pgem t easy vector  (Promega)

 
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    Name:
    pGEM-T Easy Vector Systems
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    A1360
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    Structured Review

    Promega pgem t easy vector
    Genetic map of pGEMsrdBCA. The srdBCA operon with its own promoter region was inserted into the <t>pGEM-T</t> Easy vector under the direction of the lac promoter. Annealing sites of primers used for the construction of derivative vectors are indicated: a, SRDCF;

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    Images

    1) Product Images from "Molecular Cloning and Characterization of the srdBCA Operon, Encoding the Respiratory Selenate Reductase Complex, from the Selenate-Reducing Bacterium Bacillus selenatarsenatis SF-1"

    Article Title: Molecular Cloning and Characterization of the srdBCA Operon, Encoding the Respiratory Selenate Reductase Complex, from the Selenate-Reducing Bacterium Bacillus selenatarsenatis SF-1

    Journal:

    doi: 10.1128/JB.01197-10

    Genetic map of pGEMsrdBCA. The srdBCA operon with its own promoter region was inserted into the pGEM-T Easy vector under the direction of the lac promoter. Annealing sites of primers used for the construction of derivative vectors are indicated: a, SRDCF;
    Figure Legend Snippet: Genetic map of pGEMsrdBCA. The srdBCA operon with its own promoter region was inserted into the pGEM-T Easy vector under the direction of the lac promoter. Annealing sites of primers used for the construction of derivative vectors are indicated: a, SRDCF;

    Techniques Used: Plasmid Preparation

    2) Product Images from "Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung"

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-15-23

    Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.
    Figure Legend Snippet: Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Concentration Assay, Standard Deviation

    Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .
    Figure Legend Snippet: Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Fluorescence

    BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.
    Figure Legend Snippet: BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.
    Figure Legend Snippet: Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.

    Techniques Used: Real-time Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction

    3) Product Images from "Influence of viral genes on the cell-to-cell spread of RNA silencing"

    Article Title: Influence of viral genes on the cell-to-cell spread of RNA silencing

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ern141

    A schematic representation of recombinant viral constructs. (A) Genomic organization of TCV and TCV-based local RNA silencing vectors. Viral genes and GFP coding regions are presented as open boxes. Mutated open reading frames have asterisks and are shaded. The self-cleaving 2A oligopeptide of Foot-and-mouth disease virus is shown as a dark box. (B) Gfp sense and anti-sense cassettes. The GFP gene was cloned into pGEM-T Easy vector in opposite orientations under the transcriptional control of the T7 RNA promoter. (C) Genomic organization of PVX and PVX-based gene expression constructs.
    Figure Legend Snippet: A schematic representation of recombinant viral constructs. (A) Genomic organization of TCV and TCV-based local RNA silencing vectors. Viral genes and GFP coding regions are presented as open boxes. Mutated open reading frames have asterisks and are shaded. The self-cleaving 2A oligopeptide of Foot-and-mouth disease virus is shown as a dark box. (B) Gfp sense and anti-sense cassettes. The GFP gene was cloned into pGEM-T Easy vector in opposite orientations under the transcriptional control of the T7 RNA promoter. (C) Genomic organization of PVX and PVX-based gene expression constructs.

    Techniques Used: Recombinant, Construct, Clone Assay, Plasmid Preparation, Expressing

    4) Product Images from "The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts"

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl1137

    Analysis of the 5′end of CAT mRNAs derived from the 5′UTRKPM11 region and the Pr77 promoter. (A) Outline of the method used. Cytoplasmic RNA from T. cruzi transfected with pTEX(−)pR77CAT and pTEX(−)p5′KMP11CAT (used as a control) was decapped with tobacco acid pyrophosphatase (TAP), dephosphorylated with alkaline phosphatase enzyme (AP) and phosphorylated by polynucleotide kinase (PNK) treatment. The changes produced in each case at the 5′ end of the treated RNAs are indicated. A linker RNA was ligated at the 5′ end of the transcripts using T4 RNA ligase. cDNA was synthesized by reverse transcription (RT) using a CAT gene specific primer (CAT1) that maps at position 191–208 from the CAT start codon, followed by PCR-amplification using a second CAT gene antisense primer (CAT90) mapping at position 71–90 from the CAT start codon, and finally an oligo corresponding to the sequence of the linker RNA. PCR products were cloned into the pGEM-T®-easy vector. Plasmid DNAs from the transfected bacteria selected by positive hybridization to CAT (ds DNA) and SLTc or R77 probes were sequenced. (B) Sequence at the 5′ end of processed CAT transcripts in pTEX(−)p5′KMP11CAT (a) and pTEX(−)pR77CAT (b) transfectants. The sequences shown are those downstream of the RNA linker sequence. Arrows indicate the first nucleotide of the 5′end of the CAT transcripts derived from the 5′UTRKMP11 and Pr77 sequences. The position of the first nucleotide of each transcript, with respect to the 5′end of SL and Pr77 sequences, is shown in italics. The number below the arrows corresponds to the quantity of clones starting at this position. Sequences corresponding to the splice leader sequence (SL), 5′UTRKMP11 (5′UTRKMP11CAT ), L1TcPr77 (Pr77) and multi-cloning site – CAT region (MCS-CAT) are indicated.
    Figure Legend Snippet: Analysis of the 5′end of CAT mRNAs derived from the 5′UTRKPM11 region and the Pr77 promoter. (A) Outline of the method used. Cytoplasmic RNA from T. cruzi transfected with pTEX(−)pR77CAT and pTEX(−)p5′KMP11CAT (used as a control) was decapped with tobacco acid pyrophosphatase (TAP), dephosphorylated with alkaline phosphatase enzyme (AP) and phosphorylated by polynucleotide kinase (PNK) treatment. The changes produced in each case at the 5′ end of the treated RNAs are indicated. A linker RNA was ligated at the 5′ end of the transcripts using T4 RNA ligase. cDNA was synthesized by reverse transcription (RT) using a CAT gene specific primer (CAT1) that maps at position 191–208 from the CAT start codon, followed by PCR-amplification using a second CAT gene antisense primer (CAT90) mapping at position 71–90 from the CAT start codon, and finally an oligo corresponding to the sequence of the linker RNA. PCR products were cloned into the pGEM-T®-easy vector. Plasmid DNAs from the transfected bacteria selected by positive hybridization to CAT (ds DNA) and SLTc or R77 probes were sequenced. (B) Sequence at the 5′ end of processed CAT transcripts in pTEX(−)p5′KMP11CAT (a) and pTEX(−)pR77CAT (b) transfectants. The sequences shown are those downstream of the RNA linker sequence. Arrows indicate the first nucleotide of the 5′end of the CAT transcripts derived from the 5′UTRKMP11 and Pr77 sequences. The position of the first nucleotide of each transcript, with respect to the 5′end of SL and Pr77 sequences, is shown in italics. The number below the arrows corresponds to the quantity of clones starting at this position. Sequences corresponding to the splice leader sequence (SL), 5′UTRKMP11 (5′UTRKMP11CAT ), L1TcPr77 (Pr77) and multi-cloning site – CAT region (MCS-CAT) are indicated.

    Techniques Used: Derivative Assay, Transfection, Produced, Synthesized, Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay, Plasmid Preparation, Hybridization

    5) Product Images from "Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung"

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-15-23

    Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.
    Figure Legend Snippet: Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Concentration Assay, Standard Deviation

    Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .
    Figure Legend Snippet: Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Fluorescence

    BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.
    Figure Legend Snippet: BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.
    Figure Legend Snippet: Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.

    Techniques Used: Real-time Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction

    6) Product Images from "Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus"

    Article Title: Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-018-1465-5

    The VP1 protein has DNA-binding ability with no sequence specificity. The recombinant GST and GST-fused proteins were prepared by E. coli overexpression and purified through GST affinity chromatography. Purified results were analysed by SDS-PAGE with Coomassie blue staining and Western blotting with an anti-GST monoclonal antibody or anti-C-ter-VP1 polyclonal antibody ( a ). The purified proteins were used for DNA binding ability by an agarose gel shift assay with different DNA sequences of plasmid preparation of pcDNA3.1 ( b ), of the pGEM-T easy vector ( c ), and of pCAV containing the whole CAV genome ( d ). The binding activity of the VP1 protein was determined by comparing the existence of DNA fragments for the protein-DNA complex and DNA patterns from the blank (no-protein used), negative control (GST only) and positive control (GST-VP3). To confirm the observed DNA migration results that were induced by bound recombinant proteins, the protein-DNA experimental samples were mixed with 1% SDS as a protein denaturant (underline lane-labelled 1% SDS). Lane M, DNA ladder marker. Bold triangles indicated the protein-DNA complex formed by tested proteins and plasmids. Asterisks indicated the two conformations of plasmid DNA, including the relaxed form (Form I), and another was the supercoiled form (Form II)
    Figure Legend Snippet: The VP1 protein has DNA-binding ability with no sequence specificity. The recombinant GST and GST-fused proteins were prepared by E. coli overexpression and purified through GST affinity chromatography. Purified results were analysed by SDS-PAGE with Coomassie blue staining and Western blotting with an anti-GST monoclonal antibody or anti-C-ter-VP1 polyclonal antibody ( a ). The purified proteins were used for DNA binding ability by an agarose gel shift assay with different DNA sequences of plasmid preparation of pcDNA3.1 ( b ), of the pGEM-T easy vector ( c ), and of pCAV containing the whole CAV genome ( d ). The binding activity of the VP1 protein was determined by comparing the existence of DNA fragments for the protein-DNA complex and DNA patterns from the blank (no-protein used), negative control (GST only) and positive control (GST-VP3). To confirm the observed DNA migration results that were induced by bound recombinant proteins, the protein-DNA experimental samples were mixed with 1% SDS as a protein denaturant (underline lane-labelled 1% SDS). Lane M, DNA ladder marker. Bold triangles indicated the protein-DNA complex formed by tested proteins and plasmids. Asterisks indicated the two conformations of plasmid DNA, including the relaxed form (Form I), and another was the supercoiled form (Form II)

    Techniques Used: Binding Assay, Sequencing, Recombinant, Over Expression, Purification, Affinity Chromatography, SDS Page, Staining, Western Blot, Agarose Gel Electrophoresis, Shift Assay, Plasmid Preparation, Activity Assay, Negative Control, Positive Control, Migration, Marker

    7) Product Images from "Development of a Rapid Method for Identifying Carryover Contamination of Positive Control DNA, Using a Chimeric Positive Control and Restriction Enzyme for the Diagnosis of White Spot Syndrome Virus by Nested PCR"

    Article Title: Development of a Rapid Method for Identifying Carryover Contamination of Positive Control DNA, Using a Chimeric Positive Control and Restriction Enzyme for the Diagnosis of White Spot Syndrome Virus by Nested PCR

    Journal:

    doi: 10.1007/s12088-014-0480-x

    Schematic representation of construction of the WSSV chimeric plasmid DNA positive controls AR and phage f1 genes are truncated with the WSSV 146F1 and R1 primer sets and with the WSSV 146F2 and 146R2 primer sets and cloned into a pGEM-T Easy vector
    Figure Legend Snippet: Schematic representation of construction of the WSSV chimeric plasmid DNA positive controls AR and phage f1 genes are truncated with the WSSV 146F1 and R1 primer sets and with the WSSV 146F2 and 146R2 primer sets and cloned into a pGEM-T Easy vector

    Techniques Used: Plasmid Preparation, Clone Assay

    8) Product Images from "Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung"

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-15-23

    Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.
    Figure Legend Snippet: Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Concentration Assay, Standard Deviation

    Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .
    Figure Legend Snippet: Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Fluorescence

    BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.
    Figure Legend Snippet: BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.
    Figure Legend Snippet: Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.

