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  • 99
    Promega pgem t easy vector
    Strategies for generation of dsRNAs and d-siRNAs. (A) <t>PCR</t> template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.
    Pgem T Easy Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 96119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 96119 article reviews
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    92
    Promega plasmid pgem t easy
    Construction of plasmid vehicle pGFbKAN used to inactivate flaB . pGFb was derived from both the <t>pGEM-T</t> Easy vector and amplified flaB plus flanking <t>DNA</t> by using primers P1 and P2. The kan cassette was amplified with Age I-containing restriction sites with primers KAN3 and KAN4 and inserted into Age I site of pGFb to yield plasmid pGFbKAN. After digestion with Eco RI and Ava II, the purified linear fragment containing flaBkan was used for allelic exchange. Wide arrows indicate direction of transcription. P represents PCR primers. bla is the β-lactamase gene. Plasmids are not drawn to scale.
    Plasmid Pgem T Easy, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 1100 article reviews
    Price from $9.99 to $1999.99
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    91
    Promega pgem t easy kit
    Construction of plasmid vehicle pGFbKAN used to inactivate flaB . pGFb was derived from both the <t>pGEM-T</t> Easy vector and amplified flaB plus flanking <t>DNA</t> by using primers P1 and P2. The kan cassette was amplified with Age I-containing restriction sites with primers KAN3 and KAN4 and inserted into Age I site of pGFb to yield plasmid pGFbKAN. After digestion with Eco RI and Ava II, the purified linear fragment containing flaBkan was used for allelic exchange. Wide arrows indicate direction of transcription. P represents PCR primers. bla is the β-lactamase gene. Plasmids are not drawn to scale.
    Pgem T Easy Kit, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 869 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgem t easy kit/product/Promega
    Average 91 stars, based on 869 article reviews
    Price from $9.99 to $1999.99
    pgem t easy kit - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    91
    Promega pgem t easy cloning kit
    Construction of plasmid vehicle pGFbKAN used to inactivate flaB . pGFb was derived from both the <t>pGEM-T</t> Easy vector and amplified flaB plus flanking <t>DNA</t> by using primers P1 and P2. The kan cassette was amplified with Age I-containing restriction sites with primers KAN3 and KAN4 and inserted into Age I site of pGFb to yield plasmid pGFbKAN. After digestion with Eco RI and Ava II, the purified linear fragment containing flaBkan was used for allelic exchange. Wide arrows indicate direction of transcription. P represents PCR primers. bla is the β-lactamase gene. Plasmids are not drawn to scale.
    Pgem T Easy Cloning Kit, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 657 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgem t easy cloning kit/product/Promega
    Average 91 stars, based on 657 article reviews
    Price from $9.99 to $1999.99
    pgem t easy cloning kit - by Bioz Stars, 2020-09
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    94
    Thermo Fisher pgem t easy vector
    DNA sequence of a part of FGF2 in the affected patients and unaffected individuals. A : A mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. B : Sequence of the normal allele of exon 8 subcloned into the <t>pGEM-T</t> vector used as a control. C : A heterozygous missense mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. The mutation causes the glutarnine 289 codon (CAG) to change to a proline codon (CCG).
    Pgem T Easy Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1311 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.

    Journal: PLoS ONE

    Article Title: Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans

    doi: 10.1371/journal.pone.0075443

    Figure Lengend Snippet: Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.

    Article Snippet: The sGFP gene was PCR amplified from the pMT-sGFP vector and cloned in pGEM T-easy vector.

    Techniques: Polymerase Chain Reaction, Sequencing, Amplification, Plasmid Preparation, In Vitro, Polyacrylamide Gel Electrophoresis, Purification, Generated

