pcr clean up systems  (Promega)

 
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    Name:
    Wizard SV Gel and PCR Clean-Up System
    Description:

    Catalog Number:
    A9281
    Price:
    None
    Score:
    85
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    Promega pcr clean up systems

    https://www.bioz.com/result/pcr clean up systems/product/Promega
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    pcr clean up systems - by Bioz Stars, 2019-12
    92/100 stars

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    Related Articles

    Clone Assay:

    Article Title: An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study
    Article Snippet: T4 DNA ligase, Wizard plus SV Minipreps DNA purification, and Wizard SV Gel and PCR Clean-up systems (Promega, Madison, WI, USA) were used for ligation and DNA purification. .. T4 DNA ligase, Wizard plus SV Minipreps DNA purification, and Wizard SV Gel and PCR Clean-up systems (Promega, Madison, WI, USA) were used for ligation and DNA purification.

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: The PCR conditions for amplification of bacterial 16S rRNA genes was the same as described above for the DGGE protocol, except that the primers used for PCR did not have a GC clamp. .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA). .. Purified amplicons were then ligated into the pGEM-T-Easy vector and introduced into competent Escherichia coli JM109 (Promega, USA) following the manufacturer's instructions.

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega. .. DNA sequencing was performed by Genewiz, Inc. and SeqWright, Inc. Oligonucleotides were purchased from Sigma-Aldrich and Integrated DNA Technologies (IDT) and are listed in .

    Amplification:

    Article Title: Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis
    Article Snippet: For microscopic visualization of exogenous DNA binding, we used a fluorescently-labeled 1,5 kb PCR fragment corresponding to the amplification of the amyE gene. .. Fragments were purified using Wizard SV Gel and PCR clean-up systems (Promega corporation).

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: Plasmids were amplified in E. coli Novablue (Novagen) and purified with Wizard Plus SV Minipreps DNA Purification System (Promega). .. PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega).

    Article Title: Drinking water microbiome assembly induced by water stagnation
    Article Snippet: The extracted DNA was amplified with the following bacterial specific forward 515 F and 806 R (Read1-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA; Read2-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT; Linker -ATTAGAWACCCBDGTAGTCCGGCTGACTGACT) [ ]. .. The amplified products were purified with Promega PCR clean-up systems. .. Sequencing was performed at the W.M.

    Article Title: The Unstable CCTG Repeat Responsible for Myotonic Dystrophy Type 2 Originates from an AluSx Element Insertion into an Early Primate Genome
    Article Snippet: The PCR conditions included an initial denaturing at 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec, 54°C for 30 sec, and 68°C for 1 min 30 sec, with an additional extension at 68°C for 3 min. .. The amplified PCR fragments were gel purified using Wizard SV gel and PCR clean-up systems (Promega) and directly sequenced using a CEQ 8000 DNA sequence system (Bechman Caulter). .. For samples showing ambiguous sequences and heterozygosity, the PCR products were gel purified and cloned into a pTA2 plasmid vector using the TArget Clone-plus cloning system (Toyobo) and sequenced using sequencing primers ( and ).

    Article Title: Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico
    Article Snippet: Paragraph title: 4.9.1. DNA Extraction and PCR Amplification ... PCR products were purified directly or from agarose gels using Wizard SV gel or PCR Clean-up systems (Promega: Madison, WI, USA).

    Article Title: LEMONS – A Tool for the Identification of Splice Junctions in Transcriptomes of Organisms Lacking Reference Genomes
    Article Snippet: The genes analyzed were of varying transcript length, contained different number of exons and were found at various chromosomal locations. .. PCR products were visualized on an EtBr-stained 1% agarose gel, purified using Wizard SV Gel and PCR Clean-up systems (Promega) following the manufacturer’s protocol and sequenced (ABI 3100, BGU sequencing facility) using either one or two of the amplification primers, except for KIA0020. .. Sequence analysis was performed using Sequencher 4.9 (GeneCodes) and MEGA 5 [ ].

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: The PCR conditions for amplification of bacterial 16S rRNA genes was the same as described above for the DGGE protocol, except that the primers used for PCR did not have a GC clamp. .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA).

    Article Title: Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile
    Article Snippet: Housekeeping genes for MLST were amplified by using primers for V . parahaemolyticus ( ) and from the MLST website (http://pubmlst.org/vparahaemolyticus) developed by Keith Jolley (University of Oxford, Oxford, UK) ( ). .. PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci). .. DNA sequences were analyzed individually and manually assembled.

    DNA Ligation:

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Standard protocols or manufacturers’ instructions were used to isolate and manipulate DNA, including preparation of plasmid DNA, restriction endonuclease digest, DNA ligation, and polymerase chain reaction (PCR). .. Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega.

    Synthesized:

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: Clone libraries were constructed using the cDNA synthesized from the 16S rRNA gene transcripts from the 5-day sampling (time at which the first peak of 13 C accumulation in bacterial fraction was observed, ). .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA).

    Construct:

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: In total, eight libraries were constructed, consisting of duplicates of two fractions (i.e. ‘light’ and ‘heavy’) obtained for each of the two potato lines Karnico and Modena. .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA).

    SYBR Green Assay:

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega). .. Transcription–translation reactions used the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) supplemented with 20 units of Human Placenta RNase Inhibitor (New England Biolabs).

    Microarray:

    Article Title: Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile
    Article Snippet: T3SS2 genes (VPA1335, VPA1338, VPA1339, VPA1341, VPA1342, VPA1346, VPA1349, VPA1354, VPA1355, VPA1362, and VPA1367) were amplified by using primers reported for a microarray assay at 61ºC by Meador et al ( ). .. PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci).

    Incubation:

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega). .. Five micrograms of linearized plasmid and 15 µL of Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) were each diluted separately into 500 µL of Optimem medium (Thermo Fisher Scientific) and incubated for 10 min at room temperature.

    Cell Culture:

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Human Embryonic Kidney (HEK293) cells expressing EsemOrco in combination with different EsemORs were produced and cultured as previously described ( ). .. Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega).

    Expressing:

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Human Embryonic Kidney (HEK293) cells expressing EsemOrco in combination with different EsemORs were produced and cultured as previously described ( ). .. Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega).

    BIA-KA:

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega. .. Phusion high-fidelity DNA polymerase from NEB and Kapa Biosystems High Fidelity Readymix from VWR International, LLC were used for PCR.

    Transfection:

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega). .. The mixture was then added to the culture flask containing ∼70% confluent isogenic tetracycline repressor-expressing (TREx) cells (allowing controlled expression of exogenous OR genes; ) and incubated overnight at 37 °C with 5% CO2 .

    Gas Chromatography:

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: The PCR conditions for amplification of bacterial 16S rRNA genes was the same as described above for the DGGE protocol, except that the primers used for PCR did not have a GC clamp. .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA).

    Ligation:

    Article Title: An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study
    Article Snippet: The restriction enzymes Nde I, Spe I, Xho I, and Bam HI (New England Biolabs, Ipswich, MA, USA) were used. .. T4 DNA ligase, Wizard plus SV Minipreps DNA purification, and Wizard SV Gel and PCR Clean-up systems (Promega, Madison, WI, USA) were used for ligation and DNA purification. .. Oligonucleotide primers were obtained from Hokkaido System Science Co., Ltd. (Hokkaido, Japan). pET21b was purchased from Merck KGaA/Novagen (Darmstadt, Germany).

    Infection:

    Article Title: A Rapid and Accurate Method to Evaluate Helicobacter pylori Infection, Clarithromycin Resistance, and CYP2C19 Genotypes Simultaneously From Gastric Juice
    Article Snippet: DNA from the enrolled patients’ blood samples, gastric juices that included host cells with/without H pylori , and H pylori stains were extracted with the DNeasy Blood & Tissue kit (QIAGEN, Germany), and then was purified by Wizard SV and PCR Clean-up systems (Promega, Madison, WI). .. DNA products were then subjected to PCR and PCR-RFLP.

    DNA Sequencing:

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega. .. Protein concentrations were determined using a bicinchoninic acid (BCA) assay from Thermo Scientific.