    Techniques Used: Real-time Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction

    9) Product Images from "A novel assay to detect calreticulin mutations in myeloproliferative neoplasms"

    Article Title: A novel assay to detect calreticulin mutations in myeloproliferative neoplasms

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14113

    Detection analysis by PNA directed PCR clamping of CALR type-1 (DEL) and type-2 (INS) mutations ( A ) Step I: cDNA amplification of patients with CALR wild-type (WT), type-1 (DEL) and type-2 (INS) mutations. pGEM-T- CALR wild-type (WT), pGEM-T- CALR type-1 mut (DEL), pGEM-T- CALR type-1 mut 50% vs. wild-type (DEL 50%) and pGEM- CALR type-2 mut (INS) were used as PCR positive control. 5 μL of each amplifier were loaded on 1% Agarose-TBE 1x gel with 5 μg/mL ethidium bromide (EtBr) and run at 120 V for 30 minutes. ( B ) Step II: PCR amplification of a small area of CALR gene, containing type-1 and type-2 mutations, was carried-out in absence (−) or in presence (+) of PNA probe. The plasmids amplified in the step I were used, in a dilution of 1:100, in the step II in order to interpret the results, 10 μL of each amplifier were loaded on 2% Agarose-TBE 1x gel with 5μg/mL EtBr and run at 100 V for 30 minutes and than at 65 V for 15 minutes. The arrow indicates the non-specific band present in the amplified of patients.
    Figure Legend Snippet: Detection analysis by PNA directed PCR clamping of CALR type-1 (DEL) and type-2 (INS) mutations ( A ) Step I: cDNA amplification of patients with CALR wild-type (WT), type-1 (DEL) and type-2 (INS) mutations. pGEM-T- CALR wild-type (WT), pGEM-T- CALR type-1 mut (DEL), pGEM-T- CALR type-1 mut 50% vs. wild-type (DEL 50%) and pGEM- CALR type-2 mut (INS) were used as PCR positive control. 5 μL of each amplifier were loaded on 1% Agarose-TBE 1x gel with 5 μg/mL ethidium bromide (EtBr) and run at 120 V for 30 minutes. ( B ) Step II: PCR amplification of a small area of CALR gene, containing type-1 and type-2 mutations, was carried-out in absence (−) or in presence (+) of PNA probe. The plasmids amplified in the step I were used, in a dilution of 1:100, in the step II in order to interpret the results, 10 μL of each amplifier were loaded on 2% Agarose-TBE 1x gel with 5μg/mL EtBr and run at 100 V for 30 minutes and than at 65 V for 15 minutes. The arrow indicates the non-specific band present in the amplified of patients.

    Techniques Used: Polymerase Chain Reaction, Amplification, Positive Control

    10) Product Images from "Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data"

    Article Title: Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1361

    In vitro transcription analysis of upstream deleted Alu loci. In vitro transcription reactions were performed in HeLa nuclear extract using 0.5 μg of the indicated Alu templates (lanes 5–10, 15–20, 25–30). A previously characterized Alu producing a 372-nt RNA (lanes 2, 12, 22) and a human tRNA Val gene producing a known transcript pattern due to heterogeneous transcription termination (lanes 3, 13, 23) ( 25 ) were used as positive controls for in vitro transcription and, at the same time, as a source of RNA size markers. Negative control reactions contained either empty pGEM®-T Easy vector (lanes 1, 11, 21) or no template DNA (no-template control (NTC), lanes 4, 14, 24). For each Alu , both the wild-type and a mutant version lacking most of the native 5′-flanking region ( 5 ′ del ) were tested. For each of the nine Alu s subjected to 5′-flank deletion, the extent of reduction of transcription activity, observed with respect to the corresponding wild-type Alu , is reported below the lanes corresponding to each wt-mutant pair. The values represent the average of two independent transcription experiments that differed by no more than 20% of the mean.
    Figure Legend Snippet: In vitro transcription analysis of upstream deleted Alu loci. In vitro transcription reactions were performed in HeLa nuclear extract using 0.5 μg of the indicated Alu templates (lanes 5–10, 15–20, 25–30). A previously characterized Alu producing a 372-nt RNA (lanes 2, 12, 22) and a human tRNA Val gene producing a known transcript pattern due to heterogeneous transcription termination (lanes 3, 13, 23) ( 25 ) were used as positive controls for in vitro transcription and, at the same time, as a source of RNA size markers. Negative control reactions contained either empty pGEM®-T Easy vector (lanes 1, 11, 21) or no template DNA (no-template control (NTC), lanes 4, 14, 24). For each Alu , both the wild-type and a mutant version lacking most of the native 5′-flanking region ( 5 ′ del ) were tested. For each of the nine Alu s subjected to 5′-flank deletion, the extent of reduction of transcription activity, observed with respect to the corresponding wild-type Alu , is reported below the lanes corresponding to each wt-mutant pair. The values represent the average of two independent transcription experiments that differed by no more than 20% of the mean.

    Techniques Used: In Vitro, Negative Control, Plasmid Preparation, Mutagenesis, Activity Assay

    In vitro transcription analysis of wild type and B box-mutated Alu loci. In vitro transcription reactions were performed in HeLa nuclear extract using 0.5 μg of the indicated. Alu templates (lanes 5–10, 15–20, 25–30). A previously characterized Alu producing a 372-nt RNA (lanes 2, 12, 22) and a human tRNA Val gene producing a known transcript pattern due to heterogeneous transcription termination (lanes 3, 13, 23) ( 25 ) were used as positive controls for in vitro transcription and, at the same time, as a source of RNA size markers. Negative control reactions contained either empty pGEM®-T Easy vector (lanes 1, 11, 21) or no template DNA (no-template control (NTC), lanes 4, 14, 24). For each Alu , both the wild-type and a B box-mutated ( Bmut ) version were tested.
    Figure Legend Snippet: In vitro transcription analysis of wild type and B box-mutated Alu loci. In vitro transcription reactions were performed in HeLa nuclear extract using 0.5 μg of the indicated. Alu templates (lanes 5–10, 15–20, 25–30). A previously characterized Alu producing a 372-nt RNA (lanes 2, 12, 22) and a human tRNA Val gene producing a known transcript pattern due to heterogeneous transcription termination (lanes 3, 13, 23) ( 25 ) were used as positive controls for in vitro transcription and, at the same time, as a source of RNA size markers. Negative control reactions contained either empty pGEM®-T Easy vector (lanes 1, 11, 21) or no template DNA (no-template control (NTC), lanes 4, 14, 24). For each Alu , both the wild-type and a B box-mutated ( Bmut ) version were tested.

    Techniques Used: In Vitro, Negative Control, Plasmid Preparation

    11) Product Images from "Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector"

    Article Title: Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02460-2

    Construction of the multi-gene co-expression vector with 2A sequences. ( A ) Schematic representation of the mechanism of “self-cleaving” 2A peptides. ( B ) DNA and amino acid sequences of the various 2As used. A GSG linker was added to the N-terminus of all 2As. ( C ) A simplified map of the pGEM-T-PTE2A cloning vector showing 2As and restriction sites used.
    Figure Legend Snippet: Construction of the multi-gene co-expression vector with 2A sequences. ( A ) Schematic representation of the mechanism of “self-cleaving” 2A peptides. ( B ) DNA and amino acid sequences of the various 2As used. A GSG linker was added to the N-terminus of all 2As. ( C ) A simplified map of the pGEM-T-PTE2A cloning vector showing 2As and restriction sites used.

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay

    12) Product Images from "Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae"

    Article Title: Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq583

    CPA-sRNA validation using 3′-RACE. ( A ) Total RNA from M. oryzae was used to purify 5′ methylguanosine-capped RNAs using recombinant eIF4E K119A bound to beads ( 21 ). 5′ methylguanosine-capped RNA was treated with DNase I and single-stranded cDNA synthesized using an oligo (dT) 20 VN primer. PCR amplification was performed using a forward primer to the 5′-end of specific CPA-sRNAs and reverse primer specific to the oligo (dT) 20 VN linker. PCR products were analyzed on 3% agarose gels, bands eluted, cloned into pGEM-T vectors and Sanger sequenced. PCR products were resolved on a 3% agarose gels for ( B ) protein-coding mRNA (MGG_0383.6, MGG_6594.6, MGG_0469.6, MGG_0592.6, MGG_02597.6, MGG_07928.6, MGG_10680.6, MGG_14279.6 and MGG_01210.6); ( C ) tRNAs (Ala: MGG_20297.6, Cys: MGG_20209.6, Gln: MGG_20266.6 and Leu: MGG_20218.6); ( D ) rRNAs (18S and 28S); (E ) snRNAs (U6 and U2) and ( F ) retroelements (MAGGY-LTR). A DNA ladder is shown on the left of each panel. Arrows indicate PCR products that were sequenced.
    Figure Legend Snippet: CPA-sRNA validation using 3′-RACE. ( A ) Total RNA from M. oryzae was used to purify 5′ methylguanosine-capped RNAs using recombinant eIF4E K119A bound to beads ( 21 ). 5′ methylguanosine-capped RNA was treated with DNase I and single-stranded cDNA synthesized using an oligo (dT) 20 VN primer. PCR amplification was performed using a forward primer to the 5′-end of specific CPA-sRNAs and reverse primer specific to the oligo (dT) 20 VN linker. PCR products were analyzed on 3% agarose gels, bands eluted, cloned into pGEM-T vectors and Sanger sequenced. PCR products were resolved on a 3% agarose gels for ( B ) protein-coding mRNA (MGG_0383.6, MGG_6594.6, MGG_0469.6, MGG_0592.6, MGG_02597.6, MGG_07928.6, MGG_10680.6, MGG_14279.6 and MGG_01210.6); ( C ) tRNAs (Ala: MGG_20297.6, Cys: MGG_20209.6, Gln: MGG_20266.6 and Leu: MGG_20218.6); ( D ) rRNAs (18S and 28S); (E ) snRNAs (U6 and U2) and ( F ) retroelements (MAGGY-LTR). A DNA ladder is shown on the left of each panel. Arrows indicate PCR products that were sequenced.

    Techniques Used: Recombinant, Synthesized, Polymerase Chain Reaction, Amplification, Clone Assay

    13) Product Images from "RNA-Seq Approach for Genetic Improvement of Meat Quality in Pig and Evolutionary Insight into the Substrate Specificity of Animal Carbonyl Reductases"

    Article Title: RNA-Seq Approach for Genetic Improvement of Meat Quality in Pig and Evolutionary Insight into the Substrate Specificity of Animal Carbonyl Reductases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042198

    Confirmation of the nonsense variation at the genomic, transcript, and protein levels for PTCR . The partial genomic DNA (A) and full length cDNA (B) of PTCR , including the nonsense variation locus, were obtained by PCR using specific primers as described in the ‘ Methods ’ section and subcloned into the pGEM T easy vector for Sanger sequencing. Boxes indicate different variants (G/T) in an exonic region of genome (A) and at nucleotide 951 of the transcript (B). Expression of PTCR genes with different variants (G/T) was induced in E. coli BL21 by IPTG, and total extracts were loaded onto 12% and 20% SDS-polyacrylamide gels (C). Arrows indicate the His-tagged PTCR fusion proteins of different sizes, about 32 kD or 31 kD, which correspond to PTCR(WT) and PTCR(ΔCterm), respectively.
    Figure Legend Snippet: Confirmation of the nonsense variation at the genomic, transcript, and protein levels for PTCR . The partial genomic DNA (A) and full length cDNA (B) of PTCR , including the nonsense variation locus, were obtained by PCR using specific primers as described in the ‘ Methods ’ section and subcloned into the pGEM T easy vector for Sanger sequencing. Boxes indicate different variants (G/T) in an exonic region of genome (A) and at nucleotide 951 of the transcript (B). Expression of PTCR genes with different variants (G/T) was induced in E. coli BL21 by IPTG, and total extracts were loaded onto 12% and 20% SDS-polyacrylamide gels (C). Arrows indicate the His-tagged PTCR fusion proteins of different sizes, about 32 kD or 31 kD, which correspond to PTCR(WT) and PTCR(ΔCterm), respectively.