    Recombination of BMV RNA3 with sgRNA3a in barley protoplasts. A. Restriction enzyme digestion of recombinant RNA3 cDNA clones obtained from barley protoplasts that were transfected with a mixture of wt BMV RNAs 1 and 2 and (top) Neg-RNA3 plus JS(H)-21 sgRNA3a or (bottom) mut-RNA3 and JS(H)-21 sgRNA3a. The RNA3 region representing the sgRNA3a sequence was amplified from total RNA extracts by RT-PCR, and the cDNA products were cloned into the pGEM-T Easy system. The insert sequences were released by Hind III/ Eco RI digestion and separated by electrophoresis in a 1.5% agarose gel. An intact 1.2-kb fragment reflected the lack of the Hind III marker site at nt position 780, whereas the double 0.44 kb and 0.76 kb bands indicate the presence of the Hind III marker. B. Northern blot analysis showing the accumulation in protoplasts of either (+) or (−) strands (left and right panels, respectively) of BMV RNAs. Total protoplast RNA was separated in a 1.2% denaturing agarose gel, blotted, and probed with a 3′-specific RNA probe detecting either (+) or (−) strands (see Materials and methods ). Lanes 1 and 6, virion BMV RNA used as size standards; lanes 2 and 7, negative controls from mock inoculated protoplasts; lanes 3 and 8, protoplasts transfected with equimolar amounts of BMV RNAs 1 and 2 and wt RNA3; lanes 4 and 9, protoplasts transfected with BMV RNAs 1 and 2 and (A → U) RNA3; lanes 5 and 10, protoplasts transfected with BMV RNAs 1 and 2, (A → U) RNA3, and sgRNA3a. Ribosomal RNA (rRNA) bands (after staining with ethidium bromide) are shown below. The position corresponding to the (−) RNA4 band is marked with a single asterisk on the right panel, whereas the migration position of degradation products (likely because minus strands are not encapsidated and thus less protected) is marked with two asterisks.

    Journal: Virology

    Article Title: Recombination of 5′ subgenomic RNA3a with genomic RNA3 of Brome mosaic bromovirus in vitro and in vivo

    doi: 10.1016/j.virol.2010.10.037

    Figure Lengend Snippet: Recombination of BMV RNA3 with sgRNA3a in barley protoplasts. A. Restriction enzyme digestion of recombinant RNA3 cDNA clones obtained from barley protoplasts that were transfected with a mixture of wt BMV RNAs 1 and 2 and (top) Neg-RNA3 plus JS(H)-21 sgRNA3a or (bottom) mut-RNA3 and JS(H)-21 sgRNA3a. The RNA3 region representing the sgRNA3a sequence was amplified from total RNA extracts by RT-PCR, and the cDNA products were cloned into the pGEM-T Easy system. The insert sequences were released by Hind III/ Eco RI digestion and separated by electrophoresis in a 1.5% agarose gel. An intact 1.2-kb fragment reflected the lack of the Hind III marker site at nt position 780, whereas the double 0.44 kb and 0.76 kb bands indicate the presence of the Hind III marker. B. Northern blot analysis showing the accumulation in protoplasts of either (+) or (−) strands (left and right panels, respectively) of BMV RNAs. Total protoplast RNA was separated in a 1.2% denaturing agarose gel, blotted, and probed with a 3′-specific RNA probe detecting either (+) or (−) strands (see Materials and methods ). Lanes 1 and 6, virion BMV RNA used as size standards; lanes 2 and 7, negative controls from mock inoculated protoplasts; lanes 3 and 8, protoplasts transfected with equimolar amounts of BMV RNAs 1 and 2 and wt RNA3; lanes 4 and 9, protoplasts transfected with BMV RNAs 1 and 2 and (A → U) RNA3; lanes 5 and 10, protoplasts transfected with BMV RNAs 1 and 2, (A → U) RNA3, and sgRNA3a. Ribosomal RNA (rRNA) bands (after staining with ethidium bromide) are shown below. The position corresponding to the (−) RNA4 band is marked with a single asterisk on the right panel, whereas the migration position of degradation products (likely because minus strands are not encapsidated and thus less protected) is marked with two asterisks.

    Article Snippet: The RNA sequences were amplified by RT-PCR with primers 5 and 6 ( ) and the PCR products were cloned using the pGEM-T Easy Vector System from Promega, followed by sequencing.

    Techniques: Recombinant, Clone Assay, Transfection, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Marker, Northern Blot, Staining, Migration

    Localization of Stx2 in brains of mice inoculated with pStx2. The area observed in this study is located in the mouse CA2 hippocampus. (A) Brain from control mouse inoculated with pGEMT. (B) Brain from mouse inoculated with 3 µg of pStx2 24 h after inoculation. (C) Brain from mouse inoculated with 0.25 µg of pStx2 48 h after inoculation. (D) Brain from mouse injected with 0.5 ng of Stx2 4 days after injection. (E) Brain from mouse inoculated with 0.25 µg of pStx2 48 h after inoculation, without primary antibody. (F) Number of Stx2-immunopositive cells in hippocampus per micrograph. Scale bar = 50 µm.