    Enzyme Inhibition Assay:

    Article Title: A Rapid and Accurate Method to Evaluate Helicobacter pylori Infection, Clarithromycin Resistance, and CYP2C19 Genotypes Simultaneously From Gastric Juice
    Article Snippet: Before storage, the pH of gastric juice (pH value ≤ 3) was adjusted to > 3 with culture medium DMEM (Invitrogen) for improving DNA quality in gastric juice and prevent enzyme inhibition in further PCR reaction. .. DNA from the enrolled patients’ blood samples, gastric juices that included host cells with/without H pylori , and H pylori stains were extracted with the DNeasy Blood & Tissue kit (QIAGEN, Germany), and then was purified by Wizard SV and PCR Clean-up systems (Promega, Madison, WI).

    Sequencing:

    Article Title: Drinking water microbiome assembly induced by water stagnation
    Article Snippet: Paragraph title: Illumina sequencing ... The amplified products were purified with Promega PCR clean-up systems.

    Article Title: The Unstable CCTG Repeat Responsible for Myotonic Dystrophy Type 2 Originates from an AluSx Element Insertion into an Early Primate Genome
    Article Snippet: The PCR conditions included an initial denaturing at 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec, 54°C for 30 sec, and 68°C for 1 min 30 sec, with an additional extension at 68°C for 3 min. .. The amplified PCR fragments were gel purified using Wizard SV gel and PCR clean-up systems (Promega) and directly sequenced using a CEQ 8000 DNA sequence system (Bechman Caulter). .. For samples showing ambiguous sequences and heterozygosity, the PCR products were gel purified and cloned into a pTA2 plasmid vector using the TArget Clone-plus cloning system (Toyobo) and sequenced using sequencing primers ( and ).

    Article Title: A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage
    Article Snippet: Paragraph title: Whole genome sequencing, PCR, DNA extraction and sequencing methods ... Plasmid and PCR/gel extractions were done using PureYield Plasmid Miniprep and Wizard SV Gel and PCR clean-up systems respectively (Promega).

    Article Title: LEMONS – A Tool for the Identification of Splice Junctions in Transcriptomes of Organisms Lacking Reference Genomes
    Article Snippet: The genes analyzed were of varying transcript length, contained different number of exons and were found at various chromosomal locations. .. PCR products were visualized on an EtBr-stained 1% agarose gel, purified using Wizard SV Gel and PCR Clean-up systems (Promega) following the manufacturer’s protocol and sequenced (ABI 3100, BGU sequencing facility) using either one or two of the amplification primers, except for KIA0020. .. Sequence analysis was performed using Sequencher 4.9 (GeneCodes) and MEGA 5 [ ].

    Article Title: Human papillomavirus in head and neck squamous cell carcinomas in a South African cohort
    Article Snippet: PCR products were excised from the 1% or 2.5% agarose gels and were purified using Wizard®SV Gel and PCR Clean-Up Systems according to manufacturer's instructions (Promega, Wisconsin, USA). .. Nucleotide sequences obtained for PCR positive amplicons were edited using Chromas Pro version 1.6 and aligned using Clustal Omega 1.2.1 .

    Article Title: Human papillomavirus in head and neck squamous cell carcinomas in a South African cohort
    Article Snippet: 2.3 PCR products were excised from the 1% or 2.5% agarose gels and were purified using Wizard®SV Gel and PCR Clean-Up Systems according to manufacturer's instructions (Promega, Wisconsin, USA). .. Nucleotide sequences obtained for PCR positive amplicons were edited using Chromas Pro version 1.6 and aligned using Clustal Omega 1.2.1 .

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA). .. Clones containing the insert (evaluated by blue/white colony) were subjected to colony PCR with primers M13F and M13R to determine which clones contained a correctly-sized insert.

    Article Title: Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile
    Article Snippet: PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci). .. PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci).

    Binding Assay:

    Article Title: Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis
    Article Snippet: For microscopic visualization of exogenous DNA binding, we used a fluorescently-labeled 1,5 kb PCR fragment corresponding to the amplification of the amyE gene. .. Fragments were purified using Wizard SV Gel and PCR clean-up systems (Promega corporation).

    Imaging:

    Article Title: Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival
    Article Snippet: Gel images were captured by using an Image Master VDS-CL gel documentation system (GE Healthcare Life Sciences, Little Chalfont, Buckinghampshire, UK) fitted with a Fujifilm FTI-500 Thermal Imaging system (GE Healthcare). .. DNA fragments were purified from agarose gels using Wizard® SV gel and PCR Clean-Up Systems (Promega) according to the manufacturer’s instructions.

    DNA Extraction:

    Article Title: A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage
    Article Snippet: Paragraph title: Whole genome sequencing, PCR, DNA extraction and sequencing methods ... Plasmid and PCR/gel extractions were done using PureYield Plasmid Miniprep and Wizard SV Gel and PCR clean-up systems respectively (Promega).

    Article Title: Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico
    Article Snippet: Paragraph title: 4.9.1. DNA Extraction and PCR Amplification ... PCR products were purified directly or from agarose gels using Wizard SV gel or PCR Clean-up systems (Promega: Madison, WI, USA).

    Article Title: PCR Detection of Helicobacter pylori in Clinical Samples
    Article Snippet: Paragraph title: 2.1. DNA Extraction from Stool (see ) ... Wizard SV Gel and PCR Clean-up systems (Promega, Madison, WI, USA).

    Fluorescence:

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega). .. Transcription–translation reactions used the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) supplemented with 20 units of Human Placenta RNase Inhibitor (New England Biolabs).

    Mutagenesis:

    Article Title: Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico
    Article Snippet: Two sets of primers covering all five known mutation sites in regions A (1781) and B (2027, 2041, 2078, and 2096), were designed based on the sequences of ACCase chloroplastics from other grass weeds, P. minor (AY19648) and P. paradoxa (AM745339). .. PCR products were purified directly or from agarose gels using Wizard SV gel or PCR Clean-up systems (Promega: Madison, WI, USA).

    Size-exclusion Chromatography:

    Article Title: The Unstable CCTG Repeat Responsible for Myotonic Dystrophy Type 2 Originates from an AluSx Element Insertion into an Early Primate Genome
    Article Snippet: The amplified PCR fragments were gel purified using Wizard SV gel and PCR clean-up systems (Promega) and directly sequenced using a CEQ 8000 DNA sequence system (Bechman Caulter). .. The amplified PCR fragments were gel purified using Wizard SV gel and PCR clean-up systems (Promega) and directly sequenced using a CEQ 8000 DNA sequence system (Bechman Caulter).

    Labeling:

    Article Title: Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis
    Article Snippet: Paragraph title: Fluorescent-DNA (ATTO-550-DNA) preparation and labeling ... Fragments were purified using Wizard SV Gel and PCR clean-up systems (Promega corporation).

    Purification:

    Article Title: Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis
    Article Snippet: Briefly, we used 1 µl of ExTaq enzyme from Takara in 100 µl on genomic DNA (20 ng) with 1 µM of each primer, 0.25 µM of each dNTP and 0.1 µM of ATTO550-aminoallyl-dUTP. .. Fragments were purified using Wizard SV Gel and PCR clean-up systems (Promega corporation). .. 200 ng of ATTO550-labeled DNA was mixed with 100 µl of cells grown for 2 h in SPII medium.

    Article Title: A Rapid and Accurate Method to Evaluate Helicobacter pylori Infection, Clarithromycin Resistance, and CYP2C19 Genotypes Simultaneously From Gastric Juice
    Article Snippet: The gastric juice/culture medium mixture was then centrifuged at 12,000 rpm for 10 minutes to precipitate the gastric mucus for purifying genomic DNA. .. DNA from the enrolled patients’ blood samples, gastric juices that included host cells with/without H pylori , and H pylori stains were extracted with the DNeasy Blood & Tissue kit (QIAGEN, Germany), and then was purified by Wizard SV and PCR Clean-up systems (Promega, Madison, WI). .. DNA products were then subjected to PCR and PCR-RFLP.

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: Plasmid DNA was phenol–chloroform extracted, ethanol precipitated and resuspended in deionized and diethyl pyrocarbonate-treated water. .. PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega). .. Transcription–translation reactions used the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) supplemented with 20 units of Human Placenta RNase Inhibitor (New England Biolabs).