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Expressing

    14) Product Images from "A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification"

    Article Title: A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

    Journal: Virology Journal

    doi: 10.1186/1743-422X-10-262

    Gene and protein schemes with deduced amino acid sequence. (A) The polh-6xhis fragment was amplified and cloned into the commercial vector (I), pFB1 to generate pFB1- polh-6xhis (not shown). We used Bgl II (primer added) and Not I (from the pGem-T® easy vector) restriction sites to clone the modified gene and Nco I (primer added) restriction site to use for virus coat protein fusion (II and III). These vectors were used to construct recombinant viruses, vAc- polh - 6xhis and vAc- GarMbFV-cp - polh - 6xhis by site-specific transposition in E. coli (Bac-to-bac® system, Invitrogen). The virus expressing non-fused GarMbFV-CP was constructed by homologous recombination inside insect cells co-transfected with DNA from pSyn- GarMbFV-cp and vSynVI-gal (see Methods). Deduced amino acid sequence of the (B) non-fused coat protein, GarMbFV-CP (27.9 kDa), (C) Polh-6xHis (29.9 kDa), and (D) Polh-GarMbFV-CP-6xHis recombinant protein (50.0 kDa) are shown.
    Figure Legend Snippet: Gene and protein schemes with deduced amino acid sequence. (A) The polh-6xhis fragment was amplified and cloned into the commercial vector (I), pFB1 to generate pFB1- polh-6xhis (not shown). We used Bgl II (primer added) and Not I (from the pGem-T® easy vector) restriction sites to clone the modified gene and Nco I (primer added) restriction site to use for virus coat protein fusion (II and III). These vectors were used to construct recombinant viruses, vAc- polh - 6xhis and vAc- GarMbFV-cp - polh - 6xhis by site-specific transposition in E. coli (Bac-to-bac® system, Invitrogen). The virus expressing non-fused GarMbFV-CP was constructed by homologous recombination inside insect cells co-transfected with DNA from pSyn- GarMbFV-cp and vSynVI-gal (see Methods). Deduced amino acid sequence of the (B) non-fused coat protein, GarMbFV-CP (27.9 kDa), (C) Polh-6xHis (29.9 kDa), and (D) Polh-GarMbFV-CP-6xHis recombinant protein (50.0 kDa) are shown.

    Techniques Used: Sequencing, Amplification, Clone Assay, Plasmid Preparation, Modification, Construct, Recombinant, BAC Assay, Expressing, Homologous Recombination, Transfection

    15) Product Images from "A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique"

    Article Title: A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique

    Journal: Scientific Reports

    doi: 10.1038/srep45883

    293T- SNCA -3′NL cells having the NanoLuc integration can be used to model deregulated SNCA as seen in sporadic PD. ( a ) 293T- SNCA -3′NL cells treated with 10 μM 5-AzadC for 72 hours which induced a significant increase in the NanoLuc activity as compared to the control. The NanoLuc activity was corroborated by increase in α-SYN transcript as shown in the RT-PCR. ( b ) Methylation status of 23 CpG sites on the SNCA intron1 was determined by bisulfite sequencing. Amplified PCR products were cloned into pGEM-T Easy vector and 10 clones were sequenced. (Left) Comparison of vehicle and 5-AzadC treatment (10 μM, 72 hours) showing unmethylated (open circles) and methylated (closed circles) cytosines for all 10 clones (y-axis) at each of the 23 CpGs in intron1 (x-axis). (Right) Scatter plot showing overall decrease in methylation by 31.7% compared to the control. ( c ) Similarly, 293T- SNCA -3′NL cells treated with dopamine at 100 μM concentration for 48 hours, increased NanoLuc activity significantly. Increase in the NanoLuc activity was confirmed by RT-PCR after dopamine treatment. ( d ) Following HDAC inhibitor (sodium butyrate) treatment at concentrations of 2.5 mM and 5.0 mM for 24 hours, the 293T- SNCA -3′NL cells showed a significant dose dependent increase in the NanoLuc activity. This dose-dependent increase in the NanoLuc activity was also confirmed by RT-PCR following same treatment paradigm. β-actin amplification was used as an internal control for all the PCRs. Error bars show the mean from three technical repeats. p values are given for t-test (5-AzadC, dopamine), one-way ANOVA (Sodium butyrate) where *represents p
    Figure Legend Snippet: 293T- SNCA -3′NL cells having the NanoLuc integration can be used to model deregulated SNCA as seen in sporadic PD. ( a ) 293T- SNCA -3′NL cells treated with 10 μM 5-AzadC for 72 hours which induced a significant increase in the NanoLuc activity as compared to the control. The NanoLuc activity was corroborated by increase in α-SYN transcript as shown in the RT-PCR. ( b ) Methylation status of 23 CpG sites on the SNCA intron1 was determined by bisulfite sequencing. Amplified PCR products were cloned into pGEM-T Easy vector and 10 clones were sequenced. (Left) Comparison of vehicle and 5-AzadC treatment (10 μM, 72 hours) showing unmethylated (open circles) and methylated (closed circles) cytosines for all 10 clones (y-axis) at each of the 23 CpGs in intron1 (x-axis). (Right) Scatter plot showing overall decrease in methylation by 31.7% compared to the control. ( c ) Similarly, 293T- SNCA -3′NL cells treated with dopamine at 100 μM concentration for 48 hours, increased NanoLuc activity significantly. Increase in the NanoLuc activity was confirmed by RT-PCR after dopamine treatment. ( d ) Following HDAC inhibitor (sodium butyrate) treatment at concentrations of 2.5 mM and 5.0 mM for 24 hours, the 293T- SNCA -3′NL cells showed a significant dose dependent increase in the NanoLuc activity. This dose-dependent increase in the NanoLuc activity was also confirmed by RT-PCR following same treatment paradigm. β-actin amplification was used as an internal control for all the PCRs. Error bars show the mean from three technical repeats. p values are given for t-test (5-AzadC, dopamine), one-way ANOVA (Sodium butyrate) where *represents p

    Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Methylation, Methylation Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Concentration Assay

    16) Product Images from "Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses"

    Article Title: Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-2-198

    RT-PCR analysis of Cu/ZnSOD expression in response to cold and UV stresses conditions . RNA was extracted from leaves of plants subjected to stress conditions. Plants cultivated in laboratory at 13°C (not acclimated) and at 4°C (acclimated) (Lane 1 and 2). Plants growing in the Antarctic without UV radiation and under ambient light (UV radiation) (Lane 3 and 4). Controls: genomic DNA of D. antarctica (300 ng) (Lane 5), RNA at 4°C (acclimated) (Lane 6) with primers specific to amplify psbA gene. DNA from E. coli ::pGEM-T Easy-SOD clone (50 ng) (Lane 7) was used as positive control. Deionised water, negative control (Lane 8). MW: Molecular weight marker (Fermentas). Reverse transcription of 1 μg samples of total RNA was carried out, followed by conventional PCR amplification using gene-specific primers. Reaction products (20 μl) were analyzed by gel electrophoresis.
    Figure Legend Snippet: RT-PCR analysis of Cu/ZnSOD expression in response to cold and UV stresses conditions . RNA was extracted from leaves of plants subjected to stress conditions. Plants cultivated in laboratory at 13°C (not acclimated) and at 4°C (acclimated) (Lane 1 and 2). Plants growing in the Antarctic without UV radiation and under ambient light (UV radiation) (Lane 3 and 4). Controls: genomic DNA of D. antarctica (300 ng) (Lane 5), RNA at 4°C (acclimated) (Lane 6) with primers specific to amplify psbA gene. DNA from E. coli ::pGEM-T Easy-SOD clone (50 ng) (Lane 7) was used as positive control. Deionised water, negative control (Lane 8). MW: Molecular weight marker (Fermentas). Reverse transcription of 1 μg samples of total RNA was carried out, followed by conventional PCR amplification using gene-specific primers. Reaction products (20 μl) were analyzed by gel electrophoresis.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Negative Control, Molecular Weight, Marker, Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

    17) Product Images from "The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III"

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

    Journal:

    doi: 10.1128/EC.00239-14

    Effect of tagetitoxin on tRNA-Sec gene transcription. (A) Nuclear run-on RNA was radiolabeled in the presence of tagetitoxin at 160 μM and hybridized to dot blots of double-stranded DNAs (2 μg) cloned into the pGEM-T Easy vector. The genes
    Figure Legend Snippet: Effect of tagetitoxin on tRNA-Sec gene transcription. (A) Nuclear run-on RNA was radiolabeled in the presence of tagetitoxin at 160 μM and hybridized to dot blots of double-stranded DNAs (2 μg) cloned into the pGEM-T Easy vector. The genes

    Techniques Used: Size-exclusion Chromatography, Clone Assay, Plasmid Preparation

    Nuclear run-on analysis of the tRNA-Sec gene. (A) (Top) Labeled nascent RNA from nuclei isolated from L. major promastigotes was hybridized to dot blots of double-stranded DNAs (2 μg) cloned into the pGEM-T Easy vector. (Bottom) A genomic map
    Figure Legend Snippet: Nuclear run-on analysis of the tRNA-Sec gene. (A) (Top) Labeled nascent RNA from nuclei isolated from L. major promastigotes was hybridized to dot blots of double-stranded DNAs (2 μg) cloned into the pGEM-T Easy vector. (Bottom) A genomic map

    Techniques Used: Size-exclusion Chromatography, Labeling, Isolation, Clone Assay, Plasmid Preparation

    18) Product Images from "Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation"

    Article Title: Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation

    Journal: The Journal of clinical endocrinology and metabolism

    doi: 10.1210/jc.2010-0705

    Sequencing of GR exon 6 with subsequent identification of deletion mutation Δ612GR. A–C, GR exon 6 was amplified from DNA isolated from patient blood samples using PCR. The PCR product was subsequently sequenced. The sequence trace is shown for GR (A) and the initial heterozygous Δ612GR exon 6 (B). C, After cloning into the pGEM-T–Easy vector, the sequence trace of the Δ612GR is shown with the deletion mutation highlighted in red . D, Schematic demonstrating that a receptor truncation is caused by introduction of a stop codon at residue 627. E, U2OS cells were transfected with GR or Δ612GR. After treatment with 100 n m dexamethasone for 1 h, cells were lysed in RIPA buffer containing phosphatase and protease inhibitors and analyzed by immunoblotting for GR abundance and phosphorylation on Ser211 (as indicated). Β-actin was used as a loading control. Wild-type and truncated GR is indicated with arrows . Representative images are shown.
    Figure Legend Snippet: Sequencing of GR exon 6 with subsequent identification of deletion mutation Δ612GR. A–C, GR exon 6 was amplified from DNA isolated from patient blood samples using PCR. The PCR product was subsequently sequenced. The sequence trace is shown for GR (A) and the initial heterozygous Δ612GR exon 6 (B). C, After cloning into the pGEM-T–Easy vector, the sequence trace of the Δ612GR is shown with the deletion mutation highlighted in red . D, Schematic demonstrating that a receptor truncation is caused by introduction of a stop codon at residue 627. E, U2OS cells were transfected with GR or Δ612GR. After treatment with 100 n m dexamethasone for 1 h, cells were lysed in RIPA buffer containing phosphatase and protease inhibitors and analyzed by immunoblotting for GR abundance and phosphorylation on Ser211 (as indicated). Β-actin was used as a loading control. Wild-type and truncated GR is indicated with arrows . Representative images are shown.