    Journal: mBio

    Article Title: Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo, Leading to Production of Biologically Active Stx2

    doi: 10.1128/mBio.00501-13

    Figure Lengend Snippet: Localization of Stx2 in brains of mice inoculated with pStx2. The area observed in this study is located in the mouse CA2 hippocampus. (A) Brain from control mouse inoculated with pGEMT. (B) Brain from mouse inoculated with 3 µg of pStx2 24 h after inoculation. (C) Brain from mouse inoculated with 0.25 µg of pStx2 48 h after inoculation. (D) Brain from mouse injected with 0.5 ng of Stx2 4 days after injection. (E) Brain from mouse inoculated with 0.25 µg of pStx2 48 h after inoculation, without primary antibody. (F) Number of Stx2-immunopositive cells in hippocampus per micrograph. Scale bar = 50 µm.

    Article Snippet: The pGEMT Easy vector carrying the sequence of Stx2 was digested and religated in order to delete the active site of Stx2, generating the plasmid pStx2ΔAB ( ) as a Stx2-specific biological activity control.

    Techniques: Mouse Assay, Injection

    (A) Mice displayed a lethal dose-related response to pStx2. Survival rates of mice injected with different doses of pStx2 are shown. Mice injected with pStx2ΔAB or pGEMT were used as negative controls. Mice injected with 1 LD 100 and 3 LD 100 of purified Stx2 were used as positive controls. (B) Stx2 mRNA was detected in the liver. A real-time-PCR curve using cDNA from livers of mice inoculated with pStx2 (dotted line) or pGEMT (gray line) is shown. As a positive control, 50 pg of plasmid pStx2 (bold line) was used. (C) DNA electrophoresis. Lane 1, 50-bp ladder; lane 2. cDNA from livers of mice inoculated with pStx2; lane 3, cDNA from livers of mice inoculated with pGEMT; lane 4, pStx2 was used as positive control.

    Journal: mBio

    Article Title: Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo, Leading to Production of Biologically Active Stx2

    doi: 10.1128/mBio.00501-13

    Figure Lengend Snippet: (A) Mice displayed a lethal dose-related response to pStx2. Survival rates of mice injected with different doses of pStx2 are shown. Mice injected with pStx2ΔAB or pGEMT were used as negative controls. Mice injected with 1 LD 100 and 3 LD 100 of purified Stx2 were used as positive controls. (B) Stx2 mRNA was detected in the liver. A real-time-PCR curve using cDNA from livers of mice inoculated with pStx2 (dotted line) or pGEMT (gray line) is shown. As a positive control, 50 pg of plasmid pStx2 (bold line) was used. (C) DNA electrophoresis. Lane 1, 50-bp ladder; lane 2. cDNA from livers of mice inoculated with pStx2; lane 3, cDNA from livers of mice inoculated with pGEMT; lane 4, pStx2 was used as positive control.

    Article Snippet: The pGEMT Easy vector carrying the sequence of Stx2 was digested and religated in order to delete the active site of Stx2, generating the plasmid pStx2ΔAB ( ) as a Stx2-specific biological activity control.

    Techniques: Mouse Assay, Injection, Purification, Real-time Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Nucleic Acid Electrophoresis

    pELMO and pGEM-T Easy vector cloning efficiency for different sized inserts. pELMO and pGEM-T Easy cloning efficiency regarding low, medium and large sized inserts. Cloning efficiency is expressed as the ratio of the amount of PCR positive colonies

    Journal: AMB Express

    Article Title: pELMO, an optimised in-house cloning vector

    doi: 10.1186/s13568-017-0324-2

    Figure Lengend Snippet: pELMO and pGEM-T Easy vector cloning efficiency for different sized inserts. pELMO and pGEM-T Easy cloning efficiency regarding low, medium and large sized inserts. Cloning efficiency is expressed as the ratio of the amount of PCR positive colonies

    Article Snippet: Such step is essential for plasmids having T-overhangs, like pGEM-T Easy Vector Systems (Promega Corporation MD, USA).