    Article Title: Drinking water microbiome assembly induced by water stagnation
    Article Snippet: The extracted DNA was amplified with the following bacterial specific forward 515 F and 806 R (Read1-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA; Read2-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT; Linker -ATTAGAWACCCBDGTAGTCCGGCTGACTGACT) [ ]. .. The amplified products were purified with Promega PCR clean-up systems. .. Sequencing was performed at the W.M.

    Article Title: The Unstable CCTG Repeat Responsible for Myotonic Dystrophy Type 2 Originates from an AluSx Element Insertion into an Early Primate Genome
    Article Snippet: The PCR conditions included an initial denaturing at 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec, 54°C for 30 sec, and 68°C for 1 min 30 sec, with an additional extension at 68°C for 3 min. .. The amplified PCR fragments were gel purified using Wizard SV gel and PCR clean-up systems (Promega) and directly sequenced using a CEQ 8000 DNA sequence system (Bechman Caulter). .. For samples showing ambiguous sequences and heterozygosity, the PCR products were gel purified and cloned into a pTA2 plasmid vector using the TArget Clone-plus cloning system (Toyobo) and sequenced using sequencing primers ( and ).

    Article Title: Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico
    Article Snippet: Ten individual plants from each biotype were genotyped. .. PCR products were purified directly or from agarose gels using Wizard SV gel or PCR Clean-up systems (Promega: Madison, WI, USA). .. Sequencing was performed at the central facilities of the University of Córdoba.

    Article Title: LEMONS – A Tool for the Identification of Splice Junctions in Transcriptomes of Organisms Lacking Reference Genomes
    Article Snippet: The genes analyzed were of varying transcript length, contained different number of exons and were found at various chromosomal locations. .. PCR products were visualized on an EtBr-stained 1% agarose gel, purified using Wizard SV Gel and PCR Clean-up systems (Promega) following the manufacturer’s protocol and sequenced (ABI 3100, BGU sequencing facility) using either one or two of the amplification primers, except for KIA0020. .. Sequence analysis was performed using Sequencher 4.9 (GeneCodes) and MEGA 5 [ ].

    Article Title: Human papillomavirus in head and neck squamous cell carcinomas in a South African cohort
    Article Snippet: The strips were interpreted using the HPV reference guide provided. .. PCR products were excised from the 1% or 2.5% agarose gels and were purified using Wizard®SV Gel and PCR Clean-Up Systems according to manufacturer's instructions (Promega, Wisconsin, USA). .. Determination of nucleotide sequence of the amplicon was performed using the Big Dye Terminator sequencing ready reaction kit according to manufacturer's instructions (Applied Biosystems, Foster City, CA, USA).

    Article Title: Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival
    Article Snippet: Gel images were captured by using an Image Master VDS-CL gel documentation system (GE Healthcare Life Sciences, Little Chalfont, Buckinghampshire, UK) fitted with a Fujifilm FTI-500 Thermal Imaging system (GE Healthcare). .. DNA fragments were purified from agarose gels using Wizard® SV gel and PCR Clean-Up Systems (Promega) according to the manufacturer’s instructions. .. Purified DNA was stored at −20 °C until required.

    Article Title: Human papillomavirus in head and neck squamous cell carcinomas in a South African cohort
    Article Snippet: The strips were interpreted using the HPV reference guide provided. .. 2.3 PCR products were excised from the 1% or 2.5% agarose gels and were purified using Wizard®SV Gel and PCR Clean-Up Systems according to manufacturer's instructions (Promega, Wisconsin, USA). .. Determination of nucleotide sequence of the amplicon was performed using the Big Dye Terminator sequencing ready reaction kit according to manufacturer's instructions (Applied Biosystems, Foster City, CA, USA).

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: The PCR conditions for amplification of bacterial 16S rRNA genes was the same as described above for the DGGE protocol, except that the primers used for PCR did not have a GC clamp. .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA). .. Purified amplicons were then ligated into the pGEM-T-Easy vector and introduced into competent Escherichia coli JM109 (Promega, USA) following the manufacturer's instructions.

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Enzymes were purchased from New England BioLabs, Inc. (NEB). .. Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega. .. Phusion high-fidelity DNA polymerase from NEB and Kapa Biosystems High Fidelity Readymix from VWR International, LLC were used for PCR.

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Human Embryonic Kidney (HEK293) cells expressing EsemOrco in combination with different EsemORs were produced and cultured as previously described ( ). .. Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega). .. Five micrograms of linearized plasmid and 15 µL of Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) were each diluted separately into 500 µL of Optimem medium (Thermo Fisher Scientific) and incubated for 10 min at room temperature.

    Article Title: Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile
    Article Snippet: Housekeeping genes for MLST were amplified by using primers for V . parahaemolyticus ( ) and from the MLST website (http://pubmlst.org/vparahaemolyticus) developed by Keith Jolley (University of Oxford, Oxford, UK) ( ). .. PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci). .. DNA sequences were analyzed individually and manually assembled.

    Polymerase Chain Reaction:

    Article Title: Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis
    Article Snippet: Briefly, we used 1 µl of ExTaq enzyme from Takara in 100 µl on genomic DNA (20 ng) with 1 µM of each primer, 0.25 µM of each dNTP and 0.1 µM of ATTO550-aminoallyl-dUTP. .. Fragments were purified using Wizard SV Gel and PCR clean-up systems (Promega corporation). .. 200 ng of ATTO550-labeled DNA was mixed with 100 µl of cells grown for 2 h in SPII medium.

    Article Title: A Rapid and Accurate Method to Evaluate Helicobacter pylori Infection, Clarithromycin Resistance, and CYP2C19 Genotypes Simultaneously From Gastric Juice
    Article Snippet: The gastric juice/culture medium mixture was then centrifuged at 12,000 rpm for 10 minutes to precipitate the gastric mucus for purifying genomic DNA. .. DNA from the enrolled patients’ blood samples, gastric juices that included host cells with/without H pylori , and H pylori stains were extracted with the DNeasy Blood & Tissue kit (QIAGEN, Germany), and then was purified by Wizard SV and PCR Clean-up systems (Promega, Madison, WI). .. DNA products were then subjected to PCR and PCR-RFLP.

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: Plasmid DNA was phenol–chloroform extracted, ethanol precipitated and resuspended in deionized and diethyl pyrocarbonate-treated water. .. PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega). .. Transcription–translation reactions used the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) supplemented with 20 units of Human Placenta RNase Inhibitor (New England Biolabs).

    Article Title: Drinking water microbiome assembly induced by water stagnation
    Article Snippet: The extracted DNA was amplified with the following bacterial specific forward 515 F and 806 R (Read1-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA; Read2-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT; Linker -ATTAGAWACCCBDGTAGTCCGGCTGACTGACT) [ ]. .. The amplified products were purified with Promega PCR clean-up systems. .. Sequencing was performed at the W.M.

    Article Title: The Unstable CCTG Repeat Responsible for Myotonic Dystrophy Type 2 Originates from an AluSx Element Insertion into an Early Primate Genome
    Article Snippet: The PCR conditions included an initial denaturing at 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec, 54°C for 30 sec, and 68°C for 1 min 30 sec, with an additional extension at 68°C for 3 min. .. The amplified PCR fragments were gel purified using Wizard SV gel and PCR clean-up systems (Promega) and directly sequenced using a CEQ 8000 DNA sequence system (Bechman Caulter). .. For samples showing ambiguous sequences and heterozygosity, the PCR products were gel purified and cloned into a pTA2 plasmid vector using the TArget Clone-plus cloning system (Toyobo) and sequenced using sequencing primers ( and ).

    Article Title: Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico
    Article Snippet: Ten individual plants from each biotype were genotyped. .. PCR products were purified directly or from agarose gels using Wizard SV gel or PCR Clean-up systems (Promega: Madison, WI, USA). .. Sequencing was performed at the central facilities of the University of Córdoba.

    Article Title: LEMONS – A Tool for the Identification of Splice Junctions in Transcriptomes of Organisms Lacking Reference Genomes
    Article Snippet: The genes analyzed were of varying transcript length, contained different number of exons and were found at various chromosomal locations. .. PCR products were visualized on an EtBr-stained 1% agarose gel, purified using Wizard SV Gel and PCR Clean-up systems (Promega) following the manufacturer’s protocol and sequenced (ABI 3100, BGU sequencing facility) using either one or two of the amplification primers, except for KIA0020. .. Sequence analysis was performed using Sequencher 4.9 (GeneCodes) and MEGA 5 [ ].