    Techniques Used: Sequencing, Mutagenesis, Amplification, Isolation, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection

    19) Product Images from "Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing"

    Article Title: Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing

    Journal: Infection, Genetics and Evolution

    doi: 10.1016/j.meegid.2018.07.016

    Internal transcribed spacer 1 (ITS1)–PCR analysis of Sudanese Leishmania donovani strain SD9 (LEM3472). Panel A – Parasites were cloned , and ITS-1–PCR RFLP carried out . Ethidium-bromide stained agarose gel showing the parent strain SD9 (MHOM/SD/97/LEM3472) with an extra band at 239 bp*, and another Sudanese strain SD13 (MHOM/SD/98/LEM3570) displaying typical HaeIII digestion patterns (189, 77 and 51 bp arrows). Panel B - Sequence alignment of ITS1–PCR region of the Sudanese PKDL strain MHOM/SD/97/LEM3472 (SD9). The PCR products were cloned into pGEM-T Easy and colonies picked for sequencing, and DNA sequences were compared using MultAlin . Sequences examined: Genebank – SD9_database (accession number emb|AJ634370.1| ), Parental strain – SD9_parental (this study), and SD9 amplicons subcloned into pGEM –T Easy - colony_5, _2, _14 and _18 (this study). In colony 14 a single nucleotide point mutation (C189A) is present eliminating the HaeIII restriction site (GGCC) resulting in an extra 239 bp product. Additional single nucleotide point mutations, colony 18 (T169C) and colony 2 (G318A) were noted suggesting that different ITS-1 sequences exist in the tandem repeats ( ; ) of the ITS1 regions between the SSU rRNA and the 5.8S genes on chromosome 27.
    Figure Legend Snippet: Internal transcribed spacer 1 (ITS1)–PCR analysis of Sudanese Leishmania donovani strain SD9 (LEM3472). Panel A – Parasites were cloned , and ITS-1–PCR RFLP carried out . Ethidium-bromide stained agarose gel showing the parent strain SD9 (MHOM/SD/97/LEM3472) with an extra band at 239 bp*, and another Sudanese strain SD13 (MHOM/SD/98/LEM3570) displaying typical HaeIII digestion patterns (189, 77 and 51 bp arrows). Panel B - Sequence alignment of ITS1–PCR region of the Sudanese PKDL strain MHOM/SD/97/LEM3472 (SD9). The PCR products were cloned into pGEM-T Easy and colonies picked for sequencing, and DNA sequences were compared using MultAlin . Sequences examined: Genebank – SD9_database (accession number emb|AJ634370.1| ), Parental strain – SD9_parental (this study), and SD9 amplicons subcloned into pGEM –T Easy - colony_5, _2, _14 and _18 (this study). In colony 14 a single nucleotide point mutation (C189A) is present eliminating the HaeIII restriction site (GGCC) resulting in an extra 239 bp product. Additional single nucleotide point mutations, colony 18 (T169C) and colony 2 (G318A) were noted suggesting that different ITS-1 sequences exist in the tandem repeats ( ; ) of the ITS1 regions between the SSU rRNA and the 5.8S genes on chromosome 27.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Staining, Agarose Gel Electrophoresis, Sequencing, Mutagenesis

    20) Product Images from "A protein shuttle system to target RNA into mitochondria"

    Article Title: A protein shuttle system to target RNA into mitochondria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr380

    Editing of atp9 non-edited mRNA upon RNA uptake using the pDHFR protein shuttle into isolated potato mitochondria. ( A ) Schematic representation of the atp9 RNA used for mt import. Larch mt edited tRNA His ( trnH ) precursor RNA (cloverleaf and thin lines) is fused or not to the full-length potato mt atp9 mRNA (open box: coding sequence; bold lines: 5′- and 3′-UTR). The nucleotide sequence is presented in Supplementary Figure S5 . Primers P3 and P4 used for RT–PCR analysis and indicated with arrows (see Supplemental Table S1 for sequences) are located on the sequence corresponding to the pGEM-T Easy region (dashed lines) remaining upon cloning and transcription. ( B ) 32 P-labeled RNAs corresponding to either the non-edited atp9 version of the trnH-atp9 (trnH-atp9une) or of the atp9 (atp9une) were incubated in the absence (−) or presence (+) of pDHFR. Upon incubation, nucleic acids were extracted from mitochondria (Mi) or from mitoplasts (Mtp) post-treated with RNases. T1 and T2, corresponding input RNAs (1 and 2.5 fmol, respectively). ( C ) Similar experiments to those depicted in (B) but with non-radioactive RNAs. Imported RNAs were then analyzed by RT–PCR using the primers P3/P4, specific to exogenous transcripts and in the absence (−) or presence (+) of reverse transcriptase. Negative images of ethidium-stained gels are presented. Fragment sizes were determined with a DNA ladder (M). ( D ) DNA sequence of sites partially edited upon import of atp9 mRNA non-edited version into isolated potato mitochondria. The non-edited version is compared to partially edited sequences (partial editing) obtained upon RNA uptake. Non-edited sites are indicated with gray arrows, the four edited sites with black arrows. Two types (type I and II) of partially edited sequences were found. Among the 15 sequences analyzed, two are of type I, four of type II and the other are non-edited.
    Figure Legend Snippet: Editing of atp9 non-edited mRNA upon RNA uptake using the pDHFR protein shuttle into isolated potato mitochondria. ( A ) Schematic representation of the atp9 RNA used for mt import. Larch mt edited tRNA His ( trnH ) precursor RNA (cloverleaf and thin lines) is fused or not to the full-length potato mt atp9 mRNA (open box: coding sequence; bold lines: 5′- and 3′-UTR). The nucleotide sequence is presented in Supplementary Figure S5 . Primers P3 and P4 used for RT–PCR analysis and indicated with arrows (see Supplemental Table S1 for sequences) are located on the sequence corresponding to the pGEM-T Easy region (dashed lines) remaining upon cloning and transcription. ( B ) 32 P-labeled RNAs corresponding to either the non-edited atp9 version of the trnH-atp9 (trnH-atp9une) or of the atp9 (atp9une) were incubated in the absence (−) or presence (+) of pDHFR. Upon incubation, nucleic acids were extracted from mitochondria (Mi) or from mitoplasts (Mtp) post-treated with RNases. T1 and T2, corresponding input RNAs (1 and 2.5 fmol, respectively). ( C ) Similar experiments to those depicted in (B) but with non-radioactive RNAs. Imported RNAs were then analyzed by RT–PCR using the primers P3/P4, specific to exogenous transcripts and in the absence (−) or presence (+) of reverse transcriptase. Negative images of ethidium-stained gels are presented. Fragment sizes were determined with a DNA ladder (M). ( D ) DNA sequence of sites partially edited upon import of atp9 mRNA non-edited version into isolated potato mitochondria. The non-edited version is compared to partially edited sequences (partial editing) obtained upon RNA uptake. Non-edited sites are indicated with gray arrows, the four edited sites with black arrows. Two types (type I and II) of partially edited sequences were found. Among the 15 sequences analyzed, two are of type I, four of type II and the other are non-edited.

    Techniques Used: Isolation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Labeling, Incubation, Staining

    Related Articles

    Clone Assay:

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    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: Many researchers have designed primers that span introns or intron/exon boundaries for RT-PCR analysis [ ]. .. We cloned the mouse Refs/CLPs standard DNA into the pGEM-T Easy vector (Figure A). .. To increase the distance between the AMCase/Ym1 and Ym2 fragments, we prepared a linear standard DNA containing the pGEM-T Easy sequence that contained BRP-39/Ym2/pGEM-T Easy/AMCase/Pep C/Chit1/GAPDH/β-actin/Ym1 by PCR using the BRP-39-forward (BglII_BRP-39_Fw) and Ym1 reverse (BglII_Ym1_Rv) primers (see Figure A and Additional file : Figure S2 and Table S2).

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
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    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: PCR products were separated and analyzed on 1% agarose gel electrophoresis essentially as previously described earlier (10,11). .. 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83. .. The ligated mix was then transformed into competent E. Coli JM109 cells, by CaCl2 transfection method .

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    Article Snippet: 5 In this paper, a novel antigen as candidate TB vaccine, MPT83 antigen from local strain M. tuberculosis bacteria has been successfully amplified using PCR technique, resulting in 660 bp band. .. The cloning of MPT83 gene into pGEM-T Easy vector results in recombinant plasmid with the size of 3678 bp. .. Based on the order of amino acid encoding protein in recombinant plasmid, pGEM-T Easy-Mpt83 was subcloned and expressed into E coli BL-21 cell in order to produce protein recombinant using 48 kDa molecules as GST-MPT83 fusion protein.

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: The cDNA was prepared with oligonucleotide Lmj06.0200.ME1 (5′-AGAGCGACACCCGTGACTTC), and PCR was performed with primers Lmj06.0200ME2 (5′-ACGGAACCCAGAACGCAGGA) and miniexon (5′-AACGCTATATAAGTATCAGTT). .. The final PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced. .. Pol III transcription termination sites were mapped by poly(A) tailing of total RNA.

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
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    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Paragraph title: Cloning of mTEX101 in TA vector ... Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
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    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
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    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
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    Article Title: Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation
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    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Trans -spliced KMP11 transcripts were also reverse transcribed using the kmp182 oligonucleotide, and PCR performed using the SLTc and kmp21 primers as a control of RNA integrity and proper sample processing. .. The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced. .. Soluble proteins were extracted from parasites in the logarithmic phase of growth.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: An RNA linker derived from the EcoRI-digested pBluescripts KS+ plasmid (Stratagene) was synthesized in vitro as described by Heras et al . ( ) and ligated to the previously generated monophosphate transcripts using T4 RNA ligase (Roche) following the method described by Bruderer ( ). cDNA was synthesized using the CAT1 primer and employed as a template in PCR using SK and CAT90 as primers. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega). .. Selection was performed after IPTG-X-gal induction by blue/white screening.

    Amplification:

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: The ligated fragments were amplified using the forward primer (Quant_mouse_AMCase_Fw) and the reverse primer (Ym2_Rv) (Additional file : Table S2). .. The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega). .. The plasmid containing the cDNA insert was sequenced using the ABI PRISM Big-Dye Terminator v3.1 Cycle Sequencing Kit and the 3130 Genetic Analyzer instrument (Applied Biosystems).

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: PCR led to the amplification of the attC site of the second cassette [attC(2) in ]. .. The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA).

    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83. .. 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83.

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: The second PCR amplification was performed using the first PCR product as the template and primers Tc-tRNASec-GSP2 (5′-AACGGCTGCGAGTCCAAC) and ME23. .. The nested PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced.

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: By utilizing real‐time quantitative PCR (RT‐qPCR), – positive‐sense viral RNA copy numbers present in muscle homogenates or cell lysates were determined. .. A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A). .. For the detection of the negative‐strand of viral RNA, total RNAs were isolated from samples that were not treated with RNAse and then mixed with biotinylated strand‐specific primer EV1 (for the negative‐strand).

