and in the presence of m 7 GpppG to get m 7 G-capped RNAs. The RNAs were run on an agarose gel and stained with ethidium bromide to estimate quality and quantity of RNAs.

    Article Snippet: A deletion-insertion mutation was introduced into the U6 capping signal (mU61-28 ) by PCR and the T7-U61-28 -18 nt-IRES fragment was inserted into pGL3-Basic vector between the Sac I and Bgl II sites.

    Techniques: Plasmid Preparation, Construct, Transfection, Polymerase Chain Reaction, Amplification, Clone Assay, Mutagenesis, Luciferase, In Vitro, Agarose Gel Electrophoresis, Staining

    rs1175550 and rs143702418 independently increase luciferase activity. Four DNA fragments of SMIM1 intron 2 (full sequences in Supplementary Table S2 ) corresponding to all four possible alleles of the two biallelic variants were inserted upstream of the luciferase gene in the pGL3-Basic vector and assayed for luciferase activity in K562 ( b , left panel) and HEL cells ( b , right panel). Values are fireflly/renilla ratios normalised to pGL3-Basic without insert (ctr), plotted as mean ± SEM and based on two different experiments performed in triplicate (n = 6) for each cell line. Pairwise, two-tailed unpaired t-tests were performed on all groups; selected p -values less than 0.05 are indicated. All groups were significantly different from the pGL3-Basic control vector.

    Journal: Scientific Reports

    Article Title: SMIM1 variants rs1175550 and rs143702418 independently modulate Vel blood group antigen expression

    doi: 10.1038/srep40451

    Figure Lengend Snippet: rs1175550 and rs143702418 independently increase luciferase activity. Four DNA fragments of SMIM1 intron 2 (full sequences in Supplementary Table S2 ) corresponding to all four possible alleles of the two biallelic variants were inserted upstream of the luciferase gene in the pGL3-Basic vector and assayed for luciferase activity in K562 ( b , left panel) and HEL cells ( b , right panel). Values are fireflly/renilla ratios normalised to pGL3-Basic without insert (ctr), plotted as mean ± SEM and based on two different experiments performed in triplicate (n = 6) for each cell line. Pairwise, two-tailed unpaired t-tests were performed on all groups; selected p -values less than 0.05 are indicated. All groups were significantly different from the pGL3-Basic control vector.

    Article Snippet: Luciferase assay Four DNA fragments of 117 bp or 120 bp (synthesised by Integrated DNA Technologies, USA) corresponding to the possible combinations created by the two variants rs143702418C/CGCA and rs1175550A/G ( ) were inserted upstream of the luciferase gene in the pGL3-Basic luciferase vector (Promega) using restriction enzymes Nhe I and Hind III.

    Techniques: Luciferase, Activity Assay, Plasmid Preparation, Two Tailed Test

    Mutant analysis of IGFBP-3 promoter for the induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.

    Journal: BMC Cancer

    Article Title: Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

    doi: 10.1186/1471-2407-5-9

    Figure Lengend Snippet: Mutant analysis of IGFBP-3 promoter for the induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.

    Article Snippet: A series of deletion mutant constructs, pGL2-270, pGL2-240, pGL2-210, pGL2-180, pGL2-150, pGL2-120, pGL2-90, pGL2-60, pGL2-30, and pGL2-1 containing the indicated fragments upstream of the transcription start site and 60 bp of fragments downstream of the transcription start site, were generated by PCR amplification of the promoter fragment and subsequent subcloning of the Mlu I-Bgl II fragment to pGL2-Basic (Table , Fig. ).

    Techniques: Mutagenesis, Sequencing, Transfection, Luciferase, Construct, Cotransfection, Activity Assay

    Methylation analysis of IGFBP-3 promoter for induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and a sequence of the methylated construct carrying the CpG methylation in the core consensus sequence (underlined) of pGL2-210. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter construct or methylated IGFBP-3 promoter-luciferase reporter construct with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.

    Journal: BMC Cancer

    Article Title: Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

    doi: 10.1186/1471-2407-5-9

    Figure Lengend Snippet: Methylation analysis of IGFBP-3 promoter for induction by p53 . A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and a sequence of the methylated construct carrying the CpG methylation in the core consensus sequence (underlined) of pGL2-210. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter construct or methylated IGFBP-3 promoter-luciferase reporter construct with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown.

    Article Snippet: A series of deletion mutant constructs, pGL2-270, pGL2-240, pGL2-210, pGL2-180, pGL2-150, pGL2-120, pGL2-90, pGL2-60, pGL2-30, and pGL2-1 containing the indicated fragments upstream of the transcription start site and 60 bp of fragments downstream of the transcription start site, were generated by PCR amplification of the promoter fragment and subsequent subcloning of the Mlu I-Bgl II fragment to pGL2-Basic (Table , Fig. ).

    Techniques: Methylation, Sequencing, Construct, CpG Methylation Assay, Transfection, Luciferase, Cotransfection, Activity Assay