    Article Title: An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study
    Article Snippet: The restriction enzymes Nde I, Spe I, Xho I, and Bam HI (New England Biolabs, Ipswich, MA, USA) were used. .. T4 DNA ligase, Wizard plus SV Minipreps DNA purification, and Wizard SV Gel and PCR Clean-up systems (Promega, Madison, WI, USA) were used for ligation and DNA purification. .. Oligonucleotide primers were obtained from Hokkaido System Science Co., Ltd. (Hokkaido, Japan). pET21b was purchased from Merck KGaA/Novagen (Darmstadt, Germany).

    Article Title: PCR Detection of Helicobacter pylori in Clinical Samples
    Article Snippet: 3.5 M sodium acetate (pH 5.2): Dissolve 47.6 g NaOAc·3H2 O (MW 136.08) in 80 mL ultra pure water, adjust to pH 5.2 by glacial acetic acid, and adjust volume to 100 mL. .. Wizard SV Gel and PCR Clean-up systems (Promega, Madison, WI, USA). .. DNA polymerase (see ): Ex Taq polymerase (Takara Biomedicals, Kyoto, Japan) and GoTaq® Green Master Mix is used for first PCR second PCR, respectively. dNTP Mixture (Takara Biomedicals, Kyoto, Japan).

    Article Title: Human papillomavirus in head and neck squamous cell carcinomas in a South African cohort
    Article Snippet: The strips were interpreted using the HPV reference guide provided. .. PCR products were excised from the 1% or 2.5% agarose gels and were purified using Wizard®SV Gel and PCR Clean-Up Systems according to manufacturer's instructions (Promega, Wisconsin, USA). .. Determination of nucleotide sequence of the amplicon was performed using the Big Dye Terminator sequencing ready reaction kit according to manufacturer's instructions (Applied Biosystems, Foster City, CA, USA).

    Article Title: Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival
    Article Snippet: Gel images were captured by using an Image Master VDS-CL gel documentation system (GE Healthcare Life Sciences, Little Chalfont, Buckinghampshire, UK) fitted with a Fujifilm FTI-500 Thermal Imaging system (GE Healthcare). .. DNA fragments were purified from agarose gels using Wizard® SV gel and PCR Clean-Up Systems (Promega) according to the manufacturer’s instructions. .. Purified DNA was stored at −20 °C until required.

    Article Title: Human papillomavirus in head and neck squamous cell carcinomas in a South African cohort
    Article Snippet: The strips were interpreted using the HPV reference guide provided. .. 2.3 PCR products were excised from the 1% or 2.5% agarose gels and were purified using Wizard®SV Gel and PCR Clean-Up Systems according to manufacturer's instructions (Promega, Wisconsin, USA). .. Determination of nucleotide sequence of the amplicon was performed using the Big Dye Terminator sequencing ready reaction kit according to manufacturer's instructions (Applied Biosystems, Foster City, CA, USA).

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: The PCR conditions for amplification of bacterial 16S rRNA genes was the same as described above for the DGGE protocol, except that the primers used for PCR did not have a GC clamp. .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA). .. Purified amplicons were then ligated into the pGEM-T-Easy vector and introduced into competent Escherichia coli JM109 (Promega, USA) following the manufacturer's instructions.

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Enzymes were purchased from New England BioLabs, Inc. (NEB). .. Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega. .. Phusion high-fidelity DNA polymerase from NEB and Kapa Biosystems High Fidelity Readymix from VWR International, LLC were used for PCR.

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Human Embryonic Kidney (HEK293) cells expressing EsemOrco in combination with different EsemORs were produced and cultured as previously described ( ). .. Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega). .. Five micrograms of linearized plasmid and 15 µL of Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) were each diluted separately into 500 µL of Optimem medium (Thermo Fisher Scientific) and incubated for 10 min at room temperature.

    Article Title: Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile
    Article Snippet: Housekeeping genes for MLST were amplified by using primers for V . parahaemolyticus ( ) and from the MLST website (http://pubmlst.org/vparahaemolyticus) developed by Keith Jolley (University of Oxford, Oxford, UK) ( ). .. PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci). .. DNA sequences were analyzed individually and manually assembled.

    Plasmid Preparation:

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: Plasmid DNA was phenol–chloroform extracted, ethanol precipitated and resuspended in deionized and diethyl pyrocarbonate-treated water. .. PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega).

    Article Title: A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage
    Article Snippet: DNA was extracted using the Wizard genomic DNA purification kit (Promega). .. Plasmid and PCR/gel extractions were done using PureYield Plasmid Miniprep and Wizard SV Gel and PCR clean-up systems respectively (Promega). .. Purified DNA from V. cholerae S24 was sequenced at the Wellcome Trust Sanger Institute using Illumina-based technology.

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA). .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA).

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Enzymes were purchased from New England BioLabs, Inc. (NEB). .. Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega. .. Phusion high-fidelity DNA polymerase from NEB and Kapa Biosystems High Fidelity Readymix from VWR International, LLC were used for PCR.

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega). .. The mixture was then added to the culture flask containing ∼70% confluent isogenic tetracycline repressor-expressing (TREx) cells (allowing controlled expression of exogenous OR genes; ) and incubated overnight at 37 °C with 5% CO2 .

    Software:

    Article Title: Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico
    Article Snippet: Primers were designed to amplify regions in the CT domain known to be involved in the sensitivity to ACCase herbicides using the Primer Premier 5.0 software (Premier Biosoft International: Palo Alto, CA, USA). .. PCR products were purified directly or from agarose gels using Wizard SV gel or PCR Clean-up systems (Promega: Madison, WI, USA).

    Article Title: Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile
    Article Snippet: PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci). .. PCR products were purified by using either the Wizard SV Gel or PCR Clean-Up Systems (Promega, Madison, WI, USA) and sequenced in both directions by Macrogen (Seoul, South Korea) or by McLAB (South San Francisco, CA, USA) by using forward and reverse amplification primers or primers M13F and M13R (MLST loci).

    Real-time Polymerase Chain Reaction:

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega). .. Transcription–translation reactions used the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) supplemented with 20 units of Human Placenta RNase Inhibitor (New England Biolabs).

    Denaturing Gradient Gel Electrophoresis:

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: The PCR conditions for amplification of bacterial 16S rRNA genes was the same as described above for the DGGE protocol, except that the primers used for PCR did not have a GC clamp. .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA).

    Agarose Gel Electrophoresis:

    Article Title: LEMONS – A Tool for the Identification of Splice Junctions in Transcriptomes of Organisms Lacking Reference Genomes
    Article Snippet: The genes analyzed were of varying transcript length, contained different number of exons and were found at various chromosomal locations. .. PCR products were visualized on an EtBr-stained 1% agarose gel, purified using Wizard SV Gel and PCR Clean-up systems (Promega) following the manufacturer’s protocol and sequenced (ABI 3100, BGU sequencing facility) using either one or two of the amplification primers, except for KIA0020. .. Sequence analysis was performed using Sequencher 4.9 (GeneCodes) and MEGA 5 [ ].

    Article Title: Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival
    Article Snippet: Agarose gel electrophoresis was used to fractionate digested DNA. .. DNA fragments were purified from agarose gels using Wizard® SV gel and PCR Clean-Up Systems (Promega) according to the manufacturer’s instructions.

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Human Embryonic Kidney (HEK293) cells expressing EsemOrco in combination with different EsemORs were produced and cultured as previously described ( ). .. Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega). .. Five micrograms of linearized plasmid and 15 µL of Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) were each diluted separately into 500 µL of Optimem medium (Thermo Fisher Scientific) and incubated for 10 min at room temperature.

    In Vitro:

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: Paragraph title: In vitro characterization of the riboswitch ... PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega).

    Electrophoresis:

    Article Title: Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival
    Article Snippet: Paragraph title: 4.6. Electrophoresis and Purification of Genomic DNA and PCR Products ... DNA fragments were purified from agarose gels using Wizard® SV gel and PCR Clean-Up Systems (Promega) according to the manufacturer’s instructions.