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: Washing was carried out at 55°C in 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS. .. DNA fragments from L. major chromosome 6 were amplified by PCR and cloned into the pGEM-T Easy vector. .. LmjF.06.0370 (521 bp) was amplified with oligonucleotides Lm06-0370-5′ (5′-GAAGCGATGGACTGTTCTGG) and Lm06-0370-3′ (5′-CGGTCCTTGCTGCGAATATC), and LmjF.06.0360 (539 bp) was amplified with primers Lm06-0360-5′ (5′-CTCCTCTTCTGGACATTTGCT) and Lm06-0360-3′ (5′-TTCCCTCCACTTGCAACATAG).

    Article Title: Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation
    Article Snippet: The obtained sequencing data were analyzed using Chromas Lite 2.01 ( ). .. The amplified PCR product of exon 6 was cloned into a pGEM-T-Easy vector (Promega, Madison, WI) as described by the manufacturer. .. Purified plasmid was subsequently sequenced using T7 forward or SP6 reverse sequencing primers.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Trans -spliced KMP11 transcripts were also reverse transcribed using the kmp182 oligonucleotide, and PCR performed using the SLTc and kmp21 primers as a control of RNA integrity and proper sample processing. .. The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced. .. Soluble proteins were extracted from parasites in the logarithmic phase of growth.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Paragraph title: RNA ligase-mediated amplification of the cDNA ends ... Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    Synthesized:

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: For the T. cruzi tRNA-Sec gene, the first-strand cDNA was synthesized with primer Nested(dT) (5′-CCTCTGAAGGTTCACGGATCCACATCTAGATTTTTTTTTTTTTTTTTTVN) and two PCR amplifications were performed. .. The nested PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: PCR was performed employing the above-mentioned synthesized cDNA as the DNA template and SLTc and L1Tc152 as primers. .. The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: An RNA linker derived from the EcoRI-digested pBluescripts KS+ plasmid (Stratagene) was synthesized in vitro as described by Heras et al . ( ) and ligated to the previously generated monophosphate transcripts using T4 RNA ligase (Roche) following the method described by Bruderer ( ). cDNA was synthesized using the CAT1 primer and employed as a template in PCR using SK and CAT90 as primers. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    Construct:

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: To construct the receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ), both attI and attC2 were generated by PCR using attI1-LBamH1 and attI1-RHindIII as primers and pC23 as template (see ). .. The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA).

    Article Title: Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data
    Article Snippet: Using oligonucleotides listed in Supplementary Table S4, nine human Alu loci (whose chromosome coordinates are reported in Table ), together with 5′- and 3′-flanking regions, were polymerase chain reaction (PCR)-amplified from buccal cell genomic DNA with GoTaq® DNA polymerase (Promega) and cloned into pGEM®-T Easy vector (Promega). .. Constructs containing targeted mutation of the B box internal control element were obtained by recombinant PCR through the fusion of sub-fragments overlapping in the mutated region, as previously described , followed by cloning into pGEM®-T Easy.

    Real-time Polymerase Chain Reaction:

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: By utilizing real‐time quantitative PCR (RT‐qPCR), – positive‐sense viral RNA copy numbers present in muscle homogenates or cell lysates were determined. .. A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A).

    Incubation:

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA). .. The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA).

    RNA Detection:

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: A primer pair, EV1 (5′‐CGGCCCCTGAATGCGGC‐3′) and EV2 (5′‐CACCGGATGGCCAATCCA‐3′) was used for HEV RNA detection as these primers are specific for sequences located within a highly conserved region of the 5′‐NTR of HEV. .. A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A).

    Western Blot:

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: Paragraph title: Analysis of Muscle Homogenates, Sera, and Cell Lysates by RT‐PCR, RT‐qPCR and Western Blot ... A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A).

    Transformation Assay:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product.

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. Competent cells of E. coli JM109 strain were used for transformation through heat shock method ( ).

    Activated Clotting Time Assay:

    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
    Article Snippet: All the primers synthesis and DNA sequencing in this study were serviced by Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). .. The PCR product of 680 bp from NSE-ORF sequence was obtained using NSE-probe primer-Forward 5′-gga acg gag aac aaa tcc aa-3′ and NSE-probe primer-Reverse 5′-ggg ttg gtc act gtc agg tc-3′ and identified by DNA sequencing and then was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). .. Digoxigenin (Dig)-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase, following the DIG-UTP supplier’s instructions (Roche, Mannheim, Gemany).

    Derivative Assay:

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: An RNA linker derived from the EcoRI-digested pBluescripts KS+ plasmid (Stratagene) was synthesized in vitro as described by Heras et al . ( ) and ligated to the previously generated monophosphate transcripts using T4 RNA ligase (Roche) following the method described by Bruderer ( ). cDNA was synthesized using the CAT1 primer and employed as a template in PCR using SK and CAT90 as primers. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    Transfection:

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Briefly, 15 μg of cytoplasmic RNA from each transfectant were treated with 20U of RQ1 RNAase-free DNAse (Promega) in a final volume of 200 μl. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    Sequencing:

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: Because mammalian introns are more than approximately ~3 kbp long, in general, genomic sequences are difficult to be amplified by PCR compared to target cDNA. .. Because the mouse Refs/CLPs standard DNA was cloned into the pGEM-T Easy vector (approximately ~3 kbp long, Figure A), we prepared a linearized mouse Refs/CLPs standard DNA with pGEM-T Easy sequence between Ym1 and Ym2 using PCR (Figure A and B). .. Additionally, we changed our qPCR protocol (annealing, 30 sec at 55°C; extension, 10 sec at 72°C).

    Article Title: Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data
    Article Snippet: Using oligonucleotides listed in Supplementary Table S4, nine human Alu loci (whose chromosome coordinates are reported in Table ), together with 5′- and 3′-flanking regions, were polymerase chain reaction (PCR)-amplified from buccal cell genomic DNA with GoTaq® DNA polymerase (Promega) and cloned into pGEM®-T Easy vector (Promega). .. Upstream deletion constructs employed forward PCR primers generating amplicons truncated to position −12 (or −15, in the case of Alu Sx_chr10) with respect to Alu 5′ end.

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: The numbering system is based on the published sequence for CVB1 (GeneBank Accession No. M16560.1). .. A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A).

    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
    Article Snippet: All the primers synthesis and DNA sequencing in this study were serviced by Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). .. The PCR product of 680 bp from NSE-ORF sequence was obtained using NSE-probe primer-Forward 5′-gga acg gag aac aaa tcc aa-3′ and NSE-probe primer-Reverse 5′-ggg ttg gtc act gtc agg tc-3′ and identified by DNA sequencing and then was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). .. Digoxigenin (Dig)-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase, following the DIG-UTP supplier’s instructions (Roche, Mannheim, Gemany).

    Article Title: Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation
    Article Snippet: Paragraph title: Sequencing and cloning ... The amplified PCR product of exon 6 was cloned into a pGEM-T-Easy vector (Promega, Madison, WI) as described by the manufacturer.

    Ligation:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. Competent cells of E. coli JM109 strain were used for transformation through heat shock method ( ).

    Northern Blot:

    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
    Article Snippet: Paragraph title: 3.3. Northern Blot Analysis ... The PCR product of 680 bp from NSE-ORF sequence was obtained using NSE-probe primer-Forward 5′-gga acg gag aac aaa tcc aa-3′ and NSE-probe primer-Reverse 5′-ggg ttg gtc act gtc agg tc-3′ and identified by DNA sequencing and then was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA).

    Cell Culture:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Competent cells of E. coli JM109 strain were used for transformation through heat shock method ( ).

    Generated:

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: To construct the receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ), both attI and attC2 were generated by PCR using attI1-LBamH1 and attI1-RHindIII as primers and pC23 as template (see ). .. The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA).

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: An RNA linker derived from the EcoRI-digested pBluescripts KS+ plasmid (Stratagene) was synthesized in vitro as described by Heras et al . ( ) and ligated to the previously generated monophosphate transcripts using T4 RNA ligase (Roche) following the method described by Bruderer ( ). cDNA was synthesized using the CAT1 primer and employed as a template in PCR using SK and CAT90 as primers. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    DNA Sequencing:

    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
    Article Snippet: All the primers synthesis and DNA sequencing in this study were serviced by Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). .. The PCR product of 680 bp from NSE-ORF sequence was obtained using NSE-probe primer-Forward 5′-gga acg gag aac aaa tcc aa-3′ and NSE-probe primer-Reverse 5′-ggg ttg gtc act gtc agg tc-3′ and identified by DNA sequencing and then was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). .. Digoxigenin (Dig)-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase, following the DIG-UTP supplier’s instructions (Roche, Mannheim, Gemany).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: Paragraph title: RT-PCR assays. ... The final PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced.

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: By utilizing real‐time quantitative PCR (RT‐qPCR), – positive‐sense viral RNA copy numbers present in muscle homogenates or cell lysates were determined. .. A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A). .. For the detection of the negative‐strand of viral RNA, total RNAs were isolated from samples that were not treated with RNAse and then mixed with biotinylated strand‐specific primer EV1 (for the negative‐strand).

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Paragraph title: RT-PCR ... The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced.

    Recombinant:

    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: PCR products were separated and analyzed on 1% agarose gel electrophoresis essentially as previously described earlier (10,11). .. 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83. .. The ligated mix was then transformed into competent E. Coli JM109 cells, by CaCl2 transfection method .

    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: 5 In this paper, a novel antigen as candidate TB vaccine, MPT83 antigen from local strain M. tuberculosis bacteria has been successfully amplified using PCR technique, resulting in 660 bp band. .. The cloning of MPT83 gene into pGEM-T Easy vector results in recombinant plasmid with the size of 3678 bp. .. Based on the order of amino acid encoding protein in recombinant plasmid, pGEM-T Easy-Mpt83 was subcloned and expressed into E coli BL-21 cell in order to produce protein recombinant using 48 kDa molecules as GST-MPT83 fusion protein.

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. White colonies were screened by 25 cycles of colony PCR under the aforementioned conditions.

    Isolation:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. White colonies were screened by 25 cycles of colony PCR under the aforementioned conditions.

    Labeling:

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega). .. One membrane was hybridized in situ with the CAT coding fragment obtained by PCR using CAT-32f and CAT43r as primers and the pMSGCAT vector as a template.

    Purification:

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: The ligated fragments were amplified using the forward primer (Quant_mouse_AMCase_Fw) and the reverse primer (Ym2_Rv) (Additional file : Table S2). .. The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega). .. The plasmid containing the cDNA insert was sequenced using the ABI PRISM Big-Dye Terminator v3.1 Cycle Sequencing Kit and the 3130 Genetic Analyzer instrument (Applied Biosystems).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The 750bp band was purified using QIA quick Gel Extraction Kit (QIAGEN, Germantown, MD, USA). .. The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. Competent cells of E. coli JM109 strain were used for transformation through heat shock method ( ).

    Article Title: Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation
    Article Snippet: A QIAquick Spin PCR Purification Kit (QIAGEN, Crawley, UK) was used to purify PCR product according to the manufacturer’s instructions. .. The amplified PCR product of exon 6 was cloned into a pGEM-T-Easy vector (Promega, Madison, WI) as described by the manufacturer.

    Polymerase Chain Reaction:

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: The ligated fragments were amplified using the forward primer (Quant_mouse_AMCase_Fw) and the reverse primer (Ym2_Rv) (Additional file : Table S2). .. The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega). .. The plasmid containing the cDNA insert was sequenced using the ABI PRISM Big-Dye Terminator v3.1 Cycle Sequencing Kit and the 3130 Genetic Analyzer instrument (Applied Biosystems).

