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  • 99
    New England Biolabs restricted enzymes kpni
    In vitro selection process. ( A ) RNA <t>aptamer</t> library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), <t>KpnI</t> (green triangles) and PacI (red squares), as a function of selection round.
    Restricted Enzymes Kpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher kpni digestion
    Snake plot of the adenosine A 2B R. Mutated residues within the A 2B R identified to result in increased constitutive activity are indicated in gray . These mutations originate from two previously described screens [ 2 , 3 ] and the TM4-EL2-TM5 screen described in the current paper. The putative disulfide bridges are indicated with dotted lines . The disulfide bridge conserved in many class A GPCRs links C78 3.25 and C171 EL2 . The non-conserved second disulfide bridge between EL1 and EL2 is based on an analogous bond in the crystal structures of the adenosine A 2A R (PDB: 3EML, 3QAK, 3YDO, and 3YDV); it links C72 EL1 and C167 EL2 . The restriction sites <t>KpnI</t> and <t>BglII</t> are indicated; they were used to obtain the fragment for random mutagenesis
    Kpni Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher kpni
    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major <t>LMSTI1</t> and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with <t>KpnI</t> and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).
    Kpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kpni/product/Thermo Fisher
    Average 99 stars, based on 4148 article reviews
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    94
    Millipore kpni
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Kpni, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher fastdigest kpni
    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites <t>KpnI</t> and <t>Xhol</t> ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE
    Fastdigest Kpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: In Vitro, Selection

    Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Quantitation Assay, Binding Assay, Mobility Shift

    Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Titration, Concentration Assay

    Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Binding Assay, Mobility Shift

    Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Activity Assay, Mobility Shift, Binding Assay, Inhibition, Incubation, Concentration Assay

    Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Binding Assay

    ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques:

    KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Incubation, Concentration Assay

    Snake plot of the adenosine A 2B R. Mutated residues within the A 2B R identified to result in increased constitutive activity are indicated in gray . These mutations originate from two previously described screens [ 2 , 3 ] and the TM4-EL2-TM5 screen described in the current paper. The putative disulfide bridges are indicated with dotted lines . The disulfide bridge conserved in many class A GPCRs links C78 3.25 and C171 EL2 . The non-conserved second disulfide bridge between EL1 and EL2 is based on an analogous bond in the crystal structures of the adenosine A 2A R (PDB: 3EML, 3QAK, 3YDO, and 3YDV); it links C72 EL1 and C167 EL2 . The restriction sites KpnI and BglII are indicated; they were used to obtain the fragment for random mutagenesis

    Journal: Purinergic Signalling

    Article Title: Three "hotspots" important for adenosine A2B receptor activation: a mutational analysis of transmembrane domains 4 and 5 and the second extracellular loop

    doi: 10.1007/s11302-011-9251-x

    Figure Lengend Snippet: Snake plot of the adenosine A 2B R. Mutated residues within the A 2B R identified to result in increased constitutive activity are indicated in gray . These mutations originate from two previously described screens [ 2 , 3 ] and the TM4-EL2-TM5 screen described in the current paper. The putative disulfide bridges are indicated with dotted lines . The disulfide bridge conserved in many class A GPCRs links C78 3.25 and C171 EL2 . The non-conserved second disulfide bridge between EL1 and EL2 is based on an analogous bond in the crystal structures of the adenosine A 2A R (PDB: 3EML, 3QAK, 3YDO, and 3YDV); it links C72 EL1 and C167 EL2 . The restriction sites KpnI and BglII are indicated; they were used to obtain the fragment for random mutagenesis

    Article Snippet: After the error-prone PCR, the normal fragment TM4-EL2-TM5 in the wild-type receptor was replaced by the mutated fragments using the restriction sites KpnI and BglII and transformed into DH5α Escherichia coli -competent cells (Invitrogen, San Diego, CA, USA).

    Techniques: Activity Assay, Mutagenesis

    Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Generated, Construct, Expressing, Injection, Fluorescence In Situ Hybridization, Fluorescence, Activity Assay

    Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Plasmid Preparation, Clone Assay, TA Cloning, Ligation

    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Journal: Reports of Biochemistry & Molecular Biology

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi

    doi:

    Figure Lengend Snippet: A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Article Snippet: Construction of eukaryotic expression vector for the fusion protein LmSTI1 was cloned into pcNDA3.1 (+) to produce pCLmSTI1 as follows: the TA-cloned pT-LmSTI1 plasmid and pcDNA3.1 (+) were double-digested separately with KpnI and EcoRI (Fermentas, Germany) in 1X Tango buffer.

    Techniques: Construct, Expressing, Plasmid Preparation

    A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites KpnI and Xhol ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE

    Journal: Cell Stress & Chaperones

    Article Title: Genetic polymorphism in Hsp90AA1 gene is associated with the thermotolerance in Chinese Holstein cows

    doi: 10.1007/s12192-017-0873-y

    Figure Lengend Snippet: A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites KpnI and Xhol ( a ). the plasmid was digested and visualized ( b ). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity ( c ); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele ( d ). Representative data from triple independent experiments are shown with mean ± SE

    Article Snippet: To explore whether the g.− 87 G > C SNP could affect the promoter activity of the bovine Hsp90AA1 , 296 bp of genomic DNA between − 237 and + 59 of each wild-type allele G and mutant allele C were cloned into a promoterless pGL3-basic vector (Promega, USA) KpnI and Xhol restriction sites (Fig. a, b) to construct pGL3-G308 and pGL3-C308 plasmids, which were subsequently transferred into Hela (Sigma USA).

    Techniques: Plasmid Preparation, Negative Control, Transfection, Luciferase, Activity Assay, Expressing, Mutagenesis