    Techniques: Plasmid Preparation, Clone Assay, Polymerase Chain Reaction

    Construction of full-length cDNA clone of VHSV DK-3952B genome. Six overlapping cDNA fragments covering the entire VHSV genome were assembled by ligation into a pCI vector using the EcoRI, PvuII, EcoRV, KpnI, SalI, BclI, SmaI, StuI, BsrGI and NotI restriction sites. Assembly was carried out in 3 steps; (i) cloning of six fragments (F1-F6) separately in pGEM-T vectors (see methods), and addition of hammerhead ribozyme (HHRz) at the 5′-end of F1 and hepatitis delta ribozyme (HdvRz) at the 3′-end of F6; (ii) construction of A, B and C clones into the pCI vector; (iii) assembly of the full-length clone by ligating fragments from these three clones in the pCI vector. Restriction sites artificially created (*) and naturally present in the genome are indicated. Abbreviation: CMV, cytomegalovirus immediate-early enhancer and promoter

    Journal: Virology Journal

    Article Title: The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout

    doi: 10.1186/s12985-019-1139-3

    Figure Lengend Snippet: Construction of full-length cDNA clone of VHSV DK-3952B genome. Six overlapping cDNA fragments covering the entire VHSV genome were assembled by ligation into a pCI vector using the EcoRI, PvuII, EcoRV, KpnI, SalI, BclI, SmaI, StuI, BsrGI and NotI restriction sites. Assembly was carried out in 3 steps; (i) cloning of six fragments (F1-F6) separately in pGEM-T vectors (see methods), and addition of hammerhead ribozyme (HHRz) at the 5′-end of F1 and hepatitis delta ribozyme (HdvRz) at the 3′-end of F6; (ii) construction of A, B and C clones into the pCI vector; (iii) assembly of the full-length clone by ligating fragments from these three clones in the pCI vector. Restriction sites artificially created (*) and naturally present in the genome are indicated. Abbreviation: CMV, cytomegalovirus immediate-early enhancer and promoter

    Article Snippet: The cDNA fragments obtained after RT-PCR amplification were cloned into pGEM-T or pGEM-T Easy vectors (Promega Corp., Madison, WI).

    Techniques: Ligation, Plasmid Preparation, Clone Assay

    The gene las was amplified from Hae11116 strain using the template sequence from GeneBank GI: 14994100. Initially, the upstream region of start codon of las gene was amplified using the primers lasiF and lasiR ( Table 2 ). Also the downstream region of las was amplified with the primers lasfF and lasfR. Both amplicons were cloned in pGEMT Easy (Promega), originating pLAN 75 and pLAN76. In BamHI site of pLAN75, was inserted the ermAM cassette to generate the pLAN77. A new amplification reaction was performed using the ERAM3 and lasiF primers with the pLAN77 as template. The amplicon obtained was inserted in BamHI site of pLAN76 generating the transcriptional fusion vector pLAN78. This vector was then transformed in B4 N. meningitidis strain, originating the LG2 strain.

    Journal: Brazilian Journal of Microbiology

    Article Title: Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric Fever

    doi:

    Figure Lengend Snippet: The gene las was amplified from Hae11116 strain using the template sequence from GeneBank GI: 14994100. Initially, the upstream region of start codon of las gene was amplified using the primers lasiF and lasiR ( Table 2 ). Also the downstream region of las was amplified with the primers lasfF and lasfR. Both amplicons were cloned in pGEMT Easy (Promega), originating pLAN 75 and pLAN76. In BamHI site of pLAN75, was inserted the ermAM cassette to generate the pLAN77. A new amplification reaction was performed using the ERAM3 and lasiF primers with the pLAN77 as template. The amplicon obtained was inserted in BamHI site of pLAN76 generating the transcriptional fusion vector pLAN78. This vector was then transformed in B4 N. meningitidis strain, originating the LG2 strain.

    Article Snippet: The lasi and lasf amplicons were cloned in pGEMT Easy (Promega), originating the plasmids pLAN 75 and pLAN76, respectively.