    Sampling:

    Article Title: Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
    Article Snippet: Clone libraries were constructed using the cDNA synthesized from the 16S rRNA gene transcripts from the 5-day sampling (time at which the first peak of 13 C accumulation in bacterial fraction was observed, ). .. Prior to cloning, the PCR fragments obtained were purified with Wizard® SV Gel and PCR clean-up systems (Promega, USA).

    Produced:

    Article Title: Characterization of Odorant Receptors from a Non-ditrysian Moth, Eriocrania semipurpurella Sheds Light on the Origin of Sex Pheromone Receptors in Lepidoptera
    Article Snippet: Human Embryonic Kidney (HEK293) cells expressing EsemOrco in combination with different EsemORs were produced and cultured as previously described ( ). .. Briefly, EsemOrco-expressing pcDNA4/TO was linearized using FspI (New England Biolabs), resolved on a 0.7% TAE agarose gel, and purified using the Wizard SV Gel and PCR clean-up systems (Promega).

    DNA Purification:

    Article Title: Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour
    Article Snippet: Plasmids were amplified in E. coli Novablue (Novagen) and purified with Wizard Plus SV Minipreps DNA Purification System (Promega). .. PCR products were purified with Wizard Plus SV Gel and PCR Clean-Up Systems (Promega).

    Article Title: A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage
    Article Snippet: DNA was extracted using the Wizard genomic DNA purification kit (Promega). .. Plasmid and PCR/gel extractions were done using PureYield Plasmid Miniprep and Wizard SV Gel and PCR clean-up systems respectively (Promega).

    Article Title: An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study
    Article Snippet: The restriction enzymes Nde I, Spe I, Xho I, and Bam HI (New England Biolabs, Ipswich, MA, USA) were used. .. T4 DNA ligase, Wizard plus SV Minipreps DNA purification, and Wizard SV Gel and PCR Clean-up systems (Promega, Madison, WI, USA) were used for ligation and DNA purification. .. Oligonucleotide primers were obtained from Hokkaido System Science Co., Ltd. (Hokkaido, Japan). pET21b was purchased from Merck KGaA/Novagen (Darmstadt, Germany).

    Article Title: A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface
    Article Snippet: Enzymes were purchased from New England BioLabs, Inc. (NEB). .. Plasmid DNA was purified using Wizard Plus SV miniprep DNA purification systems and DNA fragments were purified with Wizard SV gel and PCR clean-up systems from Promega. .. Phusion high-fidelity DNA polymerase from NEB and Kapa Biosystems High Fidelity Readymix from VWR International, LLC were used for PCR.

    Staining:

    Article Title: Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival
    Article Snippet: The DNA was separated in 0.8% agarose gels and stained with ethidium bromide after mixing with loading dye (50 mL glycerol, 0.25 g bromophenol blue, 5 mL of 0.5 M EDTA (pH 8.0) and 45 mL MQ-H2 O). .. DNA fragments were purified from agarose gels using Wizard® SV gel and PCR Clean-Up Systems (Promega) according to the manufacturer’s instructions.

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    Promega pcr clean up system
    <t>MHV68</t> detection in blood samples from mice infested with naturally infected nymphs or adult ticks. (A) Lanes N1-N7, blood samples of mouse 1-7 exposed to nymphs molted from larvae that had engorged on infected mice; lane N8, blood of mouse infested with uninfected nymphs. (B) Lane A1, blood of mouse infested with uninfected adult ticks; lanes A2-A14, blood samples of 13 mice exposed to adults molted from nymphs that had engorged on infected mice. All blood samples were collected 15 days after tick infestation. Lanes L1, L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene <t>PCR</t> product of 382 base pairs.
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    MHV68 detection in blood samples from mice infested with naturally infected nymphs or adult ticks. (A) Lanes N1-N7, blood samples of mouse 1-7 exposed to nymphs molted from larvae that had engorged on infected mice; lane N8, blood of mouse infested with uninfected nymphs. (B) Lane A1, blood of mouse infested with uninfected adult ticks; lanes A2-A14, blood samples of 13 mice exposed to adults molted from nymphs that had engorged on infected mice. All blood samples were collected 15 days after tick infestation. Lanes L1, L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Tick-Borne Transmission of Murine Gammaherpesvirus 68

    doi: 10.3389/fcimb.2017.00458

    Figure Lengend Snippet: MHV68 detection in blood samples from mice infested with naturally infected nymphs or adult ticks. (A) Lanes N1-N7, blood samples of mouse 1-7 exposed to nymphs molted from larvae that had engorged on infected mice; lane N8, blood of mouse infested with uninfected nymphs. (B) Lane A1, blood of mouse infested with uninfected adult ticks; lanes A2-A14, blood samples of 13 mice exposed to adults molted from nymphs that had engorged on infected mice. All blood samples were collected 15 days after tick infestation. Lanes L1, L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs.

    Article Snippet: Nested PCR products amplified from DNA isolated from the blood of three MHV68 positive mice and salivary glands of two MHV68 positive ticks were purified with Wizard® SV Gel and PCR Clean-up System (Promega, USA) according to the manufacturer's instructions.

    Techniques: Mouse Assay, Infection, Polymerase Chain Reaction

    MHV68 detection in lung and spleen samples from mice infested with F1 i adults and in F1 i female ticks. (A) Lung (a,c) and spleen (b,d) samples of mice infested with F1 i adults examined by nested PCR (a,b) and RT-PCR (c,d) . Lanes 1a 26 , 2a 17 , 3a 18 , 4a 28 , 5a 19 , 6a 20 , 7a 32 , 8a 33 , and 9a 24 sa mples of mice infested with F1 i adult ticks; A35, A36, samples of control mice infested with F1 c adults. Lanes L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs; ++ indicates MHV68 M3 gene nested RT-PCR product of 241 base pairs. (B) Semi-thin sections of frozen whole body of F1 i females fed for 4 days. (a,b) F1 i tick from mouse 3a 18 and 5a 19 stained with anti-MHV68 rabbit polyclonal serum; (c) uninfected tick (F6 generation of breeding) stained with anti-MHV68 rabbit polyclonal serum; (d) F1 i tick from mouse 3a 18 stained with rabbit polyclonal serum against PB1-F2 protein of influenza virus A (H1N1) (negative control). MD, cells of midgut diverticula; L, lumen of midgut diverculum. Scale bar, 200 μm.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Tick-Borne Transmission of Murine Gammaherpesvirus 68

    doi: 10.3389/fcimb.2017.00458

    Figure Lengend Snippet: MHV68 detection in lung and spleen samples from mice infested with F1 i adults and in F1 i female ticks. (A) Lung (a,c) and spleen (b,d) samples of mice infested with F1 i adults examined by nested PCR (a,b) and RT-PCR (c,d) . Lanes 1a 26 , 2a 17 , 3a 18 , 4a 28 , 5a 19 , 6a 20 , 7a 32 , 8a 33 , and 9a 24 sa mples of mice infested with F1 i adult ticks; A35, A36, samples of control mice infested with F1 c adults. Lanes L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs; ++ indicates MHV68 M3 gene nested RT-PCR product of 241 base pairs. (B) Semi-thin sections of frozen whole body of F1 i females fed for 4 days. (a,b) F1 i tick from mouse 3a 18 and 5a 19 stained with anti-MHV68 rabbit polyclonal serum; (c) uninfected tick (F6 generation of breeding) stained with anti-MHV68 rabbit polyclonal serum; (d) F1 i tick from mouse 3a 18 stained with rabbit polyclonal serum against PB1-F2 protein of influenza virus A (H1N1) (negative control). MD, cells of midgut diverticula; L, lumen of midgut diverculum. Scale bar, 200 μm.

    Article Snippet: Nested PCR products amplified from DNA isolated from the blood of three MHV68 positive mice and salivary glands of two MHV68 positive ticks were purified with Wizard® SV Gel and PCR Clean-up System (Promega, USA) according to the manufacturer's instructions.