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: The mouse Refs/CLPs standard DNA (1,597 nucleotides; see Figure A and Additional file : Figure S1) was prepared by PCR reamplification from the plasmid DNA using the same primers; the PCR product was purified as described above and was thereafter used as the standard DNA. .. The mouse Refs/CLPs standard DNA with pGEM-T Easy vector (4,629 nucleotides; see Figure B and Additional file : Figure S2) was prepared by PCR from the plasmid DNA using the forward primer (BglII_BRP-39_Fw) and the reverse primer (BglII_Ym1_Rv). .. The amplified DNA was purified and subsequently used as the mouse Refs/CLPs standard DNA with pGEM-T Easy.

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: Because mammalian introns are more than approximately ~3 kbp long, in general, genomic sequences are difficult to be amplified by PCR compared to target cDNA. .. Because the mouse Refs/CLPs standard DNA was cloned into the pGEM-T Easy vector (approximately ~3 kbp long, Figure A), we prepared a linearized mouse Refs/CLPs standard DNA with pGEM-T Easy sequence between Ym1 and Ym2 using PCR (Figure A and B). .. Additionally, we changed our qPCR protocol (annealing, 30 sec at 55°C; extension, 10 sec at 72°C).

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: PCR led to the amplification of the attC site of the second cassette [attC(2) in ]. .. The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA).

    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: PCR products were separated and analyzed on 1% agarose gel electrophoresis essentially as previously described earlier (10,11). .. 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83. .. The ligated mix was then transformed into competent E. Coli JM109 cells, by CaCl2 transfection method .

    Article Title: Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data
    Article Snippet: P -values for the association of TFs to expression-positive Alu s were calculated using the Fisher's exact test against total Alu s. Lists of TF-Alu interactions are reported in Supplementary Table S3. .. Using oligonucleotides listed in Supplementary Table S4, nine human Alu loci (whose chromosome coordinates are reported in Table ), together with 5′- and 3′-flanking regions, were polymerase chain reaction (PCR)-amplified from buccal cell genomic DNA with GoTaq® DNA polymerase (Promega) and cloned into pGEM®-T Easy vector (Promega). .. Constructs containing targeted mutation of the B box internal control element were obtained by recombinant PCR through the fusion of sub-fragments overlapping in the mutated region, as previously described , followed by cloning into pGEM®-T Easy.

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: The cDNA was prepared with oligonucleotide Lmj06.0200.ME1 (5′-AGAGCGACACCCGTGACTTC), and PCR was performed with primers Lmj06.0200ME2 (5′-ACGGAACCCAGAACGCAGGA) and miniexon (5′-AACGCTATATAAGTATCAGTT). .. The final PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced. .. Pol III transcription termination sites were mapped by poly(A) tailing of total RNA.

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: The second PCR amplification was performed using the first PCR product as the template and primers Tc-tRNASec-GSP2 (5′-AACGGCTGCGAGTCCAAC) and ME23. .. The nested PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced.

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The 750bp band was purified using QIA quick Gel Extraction Kit (QIAGEN, Germantown, MD, USA). .. The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Finally, the membranes were visualized using an ECL system (GE Healthcare, Biotech, Bucking-hamshire, UK). .. After cloning mTEX101 fragments in pGEM-T Easy Vector, several white colonies with probable target fragment inclusion were screened by colony PCR ( ). .. A right-sized PCR product and a pET-28a(+) vector were cut by NotI and EcoRI restriction enzymes to obtain the required fragments for the next step ( and ).

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: By utilizing real‐time quantitative PCR (RT‐qPCR), – positive‐sense viral RNA copy numbers present in muscle homogenates or cell lysates were determined. .. A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A). .. For the detection of the negative‐strand of viral RNA, total RNAs were isolated from samples that were not treated with RNAse and then mixed with biotinylated strand‐specific primer EV1 (for the negative‐strand).

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: Washing was carried out at 55°C in 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS. .. DNA fragments from L. major chromosome 6 were amplified by PCR and cloned into the pGEM-T Easy vector. .. LmjF.06.0370 (521 bp) was amplified with oligonucleotides Lm06-0370-5′ (5′-GAAGCGATGGACTGTTCTGG) and Lm06-0370-3′ (5′-CGGTCCTTGCTGCGAATATC), and LmjF.06.0360 (539 bp) was amplified with primers Lm06-0360-5′ (5′-CTCCTCTTCTGGACATTTGCT) and Lm06-0360-3′ (5′-TTCCCTCCACTTGCAACATAG).

    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
    Article Snippet: All the primers synthesis and DNA sequencing in this study were serviced by Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). .. The PCR product of 680 bp from NSE-ORF sequence was obtained using NSE-probe primer-Forward 5′-gga acg gag aac aaa tcc aa-3′ and NSE-probe primer-Reverse 5′-ggg ttg gtc act gtc agg tc-3′ and identified by DNA sequencing and then was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). .. Digoxigenin (Dig)-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase, following the DIG-UTP supplier’s instructions (Roche, Mannheim, Gemany).

    Article Title: Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation
    Article Snippet: The obtained sequencing data were analyzed using Chromas Lite 2.01 ( ). .. The amplified PCR product of exon 6 was cloned into a pGEM-T-Easy vector (Promega, Madison, WI) as described by the manufacturer. .. Purified plasmid was subsequently sequenced using T7 forward or SP6 reverse sequencing primers.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Trans -spliced KMP11 transcripts were also reverse transcribed using the kmp182 oligonucleotide, and PCR performed using the SLTc and kmp21 primers as a control of RNA integrity and proper sample processing. .. The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced. .. Soluble proteins were extracted from parasites in the logarithmic phase of growth.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: An RNA linker derived from the EcoRI-digested pBluescripts KS+ plasmid (Stratagene) was synthesized in vitro as described by Heras et al . ( ) and ligated to the previously generated monophosphate transcripts using T4 RNA ligase (Roche) following the method described by Bruderer ( ). cDNA was synthesized using the CAT1 primer and employed as a template in PCR using SK and CAT90 as primers. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega). .. Selection was performed after IPTG-X-gal induction by blue/white screening.

    Selection:

    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83. .. 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83.

    Quantitative RT-PCR:

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: Paragraph title: Analysis of Muscle Homogenates, Sera, and Cell Lysates by RT‐PCR, RT‐qPCR and Western Blot ... A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A).

    Nested PCR:

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: The second PCR amplification was performed using the first PCR product as the template and primers Tc-tRNASec-GSP2 (5′-AACGGCTGCGAGTCCAAC) and ME23. .. The nested PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced. .. To map polyadenylation sites for the tRNA-Sec genes from L. major and T. cruzi and for the LmjF.06.0210 gene from L. major , reverse transcription-PCR (RT-PCR) experiments were performed using cDNA prepared with oligonucleotide Nested(dT).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega). .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    Plasmid Preparation:

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: The ligated fragments were amplified using the forward primer (Quant_mouse_AMCase_Fw) and the reverse primer (Ym2_Rv) (Additional file : Table S2). .. The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega). .. The plasmid containing the cDNA insert was sequenced using the ABI PRISM Big-Dye Terminator v3.1 Cycle Sequencing Kit and the 3130 Genetic Analyzer instrument (Applied Biosystems).

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: The mouse Refs/CLPs standard DNA (1,597 nucleotides; see Figure A and Additional file : Figure S1) was prepared by PCR reamplification from the plasmid DNA using the same primers; the PCR product was purified as described above and was thereafter used as the standard DNA. .. The mouse Refs/CLPs standard DNA with pGEM-T Easy vector (4,629 nucleotides; see Figure B and Additional file : Figure S2) was prepared by PCR from the plasmid DNA using the forward primer (BglII_BRP-39_Fw) and the reverse primer (BglII_Ym1_Rv). .. The amplified DNA was purified and subsequently used as the mouse Refs/CLPs standard DNA with pGEM-T Easy.

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: Many researchers have designed primers that span introns or intron/exon boundaries for RT-PCR analysis [ ]. .. We cloned the mouse Refs/CLPs standard DNA into the pGEM-T Easy vector (Figure A). .. To increase the distance between the AMCase/Ym1 and Ym2 fragments, we prepared a linear standard DNA containing the pGEM-T Easy sequence that contained BRP-39/Ym2/pGEM-T Easy/AMCase/Pep C/Chit1/GAPDH/β-actin/Ym1 by PCR using the BRP-39-forward (BglII_BRP-39_Fw) and Ym1 reverse (BglII_Ym1_Rv) primers (see Figure A and Additional file : Figure S2 and Table S2).

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: Using these reference genes, we evaluated gene expression of three CLPs and chitinases in mouse tissues. .. We ligated the CLPs standard DNA with the five reference genes cDNA in a one-to-one ratio and then cloned this DNA fragment into the pGEM-T Easy vector. .. The 1,597-nucleotide-long DNA contained five reference genes (Refs) and three CLPs cDNA fragments that spanned the PCR target regions and 9-146 nucleotides of the flanking regions and contained several restriction sites (Figure A and Additional file : Figure S1).

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung
    Article Snippet: Because mammalian introns are more than approximately ~3 kbp long, in general, genomic sequences are difficult to be amplified by PCR compared to target cDNA. .. Because the mouse Refs/CLPs standard DNA was cloned into the pGEM-T Easy vector (approximately ~3 kbp long, Figure A), we prepared a linearized mouse Refs/CLPs standard DNA with pGEM-T Easy sequence between Ym1 and Ym2 using PCR (Figure A and B). .. Additionally, we changed our qPCR protocol (annealing, 30 sec at 55°C; extension, 10 sec at 72°C).

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: PCR led to the amplification of the attC site of the second cassette [attC(2) in ]. .. The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA). .. To determine its DNA binding activity, purified IntI1(his)6 (1 to 10 pmoles) was incubated either with the 5′ radiolabeled double-stranded or single-stranded attI1 fragment or with the 5′ radiolabeled double-stranded or single-stranded attC fragment for 20 min at 4°C in a total volume of 20 µl.

    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: PCR products were separated and analyzed on 1% agarose gel electrophoresis essentially as previously described earlier (10,11). .. 2.3 The PCR product of antigen MPT83 gene was eluted from the gel and cloned into pGEM-T Easy vector (Promega, USA) as per manufacturer’s instructions to yield recombinant plasmid pGEM-T Easy-Mpt83. .. The ligated mix was then transformed into competent E. Coli JM109 cells, by CaCl2 transfection method .

    Article Title: Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data
    Article Snippet: P -values for the association of TFs to expression-positive Alu s were calculated using the Fisher's exact test against total Alu s. Lists of TF-Alu interactions are reported in Supplementary Table S3. .. Using oligonucleotides listed in Supplementary Table S4, nine human Alu loci (whose chromosome coordinates are reported in Table ), together with 5′- and 3′-flanking regions, were polymerase chain reaction (PCR)-amplified from buccal cell genomic DNA with GoTaq® DNA polymerase (Promega) and cloned into pGEM®-T Easy vector (Promega). .. Constructs containing targeted mutation of the B box internal control element were obtained by recombinant PCR through the fusion of sub-fragments overlapping in the mutated region, as previously described , followed by cloning into pGEM®-T Easy.

    Article Title: Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study
    Article Snippet: 5 In this paper, a novel antigen as candidate TB vaccine, MPT83 antigen from local strain M. tuberculosis bacteria has been successfully amplified using PCR technique, resulting in 660 bp band. .. The cloning of MPT83 gene into pGEM-T Easy vector results in recombinant plasmid with the size of 3678 bp. .. Based on the order of amino acid encoding protein in recombinant plasmid, pGEM-T Easy-Mpt83 was subcloned and expressed into E coli BL-21 cell in order to produce protein recombinant using 48 kDa molecules as GST-MPT83 fusion protein.