    Techniques: Amplification, Sequencing, Clone Assay, Plasmid Preparation, Transformation Assay

    Construction and identification of recombinant adenovirus helper plasmid pHelper/recombinant adenovirus/amyloid β (Aβ)-binding alcohol dehydrogenase decoy peptide-6His (pSSHG/ABAD-DP-6His) plasmid. (A) The construction of recombinant plasmid pSSHG/ABAD-DP-6His. (B) Re-striction enzyme digestion of recombinant plasmid pGEM-T Easy/ABAD-DP. Lane 1: Recombinant plasmid pGEMT-Easy/ABAD-DP; lane 2: pGEM-T Easy/ABAD-DP cut with Eco RI; lane 3: pGEM-T Easy/ABAD-DP cut with Xho I; lane 4: HBI 1.0 kb plus DNA ladder, lane 5: λDNA/ Hin dIII marker. (C) The results of ABAD-DP-6His DNA sequencing and the comparison with our designed se-quence using DNASIS software (MiraiBio, Tokyo, Japan). File 1: Gene sequencing of ABAD-DP-6His. File 2: Gene sequencing provided by GeneBank ( http://www.ncbi.nlm.nih.gov/genbank ). (D) Recombinant plasmid pSSHG/ABAD-DP-6His was digested with Eco RI and Bam HI and confirmed that the ABAD-DO-6his fu-sion gene was 136 bp, which was consistent with the expected size. Lane 1: HBI 1.0 kb plus DNA ladder; lane 2: pSSHG/ABAD-DP-6His cut with Eco RI and Bam HI. lane 3: Recombinant plasmid pSSHG/ABAD-DP-6His. ABAD: Aβ-binding alcohol dehydrogenase; DP: decoy peptide; 6His: 6-His protein.

    Journal: Neural Regeneration Research

    Article Title: rAAV/ABAD-DP-6His attenuates oxidative stress-induced injury of PC12 cells

    doi: 10.4103/1673-5374.130065

    Figure Lengend Snippet: Construction and identification of recombinant adenovirus helper plasmid pHelper/recombinant adenovirus/amyloid β (Aβ)-binding alcohol dehydrogenase decoy peptide-6His (pSSHG/ABAD-DP-6His) plasmid. (A) The construction of recombinant plasmid pSSHG/ABAD-DP-6His. (B) Re-striction enzyme digestion of recombinant plasmid pGEM-T Easy/ABAD-DP. Lane 1: Recombinant plasmid pGEMT-Easy/ABAD-DP; lane 2: pGEM-T Easy/ABAD-DP cut with Eco RI; lane 3: pGEM-T Easy/ABAD-DP cut with Xho I; lane 4: HBI 1.0 kb plus DNA ladder, lane 5: λDNA/ Hin dIII marker. (C) The results of ABAD-DP-6His DNA sequencing and the comparison with our designed se-quence using DNASIS software (MiraiBio, Tokyo, Japan). File 1: Gene sequencing of ABAD-DP-6His. File 2: Gene sequencing provided by GeneBank ( http://www.ncbi.nlm.nih.gov/genbank ). (D) Recombinant plasmid pSSHG/ABAD-DP-6His was digested with Eco RI and Bam HI and confirmed that the ABAD-DO-6his fu-sion gene was 136 bp, which was consistent with the expected size. Lane 1: HBI 1.0 kb plus DNA ladder; lane 2: pSSHG/ABAD-DP-6His cut with Eco RI and Bam HI. lane 3: Recombinant plasmid pSSHG/ABAD-DP-6His. ABAD: Aβ-binding alcohol dehydrogenase; DP: decoy peptide; 6His: 6-His protein.

    Article Snippet: ABAD-DP cDNA was assembled in a pGEM-T Easy plasmid (Promega, Madison, WI, USA), and the proper orientation was confirmed by restriction analysis using Pma cI and Bam HI (Sino-American).