    Techniques: Mouse Assay, Nested PCR, Reverse Transcription Polymerase Chain Reaction, Staining, Negative Control

    Tick-borne virus transmission and tick infection following injection of ticks with MHV68. (A) Virus detected by nested PCR. Lanes S1-S7, salivary glands of virus-injected ticks fed on uninfected mice for 5 days; lanes B1-B5, blood samples of five mice 15 days after tick infestation; L1, HyperLadder; L2, GeneRuler DNA ladder; lane C1, MHV68 DNA (positive control); lane C2, nested PCR without template (negative control); lane C3, MHV68 DNA nested PCR 1st round product with outer primers (positive control); lane C4, 1st PCR round with outer primers without template (negative control). ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs. (B) Infectivity as determined by plaque formation in BHK-21 cells. Cells inoculated with homogenate of virus infected tick and observed by (a) light microscopy (magnification x50) and (b–d) specific immunofluorescence staining (magnification x200). (b) Single plaque shown of a maximum 5 plaques observed per tick; (c) control, uninfected cells; and (d) cells inoculated with homogenate of uninfected tick.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Tick-Borne Transmission of Murine Gammaherpesvirus 68

    doi: 10.3389/fcimb.2017.00458

    Figure Lengend Snippet: Tick-borne virus transmission and tick infection following injection of ticks with MHV68. (A) Virus detected by nested PCR. Lanes S1-S7, salivary glands of virus-injected ticks fed on uninfected mice for 5 days; lanes B1-B5, blood samples of five mice 15 days after tick infestation; L1, HyperLadder; L2, GeneRuler DNA ladder; lane C1, MHV68 DNA (positive control); lane C2, nested PCR without template (negative control); lane C3, MHV68 DNA nested PCR 1st round product with outer primers (positive control); lane C4, 1st PCR round with outer primers without template (negative control). ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs. (B) Infectivity as determined by plaque formation in BHK-21 cells. Cells inoculated with homogenate of virus infected tick and observed by (a) light microscopy (magnification x50) and (b–d) specific immunofluorescence staining (magnification x200). (b) Single plaque shown of a maximum 5 plaques observed per tick; (c) control, uninfected cells; and (d) cells inoculated with homogenate of uninfected tick.

    Article Snippet: Nested PCR products amplified from DNA isolated from the blood of three MHV68 positive mice and salivary glands of two MHV68 positive ticks were purified with Wizard® SV Gel and PCR Clean-up System (Promega, USA) according to the manufacturer's instructions.

    Techniques: Transmission Assay, Infection, Injection, Nested PCR, Mouse Assay, Positive Control, Negative Control, Polymerase Chain Reaction, Light Microscopy, Immunofluorescence, Staining

    MHV68 detection in blood samples from mice infested with F0 females or F1 nymphs. (A) Lanes A15-A34, blood samples of 20 mice infested with infected F0 females; blood collected 15 days after tick infestation. ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs. (B) Lanes 1n 26 , 2n 17 , 3n 18 , 4n 28 , 5n 19 , 6n 20 , 7n 32 , 8n 33 , and 9n 24 , blood samples of mice infested with F1 i nymphs; N9, N10 blood samples of control mice infested with F1 c nymphs. + Indicates MHV68 M3 gene one step RT-PCR product of 520 base pairs. Lanes L2, C1–C4 as for Figure 1A .

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Tick-Borne Transmission of Murine Gammaherpesvirus 68

    doi: 10.3389/fcimb.2017.00458

    Figure Lengend Snippet: MHV68 detection in blood samples from mice infested with F0 females or F1 nymphs. (A) Lanes A15-A34, blood samples of 20 mice infested with infected F0 females; blood collected 15 days after tick infestation. ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs. (B) Lanes 1n 26 , 2n 17 , 3n 18 , 4n 28 , 5n 19 , 6n 20 , 7n 32 , 8n 33 , and 9n 24 , blood samples of mice infested with F1 i nymphs; N9, N10 blood samples of control mice infested with F1 c nymphs. + Indicates MHV68 M3 gene one step RT-PCR product of 520 base pairs. Lanes L2, C1–C4 as for Figure 1A .

    Article Snippet: Nested PCR products amplified from DNA isolated from the blood of three MHV68 positive mice and salivary glands of two MHV68 positive ticks were purified with Wizard® SV Gel and PCR Clean-up System (Promega, USA) according to the manufacturer's instructions.

    Techniques: Mouse Assay, Infection, Nested PCR, Reverse Transcription Polymerase Chain Reaction

    Chromosome walking for the Cyanophage P4 and an Arabidopsis mutant using the SiteFinding-PCR method. SiteFinder-1 was used to initialize the SiteFinding reaction. ( A ) Products of the secondary round of PCR (lanes 1–2 for P4 Cyanophage and 3–4 for Arabidopsis mutant). Lanes 1–4 contained PCR products obtained with primer couples SFP2/P4-2, SFP2/P4-3, SFP2/DL2 and SFP2/DL3, respectively. ( B ) Cloned and sequenced PCR products. Lanes 1 and 2 both showed three specific products (A), and the largest one in lane 1 was cloned and sequenced as indicated in (I). There were another two GCCT sites on the 4617 bp fragment of the Cyanophage P4 sequence, which are indicated with the black arrowheads in (I) (first site and second site). These findings were consistent with the gel electrophoresis results (white arrows in lane 1). Lanes 3 and 4 both showed two specific products (A), and the larger one in lane 3 was cloned and sequenced as indicated in (II). There was another GCCT site on the 2270 bp fragment of the Arabidopsis DNA as indicated with the black arrowheads in (II) (first site), which was also consistent with the gel electrophoresis (white arrow in lane 3).

    Journal: Nucleic Acids Research

    Article Title: SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

    doi: 10.1093/nar/gni124

    Figure Lengend Snippet: Chromosome walking for the Cyanophage P4 and an Arabidopsis mutant using the SiteFinding-PCR method. SiteFinder-1 was used to initialize the SiteFinding reaction. ( A ) Products of the secondary round of PCR (lanes 1–2 for P4 Cyanophage and 3–4 for Arabidopsis mutant). Lanes 1–4 contained PCR products obtained with primer couples SFP2/P4-2, SFP2/P4-3, SFP2/DL2 and SFP2/DL3, respectively. ( B ) Cloned and sequenced PCR products. Lanes 1 and 2 both showed three specific products (A), and the largest one in lane 1 was cloned and sequenced as indicated in (I). There were another two GCCT sites on the 4617 bp fragment of the Cyanophage P4 sequence, which are indicated with the black arrowheads in (I) (first site and second site). These findings were consistent with the gel electrophoresis results (white arrows in lane 1). Lanes 3 and 4 both showed two specific products (A), and the larger one in lane 3 was cloned and sequenced as indicated in (II). There was another GCCT site on the 2270 bp fragment of the Arabidopsis DNA as indicated with the black arrowheads in (II) (first site), which was also consistent with the gel electrophoresis (white arrow in lane 3).

    Article Snippet: (i) The PCR products were directly cleaved as follows: 43 μl of PCR products were mixed with 5 μl of 10× enzyme buffer and 2 μl (20 U) of NotI (New England Biolabs, USA), and then incubated overnight at 37°C. (ii) The digested PCR products were separated by electrophoresis on 1.2% agarose gels and purified with Wizard® SV Gel and PCR Clean-Up System (Promega, USA). (iii) The purified DNA was cloned into the pBluescript SK(+) vector between the restriction sites of NotI and EcoRV.

    Techniques: Chromosome Walking, Mutagenesis, Polymerase Chain Reaction, Clone Assay, Sequencing, Nucleic Acid Electrophoresis

    Identification of T-DNA insertion sites of 14 Arabidopsis mutants with SiteFinding-PCR. Lanes II and III: the products generated by SFP2/DL2 and SFP2/DL3, respectively. Samples are labeled as S1–S14. ( A ) The products of S1–S7, initially primed by SiteFinder-1. White arrows in lane II indicated the cloned products. Digitals in boxed areas show different insertion sites. As for S3, 1-1 and 1-2 show two different products of the 1st insertion site; 2-1, 2-2 and 2-3 in S3 refer to three products of the 2nd insertion site. ( B ) The products of samples 8–14, initially primed by SiteFinder-2. White arrows in lane II indicated the cloned products. ( C ) Comparison of the 3′ end of the SiteFinder with the largest or larger fragment. Bold black line segments and grey line segments represent the T-DNA and Arabidopsis DNAs, respectively. The arrows represent SFP2. The sizes of the largest or larger specific fragment of the inserted site are indicated on the right, and the smaller ones are marked in the middle with blue bars. NNNNNN with 0–3 mismatch nt (courier) helped the 3′ ends (underlined with blue bars) of SiteFinder-1 and SiteFinder-2 to anneal accurately with 3′-CGGA-5′ (GCCT site) and 3′-CGCG-5′ (GCGC site), respectively.

    Journal: Nucleic Acids Research

    Article Title: SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

    doi: 10.1093/nar/gni124

    Figure Lengend Snippet: Identification of T-DNA insertion sites of 14 Arabidopsis mutants with SiteFinding-PCR. Lanes II and III: the products generated by SFP2/DL2 and SFP2/DL3, respectively. Samples are labeled as S1–S14. ( A ) The products of S1–S7, initially primed by SiteFinder-1. White arrows in lane II indicated the cloned products. Digitals in boxed areas show different insertion sites. As for S3, 1-1 and 1-2 show two different products of the 1st insertion site; 2-1, 2-2 and 2-3 in S3 refer to three products of the 2nd insertion site. ( B ) The products of samples 8–14, initially primed by SiteFinder-2. White arrows in lane II indicated the cloned products. ( C ) Comparison of the 3′ end of the SiteFinder with the largest or larger fragment. Bold black line segments and grey line segments represent the T-DNA and Arabidopsis DNAs, respectively. The arrows represent SFP2. The sizes of the largest or larger specific fragment of the inserted site are indicated on the right, and the smaller ones are marked in the middle with blue bars. NNNNNN with 0–3 mismatch nt (courier) helped the 3′ ends (underlined with blue bars) of SiteFinder-1 and SiteFinder-2 to anneal accurately with 3′-CGGA-5′ (GCCT site) and 3′-CGCG-5′ (GCGC site), respectively.

    Article Snippet: (i) The PCR products were directly cleaved as follows: 43 μl of PCR products were mixed with 5 μl of 10× enzyme buffer and 2 μl (20 U) of NotI (New England Biolabs, USA), and then incubated overnight at 37°C. (ii) The digested PCR products were separated by electrophoresis on 1.2% agarose gels and purified with Wizard® SV Gel and PCR Clean-Up System (Promega, USA). (iii) The purified DNA was cloned into the pBluescript SK(+) vector between the restriction sites of NotI and EcoRV.

    Techniques: Polymerase Chain Reaction, Generated, Labeling, Clone Assay

    Schematic outline of SiteFinding-PCR method for chromosome walking. Known and unknown sequences are depicted with thick and thin lines, respectively. Blue segments show the expected SiteFinder targets. Gene-specific primers (GSPs) 1–3 can anneal with known sequences (white arrows). (1) Original genomic double-strand templates, showing target molecule and non-target molecules. (2) SiteFinding reaction: after low temperature priming by a SiteFinder, one strand of the target gene was replaced by long Taq DNA polymerase, which generated double-stranded target molecules of different lengths. (3) Nested PCR: the target DNA was exponentially amplified by nested PCR with GSPs and SiteFinder primers (SFPs) 1 and 2, while non-target gene amplification was suppressed by the stem–loop structure of the DNA. (4) Cloning target molecules: after being cleaved with NotI, the PCR products (generated by GSP2 and SFP2) were purified by agarose gel electrophoresis, and then the purified DNA was cloned into a pBluescript SK(+) vector linearized by NotI and EcoRV. (5) Screening and sequencing: the clones were screened by colony-PCR with the third gene-specific primer (GSP3) and a vector primer (M13 reverse primer or T3 primer), and the target molecules were screened out and sequenced subsequently.

    Journal: Nucleic Acids Research

    Article Title: SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

    doi: 10.1093/nar/gni124

    Figure Lengend Snippet: Schematic outline of SiteFinding-PCR method for chromosome walking. Known and unknown sequences are depicted with thick and thin lines, respectively. Blue segments show the expected SiteFinder targets. Gene-specific primers (GSPs) 1–3 can anneal with known sequences (white arrows). (1) Original genomic double-strand templates, showing target molecule and non-target molecules. (2) SiteFinding reaction: after low temperature priming by a SiteFinder, one strand of the target gene was replaced by long Taq DNA polymerase, which generated double-stranded target molecules of different lengths. (3) Nested PCR: the target DNA was exponentially amplified by nested PCR with GSPs and SiteFinder primers (SFPs) 1 and 2, while non-target gene amplification was suppressed by the stem–loop structure of the DNA. (4) Cloning target molecules: after being cleaved with NotI, the PCR products (generated by GSP2 and SFP2) were purified by agarose gel electrophoresis, and then the purified DNA was cloned into a pBluescript SK(+) vector linearized by NotI and EcoRV. (5) Screening and sequencing: the clones were screened by colony-PCR with the third gene-specific primer (GSP3) and a vector primer (M13 reverse primer or T3 primer), and the target molecules were screened out and sequenced subsequently.

    Article Snippet: (i) The PCR products were directly cleaved as follows: 43 μl of PCR products were mixed with 5 μl of 10× enzyme buffer and 2 μl (20 U) of NotI (New England Biolabs, USA), and then incubated overnight at 37°C. (ii) The digested PCR products were separated by electrophoresis on 1.2% agarose gels and purified with Wizard® SV Gel and PCR Clean-Up System (Promega, USA). (iii) The purified DNA was cloned into the pBluescript SK(+) vector between the restriction sites of NotI and EcoRV.

    Techniques: Polymerase Chain Reaction, Chromosome Walking, Generated, Nested PCR, Amplification, Clone Assay, Purification, Agarose Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Sequencing

    ( A ) Sequences of two SiteFinders and their primers (SFP1 and SFP2). SiteFinder-1 and 2 differed only at their 3′ ends, and contained a rare restriction enzyme site for NotI, which facilitates cloning with commonly used vectors, such as pBluescript SK(+). The four specific nucleotides underlined with blue bar at the 3′ ends of the SiteFinders, with the help of NNNNNN, were used to anneal with the complimentary sites on genomic DNAs at low temperature and initiate SiteFinding-PCR. SFP1 and SFP2 were used in the primary and secondary reactions, respectively. ( B ) Three gene-specific primers (GSPs) for Cyanophage P4 (P4-1, P4-2 and P4-3) are indicated by black arrows. The distance from P4-2 to P4-3 was 31 bp. ( C ) Three GSPs for Arabidopsis T-DNA insertion mutants (DL1, DL2 and DL3) designed based on the T-DNA sequence of pSki015 are indicated by black arrows. The distance from DL2 to DL3 was 59 bp.

    Journal: Nucleic Acids Research

    Article Title: SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

    doi: 10.1093/nar/gni124

    Figure Lengend Snippet: ( A ) Sequences of two SiteFinders and their primers (SFP1 and SFP2). SiteFinder-1 and 2 differed only at their 3′ ends, and contained a rare restriction enzyme site for NotI, which facilitates cloning with commonly used vectors, such as pBluescript SK(+). The four specific nucleotides underlined with blue bar at the 3′ ends of the SiteFinders, with the help of NNNNNN, were used to anneal with the complimentary sites on genomic DNAs at low temperature and initiate SiteFinding-PCR. SFP1 and SFP2 were used in the primary and secondary reactions, respectively. ( B ) Three gene-specific primers (GSPs) for Cyanophage P4 (P4-1, P4-2 and P4-3) are indicated by black arrows. The distance from P4-2 to P4-3 was 31 bp. ( C ) Three GSPs for Arabidopsis T-DNA insertion mutants (DL1, DL2 and DL3) designed based on the T-DNA sequence of pSki015 are indicated by black arrows. The distance from DL2 to DL3 was 59 bp.

    Article Snippet: (i) The PCR products were directly cleaved as follows: 43 μl of PCR products were mixed with 5 μl of 10× enzyme buffer and 2 μl (20 U) of NotI (New England Biolabs, USA), and then incubated overnight at 37°C. (ii) The digested PCR products were separated by electrophoresis on 1.2% agarose gels and purified with Wizard® SV Gel and PCR Clean-Up System (Promega, USA). (iii) The purified DNA was cloned into the pBluescript SK(+) vector between the restriction sites of NotI and EcoRV.

    Techniques: Clone Assay, Polymerase Chain Reaction, Sequencing

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Following isolation from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), inserts were assembled into Hin dIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method.

    Techniques: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Following isolation from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), inserts were assembled into Hin dIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method.

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Liquid Chromatography, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Article Snippet: Following isolation from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), inserts were assembled into Hin dIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method.

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Selection, Marker, Homologous Recombination, Ligation, Transformation Assay, Sequencing

    Single cell PCR analysis of the target region within the CD2 locus. Peripheral blood lymphocytes were surface stained for CD2, CD4, CD8, TCRβ and CD19 and the following populations within live cell and lymphocyte gates single cell-sorted by flow cytometry: TCRβ + CD4 + CD2 + , TCRβ + CD8 + CD2 + , TCRβ + CD4 + CD2 − , TCRβ + CD8 + CD2 − , CD19 + . A 253 bp long region including the gRNA target was amplified by two rounds of nested PCR. Products were cloned in pGEM-T and pGEM-Teasy and sequenced. For wildtype controls the single cell PCR products were column-purified and sequenced directly. ( a ) Agarose gel showing PCR products of single cell amplicons of the target region within the CD2 gene locus from sorted peripheral blood single cells. Lanes 6 and 12 on both sides of the marker are H 2 0 negative controls. ( b ) Table showing the number of obtained mutations in amplicons of double-transgenic and wildtype cells. ( c ) Alignment of the obtained sequences from the CD2 gene amplicon. Indicated is the number (left side) and the cell type as well as the outcome of the mutation (OF: out of frame, IF: in frame) (right side) of the respective sequence. – indicates a deletion and red bases insertions.

    Journal: Scientific Reports

    Article Title: T cell-specific inactivation of mouse CD2 by CRISPR/Cas9

    doi: 10.1038/srep21377

    Figure Lengend Snippet: Single cell PCR analysis of the target region within the CD2 locus. Peripheral blood lymphocytes were surface stained for CD2, CD4, CD8, TCRβ and CD19 and the following populations within live cell and lymphocyte gates single cell-sorted by flow cytometry: TCRβ + CD4 + CD2 + , TCRβ + CD8 + CD2 + , TCRβ + CD4 + CD2 − , TCRβ + CD8 + CD2 − , CD19 + . A 253 bp long region including the gRNA target was amplified by two rounds of nested PCR. Products were cloned in pGEM-T and pGEM-Teasy and sequenced. For wildtype controls the single cell PCR products were column-purified and sequenced directly. ( a ) Agarose gel showing PCR products of single cell amplicons of the target region within the CD2 gene locus from sorted peripheral blood single cells. Lanes 6 and 12 on both sides of the marker are H 2 0 negative controls. ( b ) Table showing the number of obtained mutations in amplicons of double-transgenic and wildtype cells. ( c ) Alignment of the obtained sequences from the CD2 gene amplicon. Indicated is the number (left side) and the cell type as well as the outcome of the mutation (OF: out of frame, IF: in frame) (right side) of the respective sequence. – indicates a deletion and red bases insertions.

    Article Snippet: The Cas9-gRNA efficiency test of four different gRNAs targeting CD2 was performed in vitro on column-purified 839 bp CD2 PCR products (Wizard SV Gel and PCR Clean-UP System, Promega) which were amplified using Taq PCR Master Mix Kit (Qiagen) and following primers: CD2 cut F: 5′-cct gac aga aag aac tc-3′, CD2 cut R: 5′-ctc act gct cct agg ca-3′.

    Techniques: Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry, Amplification, Nested PCR, Clone Assay, Purification, Agarose Gel Electrophoresis, Marker, Transgenic Assay, Mutagenesis, Sequencing

    Functional analysis ( a ) In vitro test of gRNA efficiency. gRNA(CD2.0) as well as three controls (CD2.1,CD2.2, CD2.3) were incubated with a PCR product for the indicated period of time. Digests were separated on an agarose gel. ( b ) Analysis of the gRNA efficiency. The intensities of the bands resulting from gRNA/Cas9-digested PCR product of three different experiments were analyzed using ImageJ. Shown is the mean and standard error of the mean.

    Journal: Scientific Reports

    Article Title: T cell-specific inactivation of mouse CD2 by CRISPR/Cas9

    doi: 10.1038/srep21377

    Figure Lengend Snippet: Functional analysis ( a ) In vitro test of gRNA efficiency. gRNA(CD2.0) as well as three controls (CD2.1,CD2.2, CD2.3) were incubated with a PCR product for the indicated period of time. Digests were separated on an agarose gel. ( b ) Analysis of the gRNA efficiency. The intensities of the bands resulting from gRNA/Cas9-digested PCR product of three different experiments were analyzed using ImageJ. Shown is the mean and standard error of the mean.

    Article Snippet: The Cas9-gRNA efficiency test of four different gRNAs targeting CD2 was performed in vitro on column-purified 839 bp CD2 PCR products (Wizard SV Gel and PCR Clean-UP System, Promega) which were amplified using Taq PCR Master Mix Kit (Qiagen) and following primers: CD2 cut F: 5′-cct gac aga aag aac tc-3′, CD2 cut R: 5′-ctc act gct cct agg ca-3′.

    Techniques: Functional Assay, In Vitro, Incubation, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Conditional gene editing. ( a ) Scheme of the concept of conditional gene editing. In the Cas9 driver strain, the nuclease is placed under control of a cell type or lineage specific promoter. The gRNA construct is driven by the ubiquitous U6 promoter. Both transgenes are co-injected into oocytes. In double-transgenic animals, cell-type specific gene deletions are induced. ( b ) Scheme of constructs used for the CD4dsCas9/U6gRNA(CD2) mouse strain. The two used linearized plasmids are shown. First, distal and proximal enhancer, CD4 promoter followed by exon 1, part of exon 2 and Cas9 with a PolyA at the end. Second, the U6 promoter driven gRNA specific for CD2 followed by the U6 terminator (U6 T). ( c ) PCR analysis of tail biopsies for presence of CD4dsCas9 (835 bp amplicon) (lanes 1 and 5) and U6gRNA(CD2.0) (407 bp amplicon) (lanes 1 and 5) by PCR. DNA from a wildtype (WT) mouse as well as H 2 0 were run as a negative control. 2, 3 and 4 were non-transgenic litter mates.

    Journal: Scientific Reports

    Article Title: T cell-specific inactivation of mouse CD2 by CRISPR/Cas9

    doi: 10.1038/srep21377

    Figure Lengend Snippet: Conditional gene editing. ( a ) Scheme of the concept of conditional gene editing. In the Cas9 driver strain, the nuclease is placed under control of a cell type or lineage specific promoter. The gRNA construct is driven by the ubiquitous U6 promoter. Both transgenes are co-injected into oocytes. In double-transgenic animals, cell-type specific gene deletions are induced. ( b ) Scheme of constructs used for the CD4dsCas9/U6gRNA(CD2) mouse strain. The two used linearized plasmids are shown. First, distal and proximal enhancer, CD4 promoter followed by exon 1, part of exon 2 and Cas9 with a PolyA at the end. Second, the U6 promoter driven gRNA specific for CD2 followed by the U6 terminator (U6 T). ( c ) PCR analysis of tail biopsies for presence of CD4dsCas9 (835 bp amplicon) (lanes 1 and 5) and U6gRNA(CD2.0) (407 bp amplicon) (lanes 1 and 5) by PCR. DNA from a wildtype (WT) mouse as well as H 2 0 were run as a negative control. 2, 3 and 4 were non-transgenic litter mates.

    Article Snippet: The Cas9-gRNA efficiency test of four different gRNAs targeting CD2 was performed in vitro on column-purified 839 bp CD2 PCR products (Wizard SV Gel and PCR Clean-UP System, Promega) which were amplified using Taq PCR Master Mix Kit (Qiagen) and following primers: CD2 cut F: 5′-cct gac aga aag aac tc-3′, CD2 cut R: 5′-ctc act gct cct agg ca-3′.

    Techniques: Construct, Injection, Transgenic Assay, Polymerase Chain Reaction, Amplification, Negative Control