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: The cDNA was prepared with oligonucleotide Lmj06.0200.ME1 (5′-AGAGCGACACCCGTGACTTC), and PCR was performed with primers Lmj06.0200ME2 (5′-ACGGAACCCAGAACGCAGGA) and miniexon (5′-AACGCTATATAAGTATCAGTT). .. The final PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced. .. Pol III transcription termination sites were mapped by poly(A) tailing of total RNA.

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: The second PCR amplification was performed using the first PCR product as the template and primers Tc-tRNASec-GSP2 (5′-AACGGCTGCGAGTCCAAC) and ME23. .. The nested PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced. .. To map polyadenylation sites for the tRNA-Sec genes from L. major and T. cruzi and for the LmjF.06.0210 gene from L. major , reverse transcription-PCR (RT-PCR) experiments were performed using cDNA prepared with oligonucleotide Nested(dT).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The 750bp band was purified using QIA quick Gel Extraction Kit (QIAGEN, Germantown, MD, USA). .. The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The pGEM-T Easy Vector (Promega, Madison, WI, USA) was used for the cloning of the purified PCR product. .. Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. Competent cells of E. coli JM109 strain were used for transformation through heat shock method ( ).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Finally, the membranes were visualized using an ECL system (GE Healthcare, Biotech, Bucking-hamshire, UK). .. After cloning mTEX101 fragments in pGEM-T Easy Vector, several white colonies with probable target fragment inclusion were screened by colony PCR ( ). .. A right-sized PCR product and a pET-28a(+) vector were cut by NotI and EcoRI restriction enzymes to obtain the required fragments for the next step ( and ).

    Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease
    Article Snippet: By utilizing real‐time quantitative PCR (RT‐qPCR), – positive‐sense viral RNA copy numbers present in muscle homogenates or cell lysates were determined. .. A standard curve for the determination of viral RNA copy numbers was developed with a PCR‐fragment (193 bp long), amplified by RT‐PCR using a primer pair EV1/EV2, and cloned into a pGEM‐T easy vector (Promega) (Figure S3A). .. For the detection of the negative‐strand of viral RNA, total RNAs were isolated from samples that were not treated with RNAse and then mixed with biotinylated strand‐specific primer EV1 (for the negative‐strand).

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: Washing was carried out at 55°C in 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS. .. DNA fragments from L. major chromosome 6 were amplified by PCR and cloned into the pGEM-T Easy vector. .. LmjF.06.0370 (521 bp) was amplified with oligonucleotides Lm06-0370-5′ (5′-GAAGCGATGGACTGTTCTGG) and Lm06-0370-3′ (5′-CGGTCCTTGCTGCGAATATC), and LmjF.06.0360 (539 bp) was amplified with primers Lm06-0360-5′ (5′-CTCCTCTTCTGGACATTTGCT) and Lm06-0360-3′ (5′-TTCCCTCCACTTGCAACATAG).

    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
    Article Snippet: All the primers synthesis and DNA sequencing in this study were serviced by Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). .. The PCR product of 680 bp from NSE-ORF sequence was obtained using NSE-probe primer-Forward 5′-gga acg gag aac aaa tcc aa-3′ and NSE-probe primer-Reverse 5′-ggg ttg gtc act gtc agg tc-3′ and identified by DNA sequencing and then was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). .. Digoxigenin (Dig)-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase, following the DIG-UTP supplier’s instructions (Roche, Mannheim, Gemany).

    Article Title: Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation
    Article Snippet: The obtained sequencing data were analyzed using Chromas Lite 2.01 ( ). .. The amplified PCR product of exon 6 was cloned into a pGEM-T-Easy vector (Promega, Madison, WI) as described by the manufacturer. .. Purified plasmid was subsequently sequenced using T7 forward or SP6 reverse sequencing primers.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Trans -spliced KMP11 transcripts were also reverse transcribed using the kmp182 oligonucleotide, and PCR performed using the SLTc and kmp21 primers as a control of RNA integrity and proper sample processing. .. The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced. .. Soluble proteins were extracted from parasites in the logarithmic phase of growth.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: An RNA linker derived from the EcoRI-digested pBluescripts KS+ plasmid (Stratagene) was synthesized in vitro as described by Heras et al . ( ) and ligated to the previously generated monophosphate transcripts using T4 RNA ligase (Roche) following the method described by Bruderer ( ). cDNA was synthesized using the CAT1 primer and employed as a template in PCR using SK and CAT90 as primers. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega). .. Selection was performed after IPTG-X-gal induction by blue/white screening.

    In Vitro:

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: Paragraph title: In vitro strand transfer assays ... The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA).

    Article Title: Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data
    Article Snippet: Using oligonucleotides listed in Supplementary Table S4, nine human Alu loci (whose chromosome coordinates are reported in Table ), together with 5′- and 3′-flanking regions, were polymerase chain reaction (PCR)-amplified from buccal cell genomic DNA with GoTaq® DNA polymerase (Promega) and cloned into pGEM®-T Easy vector (Promega). .. Upstream deletion constructs employed forward PCR primers generating amplicons truncated to position −12 (or −15, in the case of Alu Sx_chr10) with respect to Alu 5′ end.

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: An RNA linker derived from the EcoRI-digested pBluescripts KS+ plasmid (Stratagene) was synthesized in vitro as described by Heras et al . ( ) and ligated to the previously generated monophosphate transcripts using T4 RNA ligase (Roche) following the method described by Bruderer ( ). cDNA was synthesized using the CAT1 primer and employed as a template in PCR using SK and CAT90 as primers. .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    Negative Control:

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced. .. The PCR reaction conditions were 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. All the amplified products were cloned into the pGEM T®-easy vector (Promega) and sequenced.

    Binding Assay:

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA). .. The IntI1-DNA complexes were then loaded on vertical 1% agarose gel and run at 50 V for 4 hours at 4°C.

    Agarose Gel Electrophoresis:

    Article Title: A New In Vitro Strand Transfer Assay for Monitoring Bacterial Class 1 Integron Recombinase IntI1 Activity
    Article Snippet: The receptor plasmids pGEM-T-attI1 (called pattI1 ) and pGEM-T-attC (called pattC ) were obtained by cloning the corresponding fragments into pGEM-T easy vector (PROMEGA). .. To determine its DNA binding activity, purified IntI1(his)6 (1 to 10 pmoles) was incubated either with the 5′ radiolabeled double-stranded or single-stranded attI1 fragment or with the 5′ radiolabeled double-stranded or single-stranded attC fragment for 20 min at 4°C in a total volume of 20 µl.

    Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
    Article Snippet: The PCR product of 680 bp from NSE-ORF sequence was obtained using NSE-probe primer-Forward 5′-gga acg gag aac aaa tcc aa-3′ and NSE-probe primer-Reverse 5′-ggg ttg gtc act gtc agg tc-3′ and identified by DNA sequencing and then was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). .. Digoxigenin (Dig)-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase, following the DIG-UTP supplier’s instructions (Roche, Mannheim, Gemany).

    In Situ:

    Article Title: The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts
    Article Snippet: Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega). .. Crude PCR products were directly cloned into the pGEM-T®easy vector (Promega).

    Produced:

    Article Title: The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III
    Article Snippet: For the tRNA-Pro gene ( LmjF.24.TRNAPRO.01 ) from L. major , the cDNA was produced with primer Lmc24-ProGSP1 (5′-GGGCCGCTAGGGGAATTGAA) and the PCR amplifications were performed with primer Lmc24-ProGSP2 (5′-TGACCTCCCGCACCCGAAG) and AAP. .. The nested PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced.

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    Promega pgem t easy vector
    Binding between ApoH and Hepatitis C virus-related particles. Sera from both five <t>HCV-positive</t> patients (HCV-pos) and three HCV-negative healthy controls (HC) were assayed for viral detection. (A) <t>RT-PCR</t> detection of HCV from 20-fold diluted sera, after the viral capture by ApoH-coated beads. Lanes M, C+ and C- respectively correspond to the DNA molecular mass marker (1 Kb Plus DNA Ladder), the positive control (pGEM-T easy with an HCV/PCR-insert) and the negative extraction control. (B) ApoH-ELISA immunoassay to detect HCV-related particles binding from serially-diluted sera. Aliquots from the same sera as used for RT-PCR were 10, 50, 500, and 5,000-fold diluted and subsequently detected with the anti-HCV/E2 MAb.
    Pgem T Easy Vector, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Binding between ApoH and Hepatitis C virus-related particles. Sera from both five HCV-positive patients (HCV-pos) and three HCV-negative healthy controls (HC) were assayed for viral detection. (A) RT-PCR detection of HCV from 20-fold diluted sera, after the viral capture by ApoH-coated beads. Lanes M, C+ and C- respectively correspond to the DNA molecular mass marker (1 Kb Plus DNA Ladder), the positive control (pGEM-T easy with an HCV/PCR-insert) and the negative extraction control. (B) ApoH-ELISA immunoassay to detect HCV-related particles binding from serially-diluted sera. Aliquots from the same sera as used for RT-PCR were 10, 50, 500, and 5,000-fold diluted and subsequently detected with the anti-HCV/E2 MAb.

    Journal: PLoS ONE

    Article Title: Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus

    doi: 10.1371/journal.pone.0140900

    Figure Lengend Snippet: Binding between ApoH and Hepatitis C virus-related particles. Sera from both five HCV-positive patients (HCV-pos) and three HCV-negative healthy controls (HC) were assayed for viral detection. (A) RT-PCR detection of HCV from 20-fold diluted sera, after the viral capture by ApoH-coated beads. Lanes M, C+ and C- respectively correspond to the DNA molecular mass marker (1 Kb Plus DNA Ladder), the positive control (pGEM-T easy with an HCV/PCR-insert) and the negative extraction control. (B) ApoH-ELISA immunoassay to detect HCV-related particles binding from serially-diluted sera. Aliquots from the same sera as used for RT-PCR were 10, 50, 500, and 5,000-fold diluted and subsequently detected with the anti-HCV/E2 MAb.

    Article Snippet: An HCV plasmid (pGEM-T easy, Promega, with an HCV/PCR-insert) was used as template to make the standard curve and compute HCV copies.

    Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Statistical comparisons and correlations between positive qRT-PCR HCV detection with and without the preparative ApoH-capture HCV step. (A) Scatter-plot comparing the HCV loads using the real-time RT-PCR COBAS® TaqMan® HCV Test, v2.0 alone versus the open home-made HCV real-time RT-PCR assay after the ApoH-HCV capture. Forty-eight clinical samples from HCV-infected patients were tested. The solid line represents the regression curve. (B) Bland-Altman plot depicts the correlation of the viral load figures from COBAS HCV real-time RT-PCR assay alone and the figures resulting from the HCV real-time RT-PCR assay associated with the ApoH sample pretreatment (n = 48). The graph displays a scatter diagram of the differences plotted against the averages of the two measurements. Horizontal lines are drawn at the mean difference and at the mean difference ± 1.96 times the standard deviation, SD, of the differences (95% limits of confidence).

    Journal: PLoS ONE

    Article Title: Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus

    doi: 10.1371/journal.pone.0140900

    Figure Lengend Snippet: Statistical comparisons and correlations between positive qRT-PCR HCV detection with and without the preparative ApoH-capture HCV step. (A) Scatter-plot comparing the HCV loads using the real-time RT-PCR COBAS® TaqMan® HCV Test, v2.0 alone versus the open home-made HCV real-time RT-PCR assay after the ApoH-HCV capture. Forty-eight clinical samples from HCV-infected patients were tested. The solid line represents the regression curve. (B) Bland-Altman plot depicts the correlation of the viral load figures from COBAS HCV real-time RT-PCR assay alone and the figures resulting from the HCV real-time RT-PCR assay associated with the ApoH sample pretreatment (n = 48). The graph displays a scatter diagram of the differences plotted against the averages of the two measurements. Horizontal lines are drawn at the mean difference and at the mean difference ± 1.96 times the standard deviation, SD, of the differences (95% limits of confidence).

