Journal: PLoS ONE

Article Title: Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells

doi: 10.1371/journal.pone.0139176

Figure Lengend Snippet: Agarose gel electrophoresis analysis of 3 combinant expression plasmid. The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β 2 -AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.

Article Snippet: The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA).

Techniques: Agarose Gel Electrophoresis, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Recombinant, Electrophoresis, Amplification

Journal: Nature genetics

Article Title: Selective propagation of functional mtDNA during oogenesis restricts the transmission of a deleterious mitochondrial variant

doi: 10.1038/ng.2920

Figure Lengend Snippet: mtDNA replication is reduced in 16-cell cysts of homoplasmic mt:CoI T300I germaria at 29°C. ( a ) Mutated nucleotide and amino acid residue (red) in mt:CoI T300I , including XhoI site (yellow). ( b ) Eclosion rate (mean±s.d., from total > 50 animals in 5 groups) of wt, homoplasmic and heteroplasmic mt:CoI T300I animals at 18°C and 29°C. * indicates zero mt:CoI T300I eclosed at 29°C. ( c ) COX activities of wt and mt:CoI T300I flies were examined on consecutive days shifting to 29°C. Data represent 3 biological replicates. Values were normalized with average COX activities of wt at 29°C 0 day and shown as mean±s.d. ( d ) Quantification of EdU puncta (mean±s.d.) in posterior cyst of region 2B at 18°C (wt n= 7, mt:CoI T300I n= 7) or 29°C (wt n= 5, mt:CoI T300I n= 8). The number of EdU puncta in mt:CoI T300I at 29°C is significantly less than that at 18°C or wild type at 29°C ( P

Article Snippet: Molecular confirmation and quantification of heteroplasmy A 4 kb mtDNA fragment spanning the XhoI site in mt:CoI was PCR amplified from total animal DNA as described previously , and purified using the Thermo Scientific gel purification kit.

Techniques:

Journal: Nature genetics

Article Title: Selective propagation of functional mtDNA during oogenesis restricts the transmission of a deleterious mitochondrial variant

doi: 10.1038/ng.2920

Figure Lengend Snippet: Germline selection against mt:CoI T300I at the restrictive temperature. ( a ) Frequency of mt:CoI T300I mutation in heteroplasmic flies maintained at 29°C or 18°C over generations. ( b ) XhoI digestion of PCR fragment spanning mt:CoI , amplified from single larvae produced by the same heteroplasmic mother at 18°C or 29°C. ( c ) Proportion of mutant mtDNA in 10 single larvae at 18°C or 29°C, calculated by quantifying band intensity in b . Average level of mutant mtDNA, 18°C, 83±5%; 29°C, 60±9%, n=10, P

Article Snippet: Molecular confirmation and quantification of heteroplasmy A 4 kb mtDNA fragment spanning the XhoI site in mt:CoI was PCR amplified from total animal DNA as described previously , and purified using the Thermo Scientific gel purification kit.

Techniques: Selection, Mutagenesis, Polymerase Chain Reaction, Amplification, Produced

Journal: mSphere

Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

doi: 10.1128/mSphere.00446-17

Figure Lengend Snippet: Southern blot analysis of Δ pksP mutant generated in the Af293 background. (A) Schematic representation of the genomic locus of the Af293 and Δ pksP strains. Deletion of the pksP gene was carried out using the HygR cassette. The cleavage sites of the dual in vitro -assembled Cas9 RNPs are marked by thick vertical lines. XhoI cutting sites are indicated in the pksP locus of the wild-type and Δ pksP strains. (B) Southern blot analysis of 6 arbitrarily selected colonies after digesting genomic DNA with the XhoI restriction enzyme. The wild type (WT) produced a 1.8-kb band that matches the expected wild-type banding pattern. Lanes 1, 2, 4, 5, and 6 displayed a 3.8-kb band which matches the expected pksP deletion banding pattern. The colony in lane 3 displayed a 7.6-kb band, likely containing a tandem integration of the HygR repair template at the pksP locus.

Article Snippet: Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

Techniques: Southern Blot, Mutagenesis, Generated, In Vitro, Produced

Journal: The Korean Journal of Parasitology

Article Title: Evaluation of Protective Immune Response Induced by a DNA Vaccine Encoding GRA8 against Acute Toxoplasmosis in a Murine Model

doi: 10.3347/kjp.2018.56.4.325

Figure Lengend Snippet: T. gondii GRA8 was expressed successfully in ARPE-19 cells. (A) The coding sequence of the T. gondii GRA8 gene was amplified by PCR (814 bp) from the cDNA of T. gondii strain GFP-RH. (B) The PCR products were digested with the appropriate restriction enzymes and cloned into the XhoI and EcoRI sites of the pDsRed2-N1 expression vector, which contains the DsRed open reading frame. The constructed recombinant plasmid was named pDsRed2-GRA8. (C) ARPE-19 cells transfected with pDsRed2-GRA8 showed specific expression of GRA8 mRNA. (D) pDsRed2-GRA8 and the pDsRed2-N1 empty vector both expressed the DsRed2 protein, which localized to the cytoplasm of the transfected cells.

Article Snippet: The GRA8 fragments were cleaved from pGEM-GRA8 by using the restriction endonucleases XhoI and EcoRI and then were subcloned into the corresponding sites of the pDsRed2-N1 vector (Invitrogen Life Technologies) to construct pDsRed2-N1-GRA8 (pDsRed2-GRA8).

Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Expressing, Plasmid Preparation, Construct, Recombinant, Transfection

Journal: Research in Pharmaceutical Sciences

Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression

doi: 10.4103/1735-5362.192485

Figure Lengend Snippet: (A) Digestion on extracted plasmid of one of positive clones with NheI and XhoI confirmed HBsAg fragment insertion in pcDNA vector. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested circular plasmid. Column 3: 696 bp and 5501 bp bands that confirmed cloning. (B) Digestion of recombinant plasmid with BglII enzyme. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested plasmid. Column 3: A 6197 bp linearized plasmid.

Article Snippet: The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately.

Techniques: Plasmid Preparation, Clone Assay, Recombinant

Journal: Iranian Journal of Veterinary Research

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system

doi:

Figure Lengend Snippet: Construction and cloning of HA1 protein. (A) Digestion of pUC57 vector containing HA1 gene by enzymes BamHI and XhoI. M: Molecular marker. Lanes 1 and 2: Digested plasmid and HA1 gene. (B) Digestion of pET28a-HA1 vector by enzymes BamHI and XhoI. M: Molecular marker. Lane 1: Digested pET and HA1 gene. (C) PCR analysis for detection of HA1 gene in transformed E. coli clonies contained pET28a-HA1 construct by HA1 specific primers. M: Molecular marker. Lane 1: Positive control, Lane 2: Transformed bacterial clony (The 329 bp band was clear), and Lane 3: Negative control

Article Snippet: The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector.

Techniques: Clone Assay, Plasmid Preparation, Marker, Positron Emission Tomography, Polymerase Chain Reaction, Transformation Assay, Construct, Positive Control, Negative Control