    Techniques: Recombinant, Plasmid Preparation, Binding Assay, Marker, DNA Sequencing, Software, Sequencing

    Amplification of the housekeeping genes and confirmation of cloning by EcoRI digestion of pGEM-T vector. (A) Amplification of the six target genes from cDNA of eggplant Lanes - M: 100 bp DNA Ladder/Marker, 1. 18S rRNA (416bp), 2: APRT (454 bp), 3: GAPDH (586bp), 4: Cyclophilin (265 bp), 5: Actin (333 bp), 6: RuBP (269bp). (B) Confirmation of the inserts in the recombinant pGEM-T vector by EcoRI digestion. Lanes - M: 100 bp DNA Ladder/Marker, 1. 18S rRNA (416bp), 2: APRT (454 bp), 3: Cyclophilin (265 bp), 4: GAPDH (586bp), 5: Actin (333 bp), 6: RuBP (269bp). (C) Amplification of the six target genes from genomic DNA of eggplant. Lanes- M: 100 bp DNA Ladder/Marker, 1. 18S rRNA (416bp), 2: APRT (No amplification), 3: Cyclophilin (265 bp), 4: GAPDH (1.2 kb), 5: Actin (333 bp), 6: RuBP (269bp).

    Journal: BMC Research Notes

    Article Title: Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L)

    doi: 10.1186/1756-0500-6-312

    Figure Lengend Snippet: Amplification of the housekeeping genes and confirmation of cloning by EcoRI digestion of pGEM-T vector. (A) Amplification of the six target genes from cDNA of eggplant Lanes - M: 100 bp DNA Ladder/Marker, 1. 18S rRNA (416bp), 2: APRT (454 bp), 3: GAPDH (586bp), 4: Cyclophilin (265 bp), 5: Actin (333 bp), 6: RuBP (269bp). (B) Confirmation of the inserts in the recombinant pGEM-T vector by EcoRI digestion. Lanes - M: 100 bp DNA Ladder/Marker, 1. 18S rRNA (416bp), 2: APRT (454 bp), 3: Cyclophilin (265 bp), 4: GAPDH (586bp), 5: Actin (333 bp), 6: RuBP (269bp). (C) Amplification of the six target genes from genomic DNA of eggplant. Lanes- M: 100 bp DNA Ladder/Marker, 1. 18S rRNA (416bp), 2: APRT (No amplification), 3: Cyclophilin (265 bp), 4: GAPDH (1.2 kb), 5: Actin (333 bp), 6: RuBP (269bp).

    Article Snippet: The amplified cDNA PCR products were cloned into pGEM-T Easy cloning vector (Promega) according to the manufacturer’s instructions.

    Techniques: Amplification, Clone Assay, Plasmid Preparation, Marker, Recombinant

    Transformation frequencies of H. pylori mutants. (A) H. pylori strain 26695 and derivative strains were transformed by homologous recombination with p801R (an 801-bp H. pylori rpsL fragment from a streptomycin-resistant [Str r ] strain cloned into pGEM-T

    Journal: Journal of Bacteriology

    Article Title: DprB Facilitates Inter- and Intragenomic Recombination in Helicobacter pylori

    doi: 10.1128/JB.00346-12

    Figure Lengend Snippet: Transformation frequencies of H. pylori mutants. (A) H. pylori strain 26695 and derivative strains were transformed by homologous recombination with p801R (an 801-bp H. pylori rpsL fragment from a streptomycin-resistant [Str r ] strain cloned into pGEM-T

    Article Snippet: An 801-bp DNA sequence encompassing H. pylori rpsL from Strr H. pylori strain 26695 ( , ) was obtained by PCR using primers rpsL801-F and rpsL801-R (see Table S2 in the supplemental material) and cloned into the vector pGEM-T Easy (Promega, Madison, WI) to create plasmid p801R (see Table S1), which was used as donor DNA for homologous transformation.

    Techniques: Transformation Assay, Homologous Recombination, Clone Assay

    Cross-species complementation. (A) Complementation of the E. coli ruvC mutant by the cloned H. pylori dprB and ruvC genes. E. coli cells carrying pGEM-T Easy-derived plasmids on an LB plate with ampicillin were subjected to a range of UV exposures, and

    Journal: Journal of Bacteriology

    Article Title: DprB Facilitates Inter- and Intragenomic Recombination in Helicobacter pylori

    doi: 10.1128/JB.00346-12

    Figure Lengend Snippet: Cross-species complementation. (A) Complementation of the E. coli ruvC mutant by the cloned H. pylori dprB and ruvC genes. E. coli cells carrying pGEM-T Easy-derived plasmids on an LB plate with ampicillin were subjected to a range of UV exposures, and

    Article Snippet: An 801-bp DNA sequence encompassing H. pylori rpsL from Strr H. pylori strain 26695 ( , ) was obtained by PCR using primers rpsL801-F and rpsL801-R (see Table S2 in the supplemental material) and cloned into the vector pGEM-T Easy (Promega, Madison, WI) to create plasmid p801R (see Table S1), which was used as donor DNA for homologous transformation.

    Techniques: Mutagenesis, Clone Assay, Derivative Assay

    Schematic representation of the capsule genes of W135 serogroup in transcriptional fusion of synG with ermAM cassette . The synG gene responsible for the synthesis of the W135 capsule was amplified using the 98-30 and 03-12-5 oligonucleotides (Table 3) from W135 ATCC strain. The amplified fragment was cloned into the pGEM-T Easy Vector System I (Promega, Madison, WI, USA), to generate the plasmid pLAN11. In the same conditions, another fragment was amplified using the 04.02-2/galECK29A from synG downstream sequence to generate pLAN52. The ermAM cassette was insered into Nco I site of pLAN52 to generate pLAN53. The fragment amplified from pLAN53 with the ERAM1 and galECK29A (Dolan Livengood [ 23 ] et al ., 2003) was insered into Pst I site of pLAN11 to generate pLAN13-2. This plasmid was linearised by the enzyme SphI and transformed into W135 ATCC strain to generate the synG::ermAM strain M6, erythromycin resistant.

    Journal: Journal of Nanobiotechnology

    Article Title: Effect of mesoporous silica under Neisseria meningitidis transformation process: environmental effects under meningococci transformation

    doi: 10.1186/1477-3155-9-28

    Figure Lengend Snippet: Schematic representation of the capsule genes of W135 serogroup in transcriptional fusion of synG with ermAM cassette . The synG gene responsible for the synthesis of the W135 capsule was amplified using the 98-30 and 03-12-5 oligonucleotides (Table 3) from W135 ATCC strain. The amplified fragment was cloned into the pGEM-T Easy Vector System I (Promega, Madison, WI, USA), to generate the plasmid pLAN11. In the same conditions, another fragment was amplified using the 04.02-2/galECK29A from synG downstream sequence to generate pLAN52. The ermAM cassette was insered into Nco I site of pLAN52 to generate pLAN53. The fragment amplified from pLAN53 with the ERAM1 and galECK29A (Dolan Livengood [ 23 ] et al ., 2003) was insered into Pst I site of pLAN11 to generate pLAN13-2. This plasmid was linearised by the enzyme SphI and transformed into W135 ATCC strain to generate the synG::ermAM strain M6, erythromycin resistant.

    Article Snippet: The amplified fragment was cloned into the pGEM-T Easy Vector System I (Promega, Madison, WI, USA), to generate the plasmid pLAN11.

    Techniques: Amplification, Clone Assay, Plasmid Preparation, Sequencing, Transformation Assay

    Schematic representation of the capsule genes of C serogroup in disrupted construction of NMB0065 gene with aaDA cassette . The NMB0065 gene was amplified using the 03-12-3 and 03-12-4 oligonucleotides (Table 3) from C2135 strain. This fragment was cloned into the pGEM-T Easy Vector System II (Promega Corporation, Madison, WI, USA), to generate the plasmid pLAN6. E. coli strain Z501 was transformed with plasmid pLAN6 resulting in the plasmid pLAN7. The Ω aaDA cassette was inserted into the BclI site of pLAN7 to generate plasmid pLAN45, which was transformed into the C2135 strain to generate the isogenic mutant strain M2.

    Journal: Journal of Nanobiotechnology

    Article Title: Effect of mesoporous silica under Neisseria meningitidis transformation process: environmental effects under meningococci transformation

    doi: 10.1186/1477-3155-9-28

    Figure Lengend Snippet: Schematic representation of the capsule genes of C serogroup in disrupted construction of NMB0065 gene with aaDA cassette . The NMB0065 gene was amplified using the 03-12-3 and 03-12-4 oligonucleotides (Table 3) from C2135 strain. This fragment was cloned into the pGEM-T Easy Vector System II (Promega Corporation, Madison, WI, USA), to generate the plasmid pLAN6. E. coli strain Z501 was transformed with plasmid pLAN6 resulting in the plasmid pLAN7. The Ω aaDA cassette was inserted into the BclI site of pLAN7 to generate plasmid pLAN45, which was transformed into the C2135 strain to generate the isogenic mutant strain M2.

    Article Snippet: This fragment was cloned into the pGEM-T Easy Vector System II (Promega Corporation, Madison, WI, USA), to generate the plasmid pLAN6.

    Techniques: Amplification, Clone Assay, Plasmid Preparation, Transformation Assay, Mutagenesis

    Export of constitutively expressed immunity protein. E. coli DH5α cells containing the ximB locus with an 5′-upstream promoter sequence in pGEM-T Easy vector was grown overnight. Cytosolic, periplasmic, and flagellar proteins were prepared

    Journal: Journal of Bacteriology

    Article Title: Xenocin Export by the Flagellar Type III Pathway in Xenorhabdus nematophila

    doi: 10.1128/JB.01532-12

    Figure Lengend Snippet: Export of constitutively expressed immunity protein. E. coli DH5α cells containing the ximB locus with an 5′-upstream promoter sequence in pGEM-T Easy vector was grown overnight. Cytosolic, periplasmic, and flagellar proteins were prepared

    Article Snippet: The plasmid vector pGEM-T Easy, from Promega (Madison, WI), was used for PCR cloning. pCR-XL-TOPO and pBCSK(+) were used for cloning and complementation studies.

    Techniques: Sequencing, Plasmid Preparation

    Construction of plasmid vehicle pGFbKAN used to inactivate flaB . pGFb was derived from both the pGEM-T Easy vector and amplified flaB plus flanking DNA by using primers P1 and P2. The kan cassette was amplified with Age I-containing restriction sites with primers KAN3 and KAN4 and inserted into Age I site of pGFb to yield plasmid pGFbKAN. After digestion with Eco RI and Ava II, the purified linear fragment containing flaBkan was used for allelic exchange. Wide arrows indicate direction of transcription. P represents PCR primers. bla is the β-lactamase gene. Plasmids are not drawn to scale.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions

    doi:

    Figure Lengend Snippet: Construction of plasmid vehicle pGFbKAN used to inactivate flaB . pGFb was derived from both the pGEM-T Easy vector and amplified flaB plus flanking DNA by using primers P1 and P2. The kan cassette was amplified with Age I-containing restriction sites with primers KAN3 and KAN4 and inserted into Age I site of pGFb to yield plasmid pGFbKAN. After digestion with Eco RI and Ava II, the purified linear fragment containing flaBkan was used for allelic exchange. Wide arrows indicate direction of transcription. P represents PCR primers. bla is the β-lactamase gene. Plasmids are not drawn to scale.

    Article Snippet: The flaB gene and flanking DNA were first amplified by PCR with primers P1 and P2 from chromosomal DNA of strain B31, and the product obtained was cloned into plasmid pGEM-T Easy (Promega) to yield pGFb (Fig. ).

    Techniques: Plasmid Preparation, Derivative Assay, Amplification, Antiviral Assay, Purification, Polymerase Chain Reaction

    DNA sequence of a part of FGF2 in the affected patients and unaffected individuals. A : A mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. B : Sequence of the normal allele of exon 8 subcloned into the pGEM-T vector used as a control. C : A heterozygous missense mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. The mutation causes the glutarnine 289 codon (CAG) to change to a proline codon (CCG).

    Journal: Molecular Vision

    Article Title: FGFR2 molecular analysis and related clinical findings in one Chinese family with Crouzon Syndrome

    doi:

    Figure Lengend Snippet: DNA sequence of a part of FGF2 in the affected patients and unaffected individuals. A : A mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. B : Sequence of the normal allele of exon 8 subcloned into the pGEM-T vector used as a control. C : A heterozygous missense mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. The mutation causes the glutarnine 289 codon (CAG) to change to a proline codon (CCG).

    Article Snippet: The purified PCR fragments were ligated into the pGEM-T easy vector (Invitrogen, Carlsbad, CA), and the resulting plasmids were transfected, by heat shock, into DH5a-competent Escherichia coli for propagation.

    Techniques: Sequencing, Mutagenesis, Plasmid Preparation