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    Promega pgem t easy vector
    Construction of pKS-VOTL plasmid. (A) Organisation of spo0B operon in B . subtilis and putative obg operon in M . synoviae has been shown. Putative –10 promoter region, transcription start site, ribosome binding site (RBS) of <t>vlhA</t> promoter region, and initiation codon for vlhA gene have been indicated. Length (bp) of each CDS is indicated inside the arrows. Identified stem loop structures in B . subtilis spo0B operon and putative stem loop in M . synoviae ‘ obg operon’ have also been indicated. (B) Schematic presentation of splicing by overlap extension (SOE) PCR to join vlhA gene promoter with obg CDS. Using PCR#1 and PCR#2, vlhA promoter (solid lines) and obg CDS (dotted lines) were amplified using indicated primers. Intermediate products with overlapping fragments (shown by horizontal bars) from both PCRs were amplified by SOE-PCR (PCR#3) using primers vlhA-ExtF and obg-ExtR. (C) Agarose gel electrophoresis of amplification products of vlhA promoter region PCR, obg CDS PCR and SOE-PCR. MW, DNA molecular weight marker (PCR Marker, Sigma, Missouri, USA). (D) Final product of SOE-PCR was first cloned at T-site of <t>pGEM-T</t> Easy Vector and then Pst I- Sac II fragment containing vlhA - obg was inserted between Pst I and Sac II sites of pBluescript II KS (+) vector. Apa I- Sal I restriction fragment containing LoriC and tetM , from pMAS-LoriC plasmid, was cloned at respective sites in pBluescript II KS (+) vector, harboring vlhA - obg , to generate pKS-VOTL plasmid.
    Pgem T Easy Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 71444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Construction of pKS-VOTL plasmid. (A) Organisation of spo0B operon in B . subtilis and putative obg operon in M . synoviae has been shown. Putative –10 promoter region, transcription start site, ribosome binding site (RBS) of vlhA promoter region, and initiation codon for vlhA gene have been indicated. Length (bp) of each CDS is indicated inside the arrows. Identified stem loop structures in B . subtilis spo0B operon and putative stem loop in M . synoviae ‘ obg operon’ have also been indicated. (B) Schematic presentation of splicing by overlap extension (SOE) PCR to join vlhA gene promoter with obg CDS. Using PCR#1 and PCR#2, vlhA promoter (solid lines) and obg CDS (dotted lines) were amplified using indicated primers. Intermediate products with overlapping fragments (shown by horizontal bars) from both PCRs were amplified by SOE-PCR (PCR#3) using primers vlhA-ExtF and obg-ExtR. (C) Agarose gel electrophoresis of amplification products of vlhA promoter region PCR, obg CDS PCR and SOE-PCR. MW, DNA molecular weight marker (PCR Marker, Sigma, Missouri, USA). (D) Final product of SOE-PCR was first cloned at T-site of pGEM-T Easy Vector and then Pst I- Sac II fragment containing vlhA - obg was inserted between Pst I and Sac II sites of pBluescript II KS (+) vector. Apa I- Sal I restriction fragment containing LoriC and tetM , from pMAS-LoriC plasmid, was cloned at respective sites in pBluescript II KS (+) vector, harboring vlhA - obg , to generate pKS-VOTL plasmid.

    Journal: PLoS ONE

    Article Title: Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics

    doi: 10.1371/journal.pone.0194528

    Figure Lengend Snippet: Construction of pKS-VOTL plasmid. (A) Organisation of spo0B operon in B . subtilis and putative obg operon in M . synoviae has been shown. Putative –10 promoter region, transcription start site, ribosome binding site (RBS) of vlhA promoter region, and initiation codon for vlhA gene have been indicated. Length (bp) of each CDS is indicated inside the arrows. Identified stem loop structures in B . subtilis spo0B operon and putative stem loop in M . synoviae ‘ obg operon’ have also been indicated. (B) Schematic presentation of splicing by overlap extension (SOE) PCR to join vlhA gene promoter with obg CDS. Using PCR#1 and PCR#2, vlhA promoter (solid lines) and obg CDS (dotted lines) were amplified using indicated primers. Intermediate products with overlapping fragments (shown by horizontal bars) from both PCRs were amplified by SOE-PCR (PCR#3) using primers vlhA-ExtF and obg-ExtR. (C) Agarose gel electrophoresis of amplification products of vlhA promoter region PCR, obg CDS PCR and SOE-PCR. MW, DNA molecular weight marker (PCR Marker, Sigma, Missouri, USA). (D) Final product of SOE-PCR was first cloned at T-site of pGEM-T Easy Vector and then Pst I- Sac II fragment containing vlhA - obg was inserted between Pst I and Sac II sites of pBluescript II KS (+) vector. Apa I- Sal I restriction fragment containing LoriC and tetM , from pMAS-LoriC plasmid, was cloned at respective sites in pBluescript II KS (+) vector, harboring vlhA - obg , to generate pKS-VOTL plasmid.

    Article Snippet: Amplicons of expected size were confirmed by agarose gel electrophoresis ( ) and vlhA - obg amplicon was cloned into pGEM-T Easy vector according to the manufacturer’s instructions (Promega, Alexandria, New South Wales, Australia).

    Techniques: Plasmid Preparation, Binding Assay, Overlap Extension Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker, Clone Assay

    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

    Agarose gel electrophoresis (1.2%) of the pGEM-T/CTRL-1 cDNA and pGEM-T/CTRL-1 genomic DNA after EcoR I digestion. M: 100-bp size marker; lane 1: full-length CTRL-1 cDNA PCR product; lane 2: pGEM-T/CTRL-1 cDNA; lane 3: CTRL-1 genomic DNA PCR product; lane 4: pGEM-T/CTRL-1 genomic DNA.

    Journal: Toxins

    Article Title: Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai

    doi: 10.3390/toxins8070205

    Figure Lengend Snippet: Agarose gel electrophoresis (1.2%) of the pGEM-T/CTRL-1 cDNA and pGEM-T/CTRL-1 genomic DNA after EcoR I digestion. M: 100-bp size marker; lane 1: full-length CTRL-1 cDNA PCR product; lane 2: pGEM-T/CTRL-1 cDNA; lane 3: CTRL-1 genomic DNA PCR product; lane 4: pGEM-T/CTRL-1 genomic DNA.

    Article Snippet: All the PCR products were purified with the Expin™ GeneAll® PCR SV purification kit (GeneAll, Seoul, Korea), cloned into the pGEM®-T Easy Vector System (Promega, Madison, WI, USA) and confirmed the clone by EcoR I digestion at 37 °C for 1 h. Full-length of cDNA sequence was identified by ABI PRISM 3739 Genetic Analyzer (Thermo Fisher, Waltham, MA, USA).

    Techniques: Agarose Gel Electrophoresis, Marker, Polymerase Chain Reaction

    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

    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 AnrasA and AnrasB PCR products were purified using PCR clean up system (Fermentas, USA) and cloned in pGEM-T easy (Promega, USA) vector and confirmed by automated DNA sequencing.

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

    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