    Article Snippet: An HCV plasmid (pGEM-T easy, Promega, with an HCV/PCR-insert) was used as template to make the standard curve and compute HCV copies.

    Techniques: Quantitative RT-PCR, Infection, Standard Deviation

    ApoH captures HCV/RNA-containing particles, from low-density and high-density plasma fractions. A pool of 10 HCV/RNA-positive untreated patients was separated into three fractions, respectively corresponding to the floating densities of VLDL, LDL and HDL. One hundred μL of VLDL, 10 μL of LDL and 1 μL of HDL were respectively incubated with 10 μL of ApoH-coated nanomagnetic beads and HCV/RNA was submitted to a home-made HCV RT-PCR. Lanes M, C+ and C- respectively correspond to the DNA molecular mass marker (1 Kb Plus DNA Ladder), the positive control (plasmid pGEM-T easy with an HCV/PCR-insert) and negative PCR control.

    Journal: PLoS ONE

    Article Title: Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus

    doi: 10.1371/journal.pone.0140900

    Figure Lengend Snippet: ApoH captures HCV/RNA-containing particles, from low-density and high-density plasma fractions. A pool of 10 HCV/RNA-positive untreated patients was separated into three fractions, respectively corresponding to the floating densities of VLDL, LDL and HDL. One hundred μL of VLDL, 10 μL of LDL and 1 μL of HDL were respectively incubated with 10 μL of ApoH-coated nanomagnetic beads and HCV/RNA was submitted to a home-made HCV RT-PCR. Lanes M, C+ and C- respectively correspond to the DNA molecular mass marker (1 Kb Plus DNA Ladder), the positive control (plasmid pGEM-T easy with an HCV/PCR-insert) and negative PCR control.

    Article Snippet: An HCV plasmid (pGEM-T easy, Promega, with an HCV/PCR-insert) was used as template to make the standard curve and compute HCV copies.

    Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control, Plasmid Preparation, Polymerase Chain Reaction

    Enhanced detection of HCV following HCV capture with ApoH-coated beads. (A) In the presence or in the absence of the ApoH-coated beads, HCV/RNA from 10-fold serial dilutions of a single HCV-infected patient serum was detected in gel after HCV RT-PCR amplification. HCV-negative healthy control serum (HC). (B) List of HCV sequences from 11 HCV-seropositive patients (samples #: 32, 36, 148, 169, 171, 151, 459, 450, 465, 448, 445) with prior negative COBAS HCV RT-PCR, but tested as HCV-positive with the two-step detection method consecutively including the ApoH-sample pretreatment and the home-made HCV RT-PCR.

    Journal: PLoS ONE

    Article Title: Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus

    doi: 10.1371/journal.pone.0140900

    Figure Lengend Snippet: Enhanced detection of HCV following HCV capture with ApoH-coated beads. (A) In the presence or in the absence of the ApoH-coated beads, HCV/RNA from 10-fold serial dilutions of a single HCV-infected patient serum was detected in gel after HCV RT-PCR amplification. HCV-negative healthy control serum (HC). (B) List of HCV sequences from 11 HCV-seropositive patients (samples #: 32, 36, 148, 169, 171, 151, 459, 450, 465, 448, 445) with prior negative COBAS HCV RT-PCR, but tested as HCV-positive with the two-step detection method consecutively including the ApoH-sample pretreatment and the home-made HCV RT-PCR.

    Article Snippet: An HCV plasmid (pGEM-T easy, Promega, with an HCV/PCR-insert) was used as template to make the standard curve and compute HCV copies.

    Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Amplification

    Binding specificity of ApoH and HCV. (A) One hundred microliters of sera from five HCV-positive patients were incubated with either the α1-acid glycoprotein- or the ApoH- coated plates. The HCV-related antigens were revealed with the anti-HCV/E2 MAb. The bars represent the corresponding means with SD. (B) Sera from both a single healthy control (HC) and one HCV-pos were either incubated with α1-acid glycoprotein or with the ApoH-coated magnetic beads and HCV/RNA was subsequently detected by RT-PCR. M, C+ and C- respectively correspond to the DNA molecular mass marker (1 Kb Plus DNA Ladder), the positive control (plasmid pGEM-T easy with an HCV/PCR-insert) and the negative PCR control. (C) The inhibition of the interaction between ApoH and HCV, previously shown, was assessed either by using the anti-ApoH, 8C3, MAb, or the anti-thyroglobulin, TG2, irrelevant MAb. Increasing concentrations of either 8C3 (solid line) or TG2 (hatched line) were used for the pre-incubation of the ApoH-coated plate with a 50-fold diluted HCV-positive serum.

    Journal: PLoS ONE

    Article Title: Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus

    doi: 10.1371/journal.pone.0140900

    Figure Lengend Snippet: Binding specificity of ApoH and HCV. (A) One hundred microliters of sera from five HCV-positive patients were incubated with either the α1-acid glycoprotein- or the ApoH- coated plates. The HCV-related antigens were revealed with the anti-HCV/E2 MAb. The bars represent the corresponding means with SD. (B) Sera from both a single healthy control (HC) and one HCV-pos were either incubated with α1-acid glycoprotein or with the ApoH-coated magnetic beads and HCV/RNA was subsequently detected by RT-PCR. M, C+ and C- respectively correspond to the DNA molecular mass marker (1 Kb Plus DNA Ladder), the positive control (plasmid pGEM-T easy with an HCV/PCR-insert) and the negative PCR control. (C) The inhibition of the interaction between ApoH and HCV, previously shown, was assessed either by using the anti-ApoH, 8C3, MAb, or the anti-thyroglobulin, TG2, irrelevant MAb. Increasing concentrations of either 8C3 (solid line) or TG2 (hatched line) were used for the pre-incubation of the ApoH-coated plate with a 50-fold diluted HCV-positive serum.

    Article Snippet: An HCV plasmid (pGEM-T easy, Promega, with an HCV/PCR-insert) was used as template to make the standard curve and compute HCV copies.

    Techniques: Binding Assay, Incubation, Magnetic Beads, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control, Plasmid Preparation, Polymerase Chain Reaction, Inhibition

    ApoH binds the RNA-containing HCV-related particles. (A) Isolation of HCV-related particles by ultracentrifugation. After the ultracentrifugation at 436,000 x g for 4 h at 4°C of pooled sera from HCV/RNA-positive patients, 100 μL fractions of the pellet were layered onto a 900 μL CsCl gradient ranging from 10% to 60% (w/w) (open dots) and ultracentrifuged again at 300,000 g for 18 h at 4°C. The resulting gradient fractions were ten-fold diluted and tested using the HCV-ApoH immunoassay (black dots). (B) The presence or the absence of HCV-RNA in the centrifugation pellet was revealed by RT-PCR, prior to the CsCl gradient and after the gradient, in some of the resulting gradient fractions (CsCl-fr 5, 7, 9, 13, 14, 15 and 18). (C) CsCl ultracentrifugation gradient fractions corresponding to a density of 1.45 g/mL were layered directly onto ApoH-coated electron microscopy grids to observe the purified HCV-particles as previously done for HBV [ 44 ].

    Journal: PLoS ONE

    Article Title: Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus

    doi: 10.1371/journal.pone.0140900

    Figure Lengend Snippet: ApoH binds the RNA-containing HCV-related particles. (A) Isolation of HCV-related particles by ultracentrifugation. After the ultracentrifugation at 436,000 x g for 4 h at 4°C of pooled sera from HCV/RNA-positive patients, 100 μL fractions of the pellet were layered onto a 900 μL CsCl gradient ranging from 10% to 60% (w/w) (open dots) and ultracentrifuged again at 300,000 g for 18 h at 4°C. The resulting gradient fractions were ten-fold diluted and tested using the HCV-ApoH immunoassay (black dots). (B) The presence or the absence of HCV-RNA in the centrifugation pellet was revealed by RT-PCR, prior to the CsCl gradient and after the gradient, in some of the resulting gradient fractions (CsCl-fr 5, 7, 9, 13, 14, 15 and 18). (C) CsCl ultracentrifugation gradient fractions corresponding to a density of 1.45 g/mL were layered directly onto ApoH-coated electron microscopy grids to observe the purified HCV-particles as previously done for HBV [ 44 ].

    Article Snippet: An HCV plasmid (pGEM-T easy, Promega, with an HCV/PCR-insert) was used as template to make the standard curve and compute HCV copies.

    Techniques: Isolation, Centrifugation, Reverse Transcription Polymerase Chain Reaction, Electron Microscopy, Purification

    Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.

    Journal: BMC Molecular Biology

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

    doi: 10.1186/1471-2199-15-23

    Figure Lengend Snippet: Validation of a qPCR system for the analysis of mouse tissues. The analyzed cDNAs were the following: (A) , BRP-39; (B) , Ym1; (C) , Ym2; (D) , Chit1; (E) , AMCase; (F) , GAPDH; (G) , β-actin; and (H) , pepsinogen C. Standard curves were obtained using the mouse Refs/CLPs standard DNA with pGEM-T Easy containing the eight mouse cDNA fragments (red closed circles). In addition, the quantification of the mouse entire coding cDNA was performed using the primer pairs for each gene. The target cDNA was amplified from a dilution of the entire coding cDNA with a known concentration and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were obtained for each tested dilution of the standard curve and entire coding cDNA. Data are expressed as mean ± standard deviation (SD) of three measurements.

    Article Snippet: The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Concentration Assay, Standard Deviation

    Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .

    Journal: BMC Molecular Biology

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

    doi: 10.1186/1471-2199-15-23

    Figure Lengend Snippet: Preparation and validation of the mouse Refs/CLPs standard DNA with pGEM-T Easy. (A) Schematic representation of the mouse Refs/CLPs standard DNA cloning into pGEM-T Easy vector. (B) The mouse Refs/CLPs standard DNA with pGEM-T Easy was PCR-amplified from the plasmid DNA using the BRP-39 forward (blue arrow) and Ym1 reverse (red arrow) primers. As described above, we amplified the Ym1 and Ym2 cDNAs from this standard DNA using the Ym1 (C) and Ym2 (D) primers and these PCR products were analyzed using 10% polyacrylamide gel electrophoresis (E) . The y axis was expressed as first derivative of the fluorescence as a function of temperature (C and D) .

    Article Snippet: The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega).

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Fluorescence

    BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.

    Journal: BMC Molecular Biology

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

    doi: 10.1186/1471-2199-15-23

    Figure Lengend Snippet: BRP-39, Ym1 and Ym2 mRNA expression in mouse tissues. Quantification of BRP-39 (A) , Ym1 (B) and Ym2 (C) mRNAs in mouse tissues. The expression levels of the CLPs were quantified by real-time PCR using the standard DNA containing the eight mouse genes (the mouse Refs/CLPs standard DNA with pGEM-T Easy). The y axis was expressed as molecules per 10 ng of total RNA. The upper panel indicates the actual value, and the lower panel shows each value on logarithmic scale. Data are presented as mean ± SD of three measurements.

    Article Snippet: The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.

    Journal: BMC Molecular Biology

    Article Title: Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

    doi: 10.1186/1471-2199-15-23

    Figure Lengend Snippet: Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [ 40 , 41 ] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.

    Article Snippet: The PCR product was purified, and 3’-dA was added to the amplified DNA using Takara Taq HS (Takara Bio) and the resulting fragment was cloned into the pGEM-T Easy vector (Promega).

    Techniques: Real-time Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction