Structured Review

TaKaRa ecori
(a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection <t>DNA</t> probe (b, horizontal bar) that had <t>EcoRI</t> digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.
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Images

1) Product Images from "Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria"

Article Title: Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria

Journal: Fems Microbiology Letters

doi: 10.1111/j.1574-6968.2008.01184.x

(a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection DNA probe (b, horizontal bar) that had EcoRI digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.
Figure Legend Snippet: (a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection DNA probe (b, horizontal bar) that had EcoRI digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.

Techniques Used: Plasmid Preparation, Homologous Recombination, Produced, Mutagenesis, Hybridization, Amplification, Labeling, DNA Labeling, Agarose Gel Electrophoresis, SDS Page, Staining

2) Product Images from "RUNX1-Evi-1 fusion gene inhibited differentiation and apoptosis in myelopoiesis: an in vivo study"

Article Title: RUNX1-Evi-1 fusion gene inhibited differentiation and apoptosis in myelopoiesis: an in vivo study

Journal: BMC Cancer

doi: 10.1186/s12885-015-1961-y

Generation of Tg(RE:HSE:EGFP) zebrafish line. ( a ) Schematic diagram of the structure of PSGH2/RUNX1-Evi-1 recombinant plasmid. A human-RUNX1-Evi-1 fragment was cloned into the EcoRI and EcoRV sites of the PSGH2 vector. ( b ) A schematic presentation of the eight multimerized heat shock element (HSE) promoter, which is flanked by two minimal promoters in opposed orientation (black arrowhead) to bidirectionally induce EGFP and RUNX1-Evi-1 expression. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. ( c ) Transgenic verification by PCR: M: TAKARA DL2000 marker; lane 1 and 2: wild type and Tg(RE:HSE:EGFP) zebrafish larvae at 3 dpf, respectively; lane 3: PSGH2/RUNX1-Evi-1 plasmid; lane 4: double distilled water. ( d ) EGFP expression in Tg(RE:HSE:EGFP) zebrafish F2 generation at 3dpf (×4)
Figure Legend Snippet: Generation of Tg(RE:HSE:EGFP) zebrafish line. ( a ) Schematic diagram of the structure of PSGH2/RUNX1-Evi-1 recombinant plasmid. A human-RUNX1-Evi-1 fragment was cloned into the EcoRI and EcoRV sites of the PSGH2 vector. ( b ) A schematic presentation of the eight multimerized heat shock element (HSE) promoter, which is flanked by two minimal promoters in opposed orientation (black arrowhead) to bidirectionally induce EGFP and RUNX1-Evi-1 expression. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. ( c ) Transgenic verification by PCR: M: TAKARA DL2000 marker; lane 1 and 2: wild type and Tg(RE:HSE:EGFP) zebrafish larvae at 3 dpf, respectively; lane 3: PSGH2/RUNX1-Evi-1 plasmid; lane 4: double distilled water. ( d ) EGFP expression in Tg(RE:HSE:EGFP) zebrafish F2 generation at 3dpf (×4)

Techniques Used: Recombinant, Plasmid Preparation, Clone Assay, Expressing, Transgenic Assay, Polymerase Chain Reaction, Marker

3) Product Images from "Characterization of Helicobacter pylori Bacteriophage KHP30"

Article Title: Characterization of Helicobacter pylori Bacteriophage KHP30

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03530-12

(A) Location of the intracellular phage genome in phage-producing H. pylori strain NY43. The DNAs of H. pylori strains 3401 and NY43 were separated by PFGE (left) and subjected to Southern blot analysis with a phage-specific probe (right). H. pylori strain 3401 was used as the non-phage-harboring control. Black and gray arrows, phage and H. pylori DNAs, respectively. Lanes M, 1, and 2 in both panels, bacteriophage lambda ladder, strain 3401, and strain NY43, respectively. (B) Restriction digestion analysis of phage DNAs. (Left) Phage DNAs derived from the purified particles or the phage DNAs derived from H. pylori strain NY43 were digested with EcoRI and separated electrophoretically in a 0.8% agarose gel. Bacteriophage lambda DNA digested with HindIII was also separated electrophoretically as a marker. The DNA bands are indicated by arrows. (Right) In silico EcoRI digestion patterns of phage KHP30 DNA in the linear form, the circular form, a mixture of the linear and circular forms, and the concatemeric form, which were resolved in a 0.7% agarose gel.
Figure Legend Snippet: (A) Location of the intracellular phage genome in phage-producing H. pylori strain NY43. The DNAs of H. pylori strains 3401 and NY43 were separated by PFGE (left) and subjected to Southern blot analysis with a phage-specific probe (right). H. pylori strain 3401 was used as the non-phage-harboring control. Black and gray arrows, phage and H. pylori DNAs, respectively. Lanes M, 1, and 2 in both panels, bacteriophage lambda ladder, strain 3401, and strain NY43, respectively. (B) Restriction digestion analysis of phage DNAs. (Left) Phage DNAs derived from the purified particles or the phage DNAs derived from H. pylori strain NY43 were digested with EcoRI and separated electrophoretically in a 0.8% agarose gel. Bacteriophage lambda DNA digested with HindIII was also separated electrophoretically as a marker. The DNA bands are indicated by arrows. (Right) In silico EcoRI digestion patterns of phage KHP30 DNA in the linear form, the circular form, a mixture of the linear and circular forms, and the concatemeric form, which were resolved in a 0.7% agarose gel.

Techniques Used: Southern Blot, Derivative Assay, Purification, Agarose Gel Electrophoresis, Lambda DNA Preparation, Marker, In Silico

4) Product Images from "Tay1 Protein, a Novel Telomere Binding Factor from Yarrowia lipolytica *"

Article Title: Tay1 Protein, a Novel Telomere Binding Factor from Yarrowia lipolytica *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.127605

DNA binding properties of Tay1 analyzed by EMSA. A , shown is EMSA analysis using radiolabeled YlTEL (50-bp EcoRI fragment of pMH25) as a probe. The DNA competitors indicated above the lanes were used at a 1000-fold excess over the YlTEL probe. B , Tay1p is able to bind dsDNA probes carrying ≥1.5 telomeric repeats. C , EMSA analysis using radiolabeled dsDNA oligonucleotides containing three telomeric repeats ( telomeric' YlTEL_EMSA-1) and three non-telomeric repeats ( nontelomeric YlTEL_EMSA-C), respectively. 100, 300, and 500 ng of poly(dI-dC) DNA were used as competitor DNA. The two bands ( C1 and C2 ) most likely represent complexes containing one and two Tay1 protein molecules, respectively. D , shown is an EMSA analysis of Tay1p binding to radiolabeled mutated telomeric probes containing either conserved GT and GG dinucleotides with other residues of the telomeric repeat mutated ((AATGTGTGGG) 3 ; YlTEL_EMSA-M5) or vice versa ((TTATACATTG) 3 ; YlTEL_EMSA-M6). Mutated residues are underlined . 300, 500, and 1000 ng of linearized pUC19 were used as a competitor. In all EMSA experiments the dsDNA probes were prepared by annealing ss oligonucleotides as described under “Experimental Procedures.”
Figure Legend Snippet: DNA binding properties of Tay1 analyzed by EMSA. A , shown is EMSA analysis using radiolabeled YlTEL (50-bp EcoRI fragment of pMH25) as a probe. The DNA competitors indicated above the lanes were used at a 1000-fold excess over the YlTEL probe. B , Tay1p is able to bind dsDNA probes carrying ≥1.5 telomeric repeats. C , EMSA analysis using radiolabeled dsDNA oligonucleotides containing three telomeric repeats ( telomeric' YlTEL_EMSA-1) and three non-telomeric repeats ( nontelomeric YlTEL_EMSA-C), respectively. 100, 300, and 500 ng of poly(dI-dC) DNA were used as competitor DNA. The two bands ( C1 and C2 ) most likely represent complexes containing one and two Tay1 protein molecules, respectively. D , shown is an EMSA analysis of Tay1p binding to radiolabeled mutated telomeric probes containing either conserved GT and GG dinucleotides with other residues of the telomeric repeat mutated ((AATGTGTGGG) 3 ; YlTEL_EMSA-M5) or vice versa ((TTATACATTG) 3 ; YlTEL_EMSA-M6). Mutated residues are underlined . 300, 500, and 1000 ng of linearized pUC19 were used as a competitor. In all EMSA experiments the dsDNA probes were prepared by annealing ss oligonucleotides as described under “Experimental Procedures.”

Techniques Used: Binding Assay

5) Product Images from "Obesity-induced DNA hypermethylation of the adiponectin gene mediates insulin resistance"

Article Title: Obesity-induced DNA hypermethylation of the adiponectin gene mediates insulin resistance

Journal: Nature Communications

doi: 10.1038/ncomms8585

DNMT1 regulates the DNA methylation of the adiponectin promoter R2. ( a ) Dnmt1 mRNA levels in adipocytes from NCD- ( n =4) or HFD-fed ( n =3) mice and in adipocytes from WT ( n =4) or db/db mice ( n =4). ( b ) Correlation between body mass index and DNMT1 mRNA levels in human adipocytes. mRNA levels were measured by qPCR. r 2 and P values are indicated on the graph. ( c – e ) DNMT1 was suppressed by small interfering RNA in 3T3-L1 cells ( n =3). ( c ) Dnmt1 and adiponectin mRNA levels. mRNA levels were measured by qPCR. ( d ) Adiponectin protein levels were determined by western blot analysis. ( e ) Bisulfite sequencing data at the R2. ( f – h ) DNMT1 overexpression in differentiated 3T3-L1 cells ( n =3). ( f , g ) Dnmt1 and adiponectin mRNA and protein levels were measured by qPCR and western blot analysis. ( h ) Degree of R2 DNA methylation was examined by bisulfite sequencing. ( i ) AluI restriction sites in R2. Red and grey arrows indicate the AluI restriction sites and CpG locations in the R2, respectively. Double headed arrow points to PCR amplified region. ( j ) Restriction enzyme accessibility assay in adipocytes from HFD-fed ( n =5) or NCD-fed ( n =5) mice. EcoRI and BamHI, which do not digest R2, were used as negative controls. Results are expressed as the mean±s.e.m. Similar results were obtained at least more than three independent experiments. * P
Figure Legend Snippet: DNMT1 regulates the DNA methylation of the adiponectin promoter R2. ( a ) Dnmt1 mRNA levels in adipocytes from NCD- ( n =4) or HFD-fed ( n =3) mice and in adipocytes from WT ( n =4) or db/db mice ( n =4). ( b ) Correlation between body mass index and DNMT1 mRNA levels in human adipocytes. mRNA levels were measured by qPCR. r 2 and P values are indicated on the graph. ( c – e ) DNMT1 was suppressed by small interfering RNA in 3T3-L1 cells ( n =3). ( c ) Dnmt1 and adiponectin mRNA levels. mRNA levels were measured by qPCR. ( d ) Adiponectin protein levels were determined by western blot analysis. ( e ) Bisulfite sequencing data at the R2. ( f – h ) DNMT1 overexpression in differentiated 3T3-L1 cells ( n =3). ( f , g ) Dnmt1 and adiponectin mRNA and protein levels were measured by qPCR and western blot analysis. ( h ) Degree of R2 DNA methylation was examined by bisulfite sequencing. ( i ) AluI restriction sites in R2. Red and grey arrows indicate the AluI restriction sites and CpG locations in the R2, respectively. Double headed arrow points to PCR amplified region. ( j ) Restriction enzyme accessibility assay in adipocytes from HFD-fed ( n =5) or NCD-fed ( n =5) mice. EcoRI and BamHI, which do not digest R2, were used as negative controls. Results are expressed as the mean±s.e.m. Similar results were obtained at least more than three independent experiments. * P

Techniques Used: DNA Methylation Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA, Western Blot, Methylation Sequencing, Over Expression, Polymerase Chain Reaction, Amplification

6) Product Images from "MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis"

Article Title: MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0059070

Generation of Tg( MYCN :HSE:EGFP) zebrafish line. (A) Schematic diagram of the structure of PSGH2/MYCN recombinant plasmid. A mouse-MYCN fragment was cloned from HA-MYCN plasmid and inserted into the EcoRI and EcoRV sites of the PSGH2 vector. (B) A schematic presentation of the heat shock element (HSE) promoter. The artificial promoter contains eight multimerized heat shock elements flanked by two minimal promoters in opposed orientation (black arrowhead). EGFP and MYCN are expressed from the bidirectional promoter. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. (C) Transgenic verification by qRT-PCR: M: TAKARA DL2000 marker; lane 1: Blank control (double distilled water); lane 2 and 3: WT and Tg F1 generation embryos at 3 dpf, respectively; lane 4: Positive control (plasmid). (D) Transgenic verification by westernblot: lane 1: WT embryo at 3 dpf; lane 2 and 3: Tg F1 and F2 generation embryos at 3 dpf, respectively. (E–F) EGFP (+) F0 mosaic zebrafish at 24 hours (×50) and 60 days post microinjection (×7.5). (G–H) EGFP (+) F1 Tg zebrafish at 24 hpf (×50) and 60 dpf (×10). (I) Expression of total MYCN (murine exogenous and zebrafish endogenous expression), which was increased gradually in Tg F1, F2 generation fish comparing with that in WT. (J) Expression of NDRG1 , which is negative controlled by MYCN in human, was keeping a low lever in Tg F1 and F2 generation. **, P
Figure Legend Snippet: Generation of Tg( MYCN :HSE:EGFP) zebrafish line. (A) Schematic diagram of the structure of PSGH2/MYCN recombinant plasmid. A mouse-MYCN fragment was cloned from HA-MYCN plasmid and inserted into the EcoRI and EcoRV sites of the PSGH2 vector. (B) A schematic presentation of the heat shock element (HSE) promoter. The artificial promoter contains eight multimerized heat shock elements flanked by two minimal promoters in opposed orientation (black arrowhead). EGFP and MYCN are expressed from the bidirectional promoter. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. (C) Transgenic verification by qRT-PCR: M: TAKARA DL2000 marker; lane 1: Blank control (double distilled water); lane 2 and 3: WT and Tg F1 generation embryos at 3 dpf, respectively; lane 4: Positive control (plasmid). (D) Transgenic verification by westernblot: lane 1: WT embryo at 3 dpf; lane 2 and 3: Tg F1 and F2 generation embryos at 3 dpf, respectively. (E–F) EGFP (+) F0 mosaic zebrafish at 24 hours (×50) and 60 days post microinjection (×7.5). (G–H) EGFP (+) F1 Tg zebrafish at 24 hpf (×50) and 60 dpf (×10). (I) Expression of total MYCN (murine exogenous and zebrafish endogenous expression), which was increased gradually in Tg F1, F2 generation fish comparing with that in WT. (J) Expression of NDRG1 , which is negative controlled by MYCN in human, was keeping a low lever in Tg F1 and F2 generation. **, P

Techniques Used: Recombinant, Plasmid Preparation, Clone Assay, Transgenic Assay, Quantitative RT-PCR, Marker, Positive Control, Expressing, Fluorescence In Situ Hybridization

7) Product Images from "Analysis of DNA Methylation in Various Swine Tissues"

Article Title: Analysis of DNA Methylation in Various Swine Tissues

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016229

Methylation profiles of pig tissue DNA with the primer combinations H-M+TAA/E+AGT. (A) The profile from F-MSAP; (B) Data from (A) quantitated using GeneScan Analysis software; H and M refer to digestion with EcoRI/HpaII and EcoRI/MspI. Lanes 1–7 represent seven tissues, namely muscle, heart, liver, spleen, lung, kidney and stomach; M: GeneScan™ -500ROX™ size standard (represent 200 bp and 220 bp).
Figure Legend Snippet: Methylation profiles of pig tissue DNA with the primer combinations H-M+TAA/E+AGT. (A) The profile from F-MSAP; (B) Data from (A) quantitated using GeneScan Analysis software; H and M refer to digestion with EcoRI/HpaII and EcoRI/MspI. Lanes 1–7 represent seven tissues, namely muscle, heart, liver, spleen, lung, kidney and stomach; M: GeneScan™ -500ROX™ size standard (represent 200 bp and 220 bp).

Techniques Used: Methylation, Software

Cytosine methylation patterns with the primer combination H-M+TAA/E+AGT. (A) The profile from F-MSAP; (B) The data from (A) quantitated using GeneScan Analysis software; (C) The profile from MSAP using silver stain; H and M refer to digestion with EcoRI/HpaII and EcoRI/MspI, II and III refer to unmethylated, hemi-methylated and fully methylated sites, respectively.
Figure Legend Snippet: Cytosine methylation patterns with the primer combination H-M+TAA/E+AGT. (A) The profile from F-MSAP; (B) The data from (A) quantitated using GeneScan Analysis software; (C) The profile from MSAP using silver stain; H and M refer to digestion with EcoRI/HpaII and EcoRI/MspI, II and III refer to unmethylated, hemi-methylated and fully methylated sites, respectively.

Techniques Used: Methylation, Software, Silver Staining

The results of Southern blots. (A) Hybridization pattern using probe T04 for the seven tissues; (B) Hybridization pattern using probe T72 for the seven tissues; (C) Hybridization pattern using probe T53 for the seven tissues; H and M indicate the enzyme combination of EcoRI/HpaII and EcoRI/MspI; Lanes 1–7 represent muscle, heart, liver, spleen, lung, kidney and stomach, respectively.
Figure Legend Snippet: The results of Southern blots. (A) Hybridization pattern using probe T04 for the seven tissues; (B) Hybridization pattern using probe T72 for the seven tissues; (C) Hybridization pattern using probe T53 for the seven tissues; H and M indicate the enzyme combination of EcoRI/HpaII and EcoRI/MspI; Lanes 1–7 represent muscle, heart, liver, spleen, lung, kidney and stomach, respectively.

Techniques Used: Hybridization

8) Product Images from "Cloning, expression and characterization of gE protein of Duck plague virus"

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus

Journal: Virology Journal

doi: 10.1186/1743-422X-7-120

PCR amplification of DPV gE gene and identification of the recombination vector . A. Result of PCR amplification for DPV gE gene. Lane 1, the amplified product of DPV gE (about 1490bp); Lane 2, DNA marker 2000; B. Identification of the recombination vector pMD18/DPV-gE and pET32a/DPV-gE by restriction enzymes digestion. Lane 1, DNA marker 15000; Lane 2, the recombinant plasmid pET32a/DPV-gE was digested with EcoRI and XhoI (the PCR products with 1490 bp and the pET32a vector about 5,900bp) Lane 3, the recombinant plasmid pMD18/DPV-gE was digested with EcoRI and XhoI (the PCR products with 1490 bp and the pMD18-T vector about 2,700bp); Lane 4, DNA marker 2000.
Figure Legend Snippet: PCR amplification of DPV gE gene and identification of the recombination vector . A. Result of PCR amplification for DPV gE gene. Lane 1, the amplified product of DPV gE (about 1490bp); Lane 2, DNA marker 2000; B. Identification of the recombination vector pMD18/DPV-gE and pET32a/DPV-gE by restriction enzymes digestion. Lane 1, DNA marker 15000; Lane 2, the recombinant plasmid pET32a/DPV-gE was digested with EcoRI and XhoI (the PCR products with 1490 bp and the pET32a vector about 5,900bp) Lane 3, the recombinant plasmid pMD18/DPV-gE was digested with EcoRI and XhoI (the PCR products with 1490 bp and the pMD18-T vector about 2,700bp); Lane 4, DNA marker 2000.

Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Marker, Recombinant

9) Product Images from "New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients"

Article Title: New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.159336

Analysis of the patient with the smallest EcoRI fragment of 10 kb. (a) DNA was digested with EcoRI (E) and EcoRI/BlnI (B), separated by pulsed-field gel electrophoresis and hybridized with probe p13E-11. 4q35-located alleles are resistant to BlnI, and 10q26-located alleles are sensitive to BlnI. Two 4q-type alleles of 10 kb (arrow) and 50 kb; two 10q-type alleles of 42 kb and 156 kb; (b) The same DNA was digested with Hind III (H) and hybridized with probes 4qA and 4qB. 4q-type allele of 10 kb (arrow) was identified as 4qA variant and 50 kb-allele was 4qB variant. M: MidRange PFG marker Y: Y chromosome.
Figure Legend Snippet: Analysis of the patient with the smallest EcoRI fragment of 10 kb. (a) DNA was digested with EcoRI (E) and EcoRI/BlnI (B), separated by pulsed-field gel electrophoresis and hybridized with probe p13E-11. 4q35-located alleles are resistant to BlnI, and 10q26-located alleles are sensitive to BlnI. Two 4q-type alleles of 10 kb (arrow) and 50 kb; two 10q-type alleles of 42 kb and 156 kb; (b) The same DNA was digested with Hind III (H) and hybridized with probes 4qA and 4qB. 4q-type allele of 10 kb (arrow) was identified as 4qA variant and 50 kb-allele was 4qB variant. M: MidRange PFG marker Y: Y chromosome.

Techniques Used: Pulsed-Field Gel, Electrophoresis, Variant Assay, Marker

10) Product Images from "Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy"

Article Title: Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy

Journal: Scientific Reports

doi: 10.1038/s41598-017-13712-6

( a ) Vector map of RP11-242C23 generated using Ape software. EcoRI sites are shown. The D4Z4 array with 13 repeats and flanking regions was excised using EcoRI digestion, yielding a 49877-bp product. ( b ) Agarose gel electrophoresis of the EcoRI-digested vector DNA. Arrow shows the band of the 49877-bp D4Z4 array.
Figure Legend Snippet: ( a ) Vector map of RP11-242C23 generated using Ape software. EcoRI sites are shown. The D4Z4 array with 13 repeats and flanking regions was excised using EcoRI digestion, yielding a 49877-bp product. ( b ) Agarose gel electrophoresis of the EcoRI-digested vector DNA. Arrow shows the band of the 49877-bp D4Z4 array.

Techniques Used: Plasmid Preparation, Generated, Software, Agarose Gel Electrophoresis

11) Product Images from "A Novel Binary Mixture of Helicoverpa armigera Single Nucleopolyhedrovirus Genotypic Variants Has Improved Insecticidal Characteristics for Control of Cotton Bollworms"

Article Title: A Novel Binary Mixture of Helicoverpa armigera Single Nucleopolyhedrovirus Genotypic Variants Has Improved Insecticidal Characteristics for Control of Cotton Bollworms

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00339-15

(A) EcoRI profiles of the genomic DNAs of wild-type HearSP1 and the HearSP1A and HearSP1B genotypes. A 1-kb DNA ladder (Nippon) was used as a molecular size marker (M), and fragment sizes are indicated at the left (in kilobases). (B) EcoRI restriction
Figure Legend Snippet: (A) EcoRI profiles of the genomic DNAs of wild-type HearSP1 and the HearSP1A and HearSP1B genotypes. A 1-kb DNA ladder (Nippon) was used as a molecular size marker (M), and fragment sizes are indicated at the left (in kilobases). (B) EcoRI restriction

Techniques Used: Marker

12) Product Images from "Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome"

Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh192

The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
Figure Legend Snippet: The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Mutagenesis, Expressing, Amplification, Polymerase Chain Reaction, Derivative Assay, Construct, Clone Assay

Rescue of the inhibitory activity of MxA proteins by over- expression of influenza RNA polymerase subunits and NP. ( A ) Effect of over-expression of viral RNA polymerase subunits. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0 or 0.1 µg indicated – or +, respectively), and three RNA polymerase subunits and NP (0.05 µg each). The additional amount (0.3 µg) of a plasmid encoding either PB1, PB2 or PA was added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Effect of over-expression of NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0, 0.1 or 0.5 µg), and three RNA polymerase subunits (0.05 µg each) and NP (0.15 µg). The additional amounts of the plasmid encoding NP were added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. The result is shown as the average of two independent experiments. ( C ) Titration of plasmid encoding NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), three RNA polymerase subunits (0.05 µg each) and NP (0.1, 0.3 or 0.5 µg) in the absence or presence of pVP16-MxA. The relative luciferase activity in the presence of MxA (0.1 µg, open circle; 0.3 µg, closed circle) was normalized with that in the absence of MxA, and plotted as a function of increasing amounts of NP. The result is shown as the average of two independent experiments. ( D ) Titration of VP16-MxA, and C-terminal (NPΔC) and N-terminal (NPΔN) deletion mutants of NP. The same procedure for (B) was carried out with mutant NPs. For expression of NP proteins lacking its C-terminal region (NPΔC) corresponding amino acid positions between 182 and 498 (where amino acid position 1 is set the first amino acid of the wild-type NP) and its N-terminal region (NPΔN) corresponding amino acid positions between 1 and 190, pCAGGS-NPΔC and pCAGGS-NPΔN plasmids were constructed as follows: portions of NP fragments were amplified by PCR with pCAGGS-NP as a template and a set of primers, 5′-TTGAATTCGCCACCATGGCGACCAAAGGCACC-3′ and 5′-TTGAATT CTTAAGCACCTGCGGCCCCAGACC-3′ for NPΔC and a set of primers, 5′-TTGAATTCGCCACCATGGAATTGATCAGAATG-3′ and 5′-GGGA ATTCTTAATTGTCGTACTCCTCTGCA-3′ for NPΔN. Synthesized NP fragments were digested with EcoRI and cloned into pCHA that had been digested with the same restriction enzyme. The additional amounts of the plasmid encoding deletion mutants of NP were transfected as indicated. The result is shown as the average of two independent experiments.
Figure Legend Snippet: Rescue of the inhibitory activity of MxA proteins by over- expression of influenza RNA polymerase subunits and NP. ( A ) Effect of over-expression of viral RNA polymerase subunits. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0 or 0.1 µg indicated – or +, respectively), and three RNA polymerase subunits and NP (0.05 µg each). The additional amount (0.3 µg) of a plasmid encoding either PB1, PB2 or PA was added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Effect of over-expression of NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0, 0.1 or 0.5 µg), and three RNA polymerase subunits (0.05 µg each) and NP (0.15 µg). The additional amounts of the plasmid encoding NP were added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. The result is shown as the average of two independent experiments. ( C ) Titration of plasmid encoding NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), three RNA polymerase subunits (0.05 µg each) and NP (0.1, 0.3 or 0.5 µg) in the absence or presence of pVP16-MxA. The relative luciferase activity in the presence of MxA (0.1 µg, open circle; 0.3 µg, closed circle) was normalized with that in the absence of MxA, and plotted as a function of increasing amounts of NP. The result is shown as the average of two independent experiments. ( D ) Titration of VP16-MxA, and C-terminal (NPΔC) and N-terminal (NPΔN) deletion mutants of NP. The same procedure for (B) was carried out with mutant NPs. For expression of NP proteins lacking its C-terminal region (NPΔC) corresponding amino acid positions between 182 and 498 (where amino acid position 1 is set the first amino acid of the wild-type NP) and its N-terminal region (NPΔN) corresponding amino acid positions between 1 and 190, pCAGGS-NPΔC and pCAGGS-NPΔN plasmids were constructed as follows: portions of NP fragments were amplified by PCR with pCAGGS-NP as a template and a set of primers, 5′-TTGAATTCGCCACCATGGCGACCAAAGGCACC-3′ and 5′-TTGAATT CTTAAGCACCTGCGGCCCCAGACC-3′ for NPΔC and a set of primers, 5′-TTGAATTCGCCACCATGGAATTGATCAGAATG-3′ and 5′-GGGA ATTCTTAATTGTCGTACTCCTCTGCA-3′ for NPΔN. Synthesized NP fragments were digested with EcoRI and cloned into pCHA that had been digested with the same restriction enzyme. The additional amounts of the plasmid encoding deletion mutants of NP were transfected as indicated. The result is shown as the average of two independent experiments.

Techniques Used: Activity Assay, Over Expression, Transfection, Plasmid Preparation, Luciferase, Standard Deviation, Titration, Mutagenesis, Expressing, Construct, Amplification, Polymerase Chain Reaction, Synthesized, Clone Assay

13) Product Images from "Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis"

Article Title: Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-300

The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.
Figure Legend Snippet: The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.

Techniques Used: Selection, Marker, Isolation, Labeling, Homologous Recombination, Plasmid Preparation, Southern Blot, Lambda DNA Preparation

Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.
Figure Legend Snippet: Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.

Techniques Used: Clone Assay, BAC Assay, Plasmid Preparation, Southern Blot, Purification, Lambda DNA Preparation, Marker

14) Product Images from "Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis"

Article Title: Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-300

The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.
Figure Legend Snippet: The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.

Techniques Used: Selection, Marker, Isolation, Labeling, Homologous Recombination, Plasmid Preparation, Southern Blot, Lambda DNA Preparation

Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.
Figure Legend Snippet: Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.

Techniques Used: Clone Assay, BAC Assay, Plasmid Preparation, Southern Blot, Purification, Lambda DNA Preparation, Marker

15) Product Images from "Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus"

Article Title: Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

Journal: Journal of Bacteriology

doi: 10.1128/JB.01344-12

(A) 5′-RACE analysis of sag SA . Total RNA from early-logarithmic-phase cells was prepared and used for 5′-RACE analysis. (A) The resulting DNA fragments containing the 5′ terminus of cDNA from mRNA sequence(s) of sag SA were amplified by nested PCR and analyzed by agarose-gel electrophoresis. M, hundred-base-pair ladder marker. (B) Cloned 5′-RACE products for sequencing. The 5′-RACE product cloned in the SmaI site of pUC18 was obtained by double digestion with EcoRI and SalI and separated by agarose gel electrophoresis. M, hundred-base-pair ladder size marker. Lanes 1 to 4 show the 5′-end part of cDNA deduced to be the transcripts from sagA1p (lane 1), sagA2p (lanes 2 and 3), and sagBp (lane 4).
Figure Legend Snippet: (A) 5′-RACE analysis of sag SA . Total RNA from early-logarithmic-phase cells was prepared and used for 5′-RACE analysis. (A) The resulting DNA fragments containing the 5′ terminus of cDNA from mRNA sequence(s) of sag SA were amplified by nested PCR and analyzed by agarose-gel electrophoresis. M, hundred-base-pair ladder marker. (B) Cloned 5′-RACE products for sequencing. The 5′-RACE product cloned in the SmaI site of pUC18 was obtained by double digestion with EcoRI and SalI and separated by agarose gel electrophoresis. M, hundred-base-pair ladder size marker. Lanes 1 to 4 show the 5′-end part of cDNA deduced to be the transcripts from sagA1p (lane 1), sagA2p (lanes 2 and 3), and sagBp (lane 4).

Techniques Used: Sequencing, Amplification, Nested PCR, Agarose Gel Electrophoresis, Marker, Clone Assay

16) Product Images from "Spread of CTX-M-15 Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates through Household Contact and Plasmid Transfer"

Article Title: Spread of CTX-M-15 Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates through Household Contact and Plasmid Transfer

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.03342-13

Patterns of EcoRI- or SacI-restricted fragments of IncI1-Iγ plasmid harboring bla CTX-M-15 . Lane 1, EcoRI-restricted fragments of plasmid from TUM11486; lane 2, EcoRI-restricted fragments of plasmid from TUM11488; lane 3, SacI-restricted fragments of plasmid from TUM11486; lane 4, SacI-restricted fragments of plasmid from TUM11488.
Figure Legend Snippet: Patterns of EcoRI- or SacI-restricted fragments of IncI1-Iγ plasmid harboring bla CTX-M-15 . Lane 1, EcoRI-restricted fragments of plasmid from TUM11486; lane 2, EcoRI-restricted fragments of plasmid from TUM11488; lane 3, SacI-restricted fragments of plasmid from TUM11486; lane 4, SacI-restricted fragments of plasmid from TUM11488.

Techniques Used: Plasmid Preparation

17) Product Images from "Identification of human sperm proteins that interact with human zona pellucida3 (ZP3) using yeast two-hybrid system"

Article Title: Identification of human sperm proteins that interact with human zona pellucida3 (ZP3) using yeast two-hybrid system

Journal: Journal of reproductive immunology

doi: 10.1016/j.jri.2009.10.006

Confirmation of ZP3 insert cloned into yeast pAS2-1 vector (8.4 kb) at EcoRI and BamHI sites. (A) The ZP3 insert of 1 kb was PCR-amplified from pME18SFL3 using specific primers (lane a). The negative control (lane b) and the DNA molecular weight ladder
Figure Legend Snippet: Confirmation of ZP3 insert cloned into yeast pAS2-1 vector (8.4 kb) at EcoRI and BamHI sites. (A) The ZP3 insert of 1 kb was PCR-amplified from pME18SFL3 using specific primers (lane a). The negative control (lane b) and the DNA molecular weight ladder

Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Negative Control, Molecular Weight

Confirmation of ZP3 insert cloned into mammalian pM vector (3.5 kb) at EcoRI and BamHI sites. The pM-ZP3 mammalian clone digested with EcoRI (lane b) or BamHI (lane c) alone did not show, but the clone digested with both the EcoRI and BamHI restriction
Figure Legend Snippet: Confirmation of ZP3 insert cloned into mammalian pM vector (3.5 kb) at EcoRI and BamHI sites. The pM-ZP3 mammalian clone digested with EcoRI (lane b) or BamHI (lane c) alone did not show, but the clone digested with both the EcoRI and BamHI restriction

Techniques Used: Clone Assay, Plasmid Preparation

18) Product Images from "PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB"

Article Title: PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.10.3015-3021.2004

DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.
Figure Legend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.
Figure Legend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

19) Product Images from "Five New Genome Types of Adenovirus Type 37 Caused Epidemic Keratoconjunctivitis in Sapporo, Japan, for More Than 10 Years"

Article Title: Five New Genome Types of Adenovirus Type 37 Caused Epidemic Keratoconjunctivitis in Sapporo, Japan, for More Than 10 Years

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.43.2.726-732.2005

Restriction patterns of each genome type based on five restriction endonucleases: BglI (A), EcoRI (B), SacI (C), SmaI (D), and XhoI (E). Lane m, 2-log DNA ladder marker (New England Biolabs); lane p, adenovirus type 37 prototype; lanes a to h, representative
Figure Legend Snippet: Restriction patterns of each genome type based on five restriction endonucleases: BglI (A), EcoRI (B), SacI (C), SmaI (D), and XhoI (E). Lane m, 2-log DNA ladder marker (New England Biolabs); lane p, adenovirus type 37 prototype; lanes a to h, representative

Techniques Used: Marker

20) Product Images from "An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments"

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

Journal: BMC Genomics

doi: 10.1186/s12864-015-1425-4

Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. Nm S , neomycin sensitive; Nm R , neomycin resistant; Spc S , spectinomycin sensitive; Spc R , spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.
Figure Legend Snippet: Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. Nm S , neomycin sensitive; Nm R , neomycin resistant; Spc S , spectinomycin sensitive; Spc R , spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.

Techniques Used: Clone Assay, Sequencing, Nucleic Acid Electrophoresis, Plasmid Preparation, Lambda DNA Preparation, Marker, Recombinant, Derivative Assay, BAC Assay

21) Product Images from "Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product"

Article Title: Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200104083

Disruption of the murine coil locus. (A) Maps of the targeting vector, wild-type, and targeted alleles. Open boxes indicate exons, and filled boxes represent selectable markers. Thick horizontal lines indicate intronic sequences. Targeting vector sequences are represented by thin horizontal lines. Large arrows indicate the start and direction of transcription for each gene. The shaded box corresponds to the probe used for confirmation of targeting. PCR primers for use in screening are indicated by small arrows (a, b, and c). NEO, neomycin resistance cassette gene; TK, thymidine kinase cassette gene B, BamHI; E, EcoRI. (B) Southern blot (top) and PCR (bottom) genotyping of progeny. Genomic DNAs for Southern blot analysis were obtained from +/+, +/−, or −/− MEFs. Control DNAs were from 129Sv/J mice (129), NIH 3T3 cells (3T3), or the targeted embryonic stem cell line 3G8 (ES). The positions of the wild-type (WT) and knockout (KO) alleles are shown along with a marker (M). Primers shown in A were used for PCR. (C) Schematic of the coilin protein. Highly conserved regions are indicated as black boxes. Also depicted are NLSs (light gray boxes), acidic patches (hatched boxes), and the putative nucleolar localization signal (dark gray box). A region rich in arginine and glycine dipeptides (RG box) is also indicated. The portion of the protein corresponding to the region deleted in the knockout is indicated by the bar.
Figure Legend Snippet: Disruption of the murine coil locus. (A) Maps of the targeting vector, wild-type, and targeted alleles. Open boxes indicate exons, and filled boxes represent selectable markers. Thick horizontal lines indicate intronic sequences. Targeting vector sequences are represented by thin horizontal lines. Large arrows indicate the start and direction of transcription for each gene. The shaded box corresponds to the probe used for confirmation of targeting. PCR primers for use in screening are indicated by small arrows (a, b, and c). NEO, neomycin resistance cassette gene; TK, thymidine kinase cassette gene B, BamHI; E, EcoRI. (B) Southern blot (top) and PCR (bottom) genotyping of progeny. Genomic DNAs for Southern blot analysis were obtained from +/+, +/−, or −/− MEFs. Control DNAs were from 129Sv/J mice (129), NIH 3T3 cells (3T3), or the targeted embryonic stem cell line 3G8 (ES). The positions of the wild-type (WT) and knockout (KO) alleles are shown along with a marker (M). Primers shown in A were used for PCR. (C) Schematic of the coilin protein. Highly conserved regions are indicated as black boxes. Also depicted are NLSs (light gray boxes), acidic patches (hatched boxes), and the putative nucleolar localization signal (dark gray box). A region rich in arginine and glycine dipeptides (RG box) is also indicated. The portion of the protein corresponding to the region deleted in the knockout is indicated by the bar.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Southern Blot, Mouse Assay, Knock-Out, Marker

22) Product Images from "Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids"

Article Title: Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

Journal: Environmental Microbiology

doi: 10.1111/j.1462-2920.2007.01518.x

Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).
Figure Legend Snippet: Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).

Techniques Used: Polymerase Chain Reaction, Amplification

23) Product Images from "Lacanobia oleracea nucleopolyhedrovirus (LaolNPV): A new European species of alphabaculovirus with a narrow host range"

Article Title: Lacanobia oleracea nucleopolyhedrovirus (LaolNPV): A new European species of alphabaculovirus with a narrow host range

Journal: PLoS ONE

doi: 10.1371/journal.pone.0176171

Restriction endonuclease analysis of LaolNPV DNA. Restriction endonuclease profiles following digestion of A) genomic DNAs of the seven isolates of LaolNPV collected in alfalfa crops nearby Montpellier with BglII, EcoRI and PstI enzymes, B) genomic DNAs of Chrysodeixis chalcites NPV (Chch), Helicoverpa armigera SNPV (Hear), Spodoptera littoralis (Spli), Spodopera exigua MNPV (Se) and the virus under study (LaolNPV) with BamHI, BglII, EcoRI and PstI, and C) genomic DNAs of Mamestra brassicae MNPV (Mb), Mamestra configurata NPV-A (MacoA), Mamestra configurata NPV-B (MacoB) and LaolNPV with BglII, EcoRI and PstI.
Figure Legend Snippet: Restriction endonuclease analysis of LaolNPV DNA. Restriction endonuclease profiles following digestion of A) genomic DNAs of the seven isolates of LaolNPV collected in alfalfa crops nearby Montpellier with BglII, EcoRI and PstI enzymes, B) genomic DNAs of Chrysodeixis chalcites NPV (Chch), Helicoverpa armigera SNPV (Hear), Spodoptera littoralis (Spli), Spodopera exigua MNPV (Se) and the virus under study (LaolNPV) with BamHI, BglII, EcoRI and PstI, and C) genomic DNAs of Mamestra brassicae MNPV (Mb), Mamestra configurata NPV-A (MacoA), Mamestra configurata NPV-B (MacoB) and LaolNPV with BglII, EcoRI and PstI.

Techniques Used:

24) Product Images from "Self-assembled HCV core virus-like particles targeted and inhibited tumor cell migration and invasion"

Article Title: Self-assembled HCV core virus-like particles targeted and inhibited tumor cell migration and invasion

Journal: Nanoscale Research Letters

doi: 10.1186/1556-276X-8-401

Transcription and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) RT-PCR results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay.
Figure Legend Snippet: Transcription and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) RT-PCR results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay.

Techniques Used: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Infection, Synthesized, Quantitative RT-PCR, SYBR Green Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Marker, SDS Page, Western Blot

RGD-core-IFN-α2a fusion proteins bind breast cancer cells MDA-MB231 in vitro. (A) Recombinant bacmid constructs, showing the strategy for insertion of the gene cassettes into the polyhedrin locus of the AcMNPV bacmid. RGD-HCV core was fused with IFN-α2a. Both cassettes depicted were inserted into the attb site (indicated by the right and left insertion sites, Tn7R and Tn7L) in the polyhedrin locus by Tn-based transposition and generated the recombinant Bacmid: AcH1, AcH2, AcH3, and AcH4. (B) Identification of pH1 and pH2. M: 1Kb Plus DNA ladder; pH1 and pH2 samples were digested by BamHI and EcoRI. (C) Identification of pH3 and pH4. M: O’Gene Ruler 1Kb DNA ladder; pH3 and pH4 samples were digested by BamHI and EcoRI. (D) Purification of RGD-core-IFN-α2a fusion protein. M: protein marker; 1: His-H1; 2: His-H2; 3: His-H3; 4: His-H4. The recombinant bacmids AcH1, AcH2, AcH3, and AcH4 were introduced by transfection into Sf9 cells to produce the recombinant proteins His-H1, His-H2, His-H3, and His-H4. The fusion proteins were purified from the supernatants of cell lysates using Ni-NTA affinity resin. (E , G) Electron micrograph images and Western blotting result of VLP H1. Purified VLPs were attached onto a carbon-coated grid for 5 min at room temperature. The grid was rinsed with distilled water and stained with 1% phosphotungstic acid for 3 min before air drying on filter paper. The specimens were viewed using a Tecnai G2 transmission electron microscopy at 75 keV. For Western blot, 10 μg purified VLPs were separated by SDS-PAGE electrophoresis and subjected to Western blot assay. (F , H) Electron micrograph images and Western blotting result of VLP H2. (I) RGD-core-IFN-α2a fusion protein bind with breast cancer cells MDA-MB231. Then, 0.2, 0.5, 2, 5, and 10 μM fusion proteins His-H1, His-H2, His-H3, and His-H4 were co-incubated with MDA-MB231 at 37° under 5% CO 2 . After 2 h, the cells were washed three times with PBS, and green fluorescence was observed under the fluorescence microscope. Scale bar = 100 μm.
Figure Legend Snippet: RGD-core-IFN-α2a fusion proteins bind breast cancer cells MDA-MB231 in vitro. (A) Recombinant bacmid constructs, showing the strategy for insertion of the gene cassettes into the polyhedrin locus of the AcMNPV bacmid. RGD-HCV core was fused with IFN-α2a. Both cassettes depicted were inserted into the attb site (indicated by the right and left insertion sites, Tn7R and Tn7L) in the polyhedrin locus by Tn-based transposition and generated the recombinant Bacmid: AcH1, AcH2, AcH3, and AcH4. (B) Identification of pH1 and pH2. M: 1Kb Plus DNA ladder; pH1 and pH2 samples were digested by BamHI and EcoRI. (C) Identification of pH3 and pH4. M: O’Gene Ruler 1Kb DNA ladder; pH3 and pH4 samples were digested by BamHI and EcoRI. (D) Purification of RGD-core-IFN-α2a fusion protein. M: protein marker; 1: His-H1; 2: His-H2; 3: His-H3; 4: His-H4. The recombinant bacmids AcH1, AcH2, AcH3, and AcH4 were introduced by transfection into Sf9 cells to produce the recombinant proteins His-H1, His-H2, His-H3, and His-H4. The fusion proteins were purified from the supernatants of cell lysates using Ni-NTA affinity resin. (E , G) Electron micrograph images and Western blotting result of VLP H1. Purified VLPs were attached onto a carbon-coated grid for 5 min at room temperature. The grid was rinsed with distilled water and stained with 1% phosphotungstic acid for 3 min before air drying on filter paper. The specimens were viewed using a Tecnai G2 transmission electron microscopy at 75 keV. For Western blot, 10 μg purified VLPs were separated by SDS-PAGE electrophoresis and subjected to Western blot assay. (F , H) Electron micrograph images and Western blotting result of VLP H2. (I) RGD-core-IFN-α2a fusion protein bind with breast cancer cells MDA-MB231. Then, 0.2, 0.5, 2, 5, and 10 μM fusion proteins His-H1, His-H2, His-H3, and His-H4 were co-incubated with MDA-MB231 at 37° under 5% CO 2 . After 2 h, the cells were washed three times with PBS, and green fluorescence was observed under the fluorescence microscope. Scale bar = 100 μm.

Techniques Used: Multiple Displacement Amplification, In Vitro, Recombinant, Construct, Generated, Purification, Marker, Transfection, Western Blot, Staining, Transmission Assay, Electron Microscopy, SDS Page, Electrophoresis, Incubation, Fluorescence, Microscopy

25) Product Images from "PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB"

Article Title: PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.10.3015-3021.2004

DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.
Figure Legend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.
Figure Legend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

26) Product Images from "Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †"

Article Title: Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02946-09

Dendrogram of Proteus O antigen gene clusters generated by using the BioNumerics software program following restriction digestion by HindIII and EcoRI.
Figure Legend Snippet: Dendrogram of Proteus O antigen gene clusters generated by using the BioNumerics software program following restriction digestion by HindIII and EcoRI.

Techniques Used: Generated, Software

PCR-RFLP analyses of putative O antigen gene clusters following restriction with HindIII and EcoRI. Lane M, DNA marker; lane 1, O69 (G2667); lane 2, O71 (G2669); lane 3, O72a (G2670); lane 4, O73ac (G2673); lane 5, O74 (G2674); lane 6, O75 (G2675).
Figure Legend Snippet: PCR-RFLP analyses of putative O antigen gene clusters following restriction with HindIII and EcoRI. Lane M, DNA marker; lane 1, O69 (G2667); lane 2, O71 (G2669); lane 3, O72a (G2670); lane 4, O73ac (G2673); lane 5, O74 (G2674); lane 6, O75 (G2675).

Techniques Used: Polymerase Chain Reaction, Marker

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Incubation:

Article Title: Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy
Article Snippet: Preparation of D4Z4 repeats from the BAC clone RP11-242C23 was digested using EcoRI and treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 20 min. DNA was subjected to electrophoresis on a 0.5% agarose gel. .. Agarose gels were soaked in phenol and incubated for 30 min at −80 °C.

Article Title: Obesity-induced DNA hypermethylation of the adiponectin gene mediates insulin resistance
Article Snippet: In brief, adipocytes from epididymal adipose tissue and 3T3-L1 cells were harvested with RSB buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2 , 0.1% Nonidet P-40 (NP-40), 5 mM butyrate, 10 mM NaF and 1 mM NaVO7 , supplemented with protease inhibitors) and incubated for 20 min on ice. .. Resuspended gDNA was digested with 100 U of AluI, EcoRI and BamHI (TaKaRa, 1004A, 1040A and 1010A).

In Silico:

Article Title: Characterization of Helicobacter pylori Bacteriophage KHP30
Article Snippet: In silico restriction digestion of the phage KHP30 genome was conducted using the NEBcutter (v2.0) program (New England BioLabs) ( ). .. The phage DNAs (1 μg) were digested with EcoRI (TaKaRa Bio) and then resolved electrophoretically in a 0.8% agarose gel.

Expressing:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: .. The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn. .. Cell culture and transfection Chinese hamster ovary (CHO) cells (Shanghai Institute of Cell Bank, China) were cultured in DMEM/F12K medium containing 10% FBS supplemented with penicillin (50 U/mL, Gibco) and streptomycin (50 U/mL, Gibco) at 37℃ in a humidified atmosphere with 5% CO2 .

Article Title: RUNX1-Evi-1 fusion gene inhibited differentiation and apoptosis in myelopoiesis: an in vivo study
Article Snippet: It was subcloned into EcoRI and EcoRV (Takara, Japan) sites of the pSGH2 vector [ ], which contains eight HSE sequence (AGAACGTTCTAGAAC) and EGFP segment. .. Then, we obtain the hRUNX1-Evi-1-HSE-EGFP insert construct (Fig. ), HSE allows the symmetrical addition of a CMV minimal promoter to both ends in order to drive the expression of two interested genes (EGFP at one side and hRUNX1-Evi-1 at the other side) flanked by 5 V and 3 V globin UTRs and SV40 polyadenylation (pA) signal (I-SceI meganuclease recognition sites) (Fig. ).

Article Title: In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate
Article Snippet: Some unique sequences that give lower expression such as GCTGGTGG (Chi site) GTTGTAAC (ter site core), AATAAA dan ATTAAA (polyA sites), TATA (consensus eukaryotic promoter core), TTAGGG (immunosuppressive telomeric motif), GCCGTCTGAA, AAGTGCGGT and ACAAGCGGTC (DNA uptake sequences), AGGT (consensus splice donor), CAGG (consensus splice acceptor), AAAAA (polyA binding proteins consensus) TTTTT (polyT binding proteins consensus), and TTATCCACA (DnaA binding site) were removed using codon optimisation ( ). .. Then, the transgene was chemically synthesised with restriction sites of Bgl II and EcoR I and cloned into the pEGFP-C1 vector (Clontech) by Gene Universal Inc., resulting in pEGFP-env-tm JDV ( ).

Article Title: MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis
Article Snippet: Construction of PSGH2/MYCN plasmid A mouse-MYCN fragment was extracted from HA-MYCN plasmid and subcloned into the EcoRI and EcoRV (Takara, Japan) sites of the PSGH2 vector to obtain the mMYCN -HSE-EGFP construct ( , ). .. The PSGH2 vector contains eight HSE sequence ( AGAACGTTCTAGAAC ) allows the symmetrical addition of a CMV minimal promoter to both ends in order to drive the expression of the genes of interest (one side is EGFP and the other side is MYCN ) flanked by 5V and 3V globin UTRs and SV40 polyadenylation (pA) signal (I-SceI meganuclease recognition sites) ( ).

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus
Article Snippet: The expression vector pET32a(+) and the host strain Escherichia coli BL21(DE3), BL21(pLysS) and Rosseta were purchased from Novagen. .. Restriction enzymes, EcoRI and XhoI, pMD18-T vector, the Total RNA Isolation System and RNase-free DNase I were purchased from TaKaRa Biotechnology Co. Ltd.

Transformation Assay:

Article Title: In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate
Article Snippet: Then, the transgene was chemically synthesised with restriction sites of Bgl II and EcoR I and cloned into the pEGFP-C1 vector (Clontech) by Gene Universal Inc., resulting in pEGFP-env-tm JDV ( ). .. The plasmid pEGFP-env-tm JDV was transformed into chemically competent E. coli DH5α using a heat-shock procedure and then screened in Luria–Bertani agar selective medium (HiMedia, India) containing 50 μg/mL of kanamycin.

Over Expression:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: Paragraph title: Construction of the overexpression vector ... The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn.

Derivative Assay:

Article Title: New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients
Article Snippet: DNA isolation, analysis of D4Z4 repeats, and 4qA/4qB variant determination High molecular weight DNA was extracted from peripheral lymphocytes derived from freshly collected blood using the standard protocols. .. Five micrograms of DNA were digested with EcoRI, EcoRI/BlnI, or HindIII (Takara, Japan), and separated by pulsed-field gel electrophoresis on a 1.2% agarose gel (Sangon, China) in × 0.5 TBE for 39 h. Nytran + membranes (GE Healthcare, USA) for allele sizing were hybridized with probe p13E-11, and those for allele typing were hybridized with probe 4qA and 4qB as previously described.

Ligation:

Article Title: Analysis of DNA Methylation in Various Swine Tissues
Article Snippet: The digestion-ligation reaction was performed in a volume of 25 uL containing 500 ng DNA template, 3 U EcoRI, 3 U HpaII (or MspI), 1.5 U T4 DNA ligase (TaKaRa, Dalian, China), 5 pmol EcoRI adapter, 50 pmol HpaII/Msp I adapter and 2.5 uL 10× T4 ligase buffer. .. Preamplified PCR reactions were performed in a final volume of 20 uL containing 2 uL of ligation products, 40 ng of E+1 and H-M+1 preamplified primers , 0.1 uL of Ex Taq polymerase, 1.6 uL of dNTPs (2.5 mM), 1.2 uL of MgCl2 (25 mM), 2 uL of 10× PCR buffer and 14.1 uL of sterile distilled H2 O.

Article Title: Tay1 Protein, a Novel Telomere Binding Factor from Yarrowia lipolytica *
Article Snippet: .. The plasmid pMH25 carrying ten Y. lipolytica telomeric repeats was constructed by annealing oligonucleotides YlTEL-sense and YlTEL-antisense followed by the digestion of the resulting dsDNA with EcoRI and ligation into the EcoRI-digested and -dephosphorylated plasmid pHIS2 (Clontech). .. We obtained the desired recombinant plasmid carrying 2 × 5 telomeric repeats separated by an EcoRI site and named it pMH25.

Cell Culture:

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus
Article Snippet: Restriction enzymes, EcoRI and XhoI, pMD18-T vector, the Total RNA Isolation System and RNase-free DNase I were purchased from TaKaRa Biotechnology Co. Ltd. .. Duck embryo fibroblasts (DEF) were cultured in MEM medium (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL) at 37°C.

Article Title: Characterization of Helicobacter pylori Bacteriophage KHP30
Article Snippet: Moreover, H. pylori strain NY43, which releases phage KHP30, was cultured in BEV medium and then pelleted. .. The phage DNAs (1 μg) were digested with EcoRI (TaKaRa Bio) and then resolved electrophoretically in a 0.8% agarose gel.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: The Fyn gene was isolated from mouse mRNA by reverse transcription PCR (RT-PCR) using Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer's instructions from the Fermentas (USA). .. The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn.

Sequencing:

Article Title: Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria
Article Snippet: Preparation of asp -disrupted or -introduced strain Fragments of strain 288 chromosomal DNA digested with the EcoRI were inserted into the EcoRI site of pUC119 ( ) and introduced into E. coli HB101 (TaKaRa Co., Kyoto, Japan). .. Sequence analysis of the cloned insert DNA (5528 bp) in pUC119-5528 ( ) identified an operon composed of two structural genes, asp and orf2 , which codes for a specific chaperone required for ASP maturation (GenBank accession no. AF253471).

Article Title: RUNX1-Evi-1 fusion gene inhibited differentiation and apoptosis in myelopoiesis: an in vivo study
Article Snippet: .. It was subcloned into EcoRI and EcoRV (Takara, Japan) sites of the pSGH2 vector [ ], which contains eight HSE sequence (AGAACGTTCTAGAAC) and EGFP segment. .. Then, we obtain the hRUNX1-Evi-1-HSE-EGFP insert construct (Fig. ), HSE allows the symmetrical addition of a CMV minimal promoter to both ends in order to drive the expression of two interested genes (EGFP at one side and hRUNX1-Evi-1 at the other side) flanked by 5 V and 3 V globin UTRs and SV40 polyadenylation (pA) signal (I-SceI meganuclease recognition sites) (Fig. ).

Article Title: In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate
Article Snippet: Codon optimisation is done without changing the amino acid sequence to prevent the changing of antigenicity from native protein. .. Then, the transgene was chemically synthesised with restriction sites of Bgl II and EcoR I and cloned into the pEGFP-C1 vector (Clontech) by Gene Universal Inc., resulting in pEGFP-env-tm JDV ( ).

Article Title: MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis
Article Snippet: Construction of PSGH2/MYCN plasmid A mouse-MYCN fragment was extracted from HA-MYCN plasmid and subcloned into the EcoRI and EcoRV (Takara, Japan) sites of the PSGH2 vector to obtain the mMYCN -HSE-EGFP construct ( , ). .. The PSGH2 vector contains eight HSE sequence ( AGAACGTTCTAGAAC ) allows the symmetrical addition of a CMV minimal promoter to both ends in order to drive the expression of the genes of interest (one side is EGFP and the other side is MYCN ) flanked by 5V and 3V globin UTRs and SV40 polyadenylation (pA) signal (I-SceI meganuclease recognition sites) ( ).

Binding Assay:

Article Title: In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate
Article Snippet: Some unique sequences that give lower expression such as GCTGGTGG (Chi site) GTTGTAAC (ter site core), AATAAA dan ATTAAA (polyA sites), TATA (consensus eukaryotic promoter core), TTAGGG (immunosuppressive telomeric motif), GCCGTCTGAA, AAGTGCGGT and ACAAGCGGTC (DNA uptake sequences), AGGT (consensus splice donor), CAGG (consensus splice acceptor), AAAAA (polyA binding proteins consensus) TTTTT (polyT binding proteins consensus), and TTATCCACA (DnaA binding site) were removed using codon optimisation ( ). .. Then, the transgene was chemically synthesised with restriction sites of Bgl II and EcoR I and cloned into the pEGFP-C1 vector (Clontech) by Gene Universal Inc., resulting in pEGFP-env-tm JDV ( ).

Article Title: Tay1 Protein, a Novel Telomere Binding Factor from Yarrowia lipolytica *
Article Snippet: The plasmid pMH25 carrying ten Y. lipolytica telomeric repeats was constructed by annealing oligonucleotides YlTEL-sense and YlTEL-antisense followed by the digestion of the resulting dsDNA with EcoRI and ligation into the EcoRI-digested and -dephosphorylated plasmid pHIS2 (Clontech). .. Digestion of pMH25 with EcoRI yields a dsDNA fragment containing five telomeric repeats used in initial DNA binding assays.

Pulsed-Field Gel:

Article Title: New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients
Article Snippet: .. Five micrograms of DNA were digested with EcoRI, EcoRI/BlnI, or HindIII (Takara, Japan), and separated by pulsed-field gel electrophoresis on a 1.2% agarose gel (Sangon, China) in × 0.5 TBE for 39 h. Nytran + membranes (GE Healthcare, USA) for allele sizing were hybridized with probe p13E-11, and those for allele typing were hybridized with probe 4qA and 4qB as previously described. ..

DNA Extraction:

Article Title: New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients
Article Snippet: Paragraph title: DNA isolation, analysis of D4Z4 repeats, and 4qA/4qB variant determination ... Five micrograms of DNA were digested with EcoRI, EcoRI/BlnI, or HindIII (Takara, Japan), and separated by pulsed-field gel electrophoresis on a 1.2% agarose gel (Sangon, China) in × 0.5 TBE for 39 h. Nytran + membranes (GE Healthcare, USA) for allele sizing were hybridized with probe p13E-11, and those for allele typing were hybridized with probe 4qA and 4qB as previously described.

Fluorescence:

Article Title: Analysis of DNA Methylation in Various Swine Tissues
Article Snippet: Paragraph title: Fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) assay ... The digestion-ligation reaction was performed in a volume of 25 uL containing 500 ng DNA template, 3 U EcoRI, 3 U HpaII (or MspI), 1.5 U T4 DNA ligase (TaKaRa, Dalian, China), 5 pmol EcoRI adapter, 50 pmol HpaII/Msp I adapter and 2.5 uL 10× T4 ligase buffer.

Methylation:

Article Title: Analysis of DNA Methylation in Various Swine Tissues
Article Snippet: Paragraph title: Fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) assay ... The digestion-ligation reaction was performed in a volume of 25 uL containing 500 ng DNA template, 3 U EcoRI, 3 U HpaII (or MspI), 1.5 U T4 DNA ligase (TaKaRa, Dalian, China), 5 pmol EcoRI adapter, 50 pmol HpaII/Msp I adapter and 2.5 uL 10× T4 ligase buffer.

Isolation:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: The Fyn gene was isolated from mouse mRNA by reverse transcription PCR (RT-PCR) using Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer's instructions from the Fermentas (USA). .. The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn.

Article Title: In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate
Article Snippet: The isolated gene of env-tm was optimised to be expressed in a Bos taurus host using codon optimisation. .. Then, the transgene was chemically synthesised with restriction sites of Bgl II and EcoR I and cloned into the pEGFP-C1 vector (Clontech) by Gene Universal Inc., resulting in pEGFP-env-tm JDV ( ).

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus
Article Snippet: .. Restriction enzymes, EcoRI and XhoI, pMD18-T vector, the Total RNA Isolation System and RNase-free DNase I were purchased from TaKaRa Biotechnology Co. Ltd. .. The Gel extraction kit purification, and the real-time PCR Master Mix SYBR Green I were purchased from Tiangen Biotechnology Co. Ltd. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, the fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit) and DAB (3'3'-Diaminobenzidine tetrahydrochloride peroxidase) were from Beijing Zhongshan Co. Ltd.

Purification:

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus
Article Snippet: Restriction enzymes, EcoRI and XhoI, pMD18-T vector, the Total RNA Isolation System and RNase-free DNase I were purchased from TaKaRa Biotechnology Co. Ltd. .. The Gel extraction kit purification, and the real-time PCR Master Mix SYBR Green I were purchased from Tiangen Biotechnology Co. Ltd. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, the fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit) and DAB (3'3'-Diaminobenzidine tetrahydrochloride peroxidase) were from Beijing Zhongshan Co. Ltd.

Polymerase Chain Reaction:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: .. The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn. .. Cell culture and transfection Chinese hamster ovary (CHO) cells (Shanghai Institute of Cell Bank, China) were cultured in DMEM/F12K medium containing 10% FBS supplemented with penicillin (50 U/mL, Gibco) and streptomycin (50 U/mL, Gibco) at 37℃ in a humidified atmosphere with 5% CO2 .

Article Title: Analysis of DNA Methylation in Various Swine Tissues
Article Snippet: The digestion-ligation reaction was performed in a volume of 25 uL containing 500 ng DNA template, 3 U EcoRI, 3 U HpaII (or MspI), 1.5 U T4 DNA ligase (TaKaRa, Dalian, China), 5 pmol EcoRI adapter, 50 pmol HpaII/Msp I adapter and 2.5 uL 10× T4 ligase buffer. .. Preamplified PCR reactions were performed in a final volume of 20 uL containing 2 uL of ligation products, 40 ng of E+1 and H-M+1 preamplified primers , 0.1 uL of Ex Taq polymerase, 1.6 uL of dNTPs (2.5 mM), 1.2 uL of MgCl2 (25 mM), 2 uL of 10× PCR buffer and 14.1 uL of sterile distilled H2 O.

Gel Extraction:

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus
Article Snippet: Restriction enzymes, EcoRI and XhoI, pMD18-T vector, the Total RNA Isolation System and RNase-free DNase I were purchased from TaKaRa Biotechnology Co. Ltd. .. The Gel extraction kit purification, and the real-time PCR Master Mix SYBR Green I were purchased from Tiangen Biotechnology Co. Ltd. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, the fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit) and DAB (3'3'-Diaminobenzidine tetrahydrochloride peroxidase) were from Beijing Zhongshan Co. Ltd.

Mouse Assay:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: Construction of the overexpression vector Total mRNA of brain (embryo 18 days) was isolated from Kunming mice (Xi'an Jiao Tong university, China) using a Trizol kit according to the manufacturer's instructions from the Invitrogen (USA). .. The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn.

Plasmid Preparation:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: .. The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn. .. Cell culture and transfection Chinese hamster ovary (CHO) cells (Shanghai Institute of Cell Bank, China) were cultured in DMEM/F12K medium containing 10% FBS supplemented with penicillin (50 U/mL, Gibco) and streptomycin (50 U/mL, Gibco) at 37℃ in a humidified atmosphere with 5% CO2 .

Article Title: Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria
Article Snippet: Preparation of asp -disrupted or -introduced strain Fragments of strain 288 chromosomal DNA digested with the EcoRI were inserted into the EcoRI site of pUC119 ( ) and introduced into E. coli HB101 (TaKaRa Co., Kyoto, Japan). .. Disruption of asp and orf2 was carried out according to the homologous recombination technique ( ) using a suicide vector pXAC623 ( ; ).

Article Title: In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate
Article Snippet: .. Then, the transgene was chemically synthesised with restriction sites of Bgl II and EcoR I and cloned into the pEGFP-C1 vector (Clontech) by Gene Universal Inc., resulting in pEGFP-env-tm JDV ( ). ..

Article Title: MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis
Article Snippet: .. Construction of PSGH2/MYCN plasmid A mouse-MYCN fragment was extracted from HA-MYCN plasmid and subcloned into the EcoRI and EcoRV (Takara, Japan) sites of the PSGH2 vector to obtain the mMYCN -HSE-EGFP construct ( , ). .. The PSGH2 vector contains eight HSE sequence ( AGAACGTTCTAGAAC ) allows the symmetrical addition of a CMV minimal promoter to both ends in order to drive the expression of the genes of interest (one side is EGFP and the other side is MYCN ) flanked by 5V and 3V globin UTRs and SV40 polyadenylation (pA) signal (I-SceI meganuclease recognition sites) ( ).

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus
Article Snippet: .. Restriction enzymes, EcoRI and XhoI, pMD18-T vector, the Total RNA Isolation System and RNase-free DNase I were purchased from TaKaRa Biotechnology Co. Ltd. .. The Gel extraction kit purification, and the real-time PCR Master Mix SYBR Green I were purchased from Tiangen Biotechnology Co. Ltd. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, the fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit) and DAB (3'3'-Diaminobenzidine tetrahydrochloride peroxidase) were from Beijing Zhongshan Co. Ltd.

Article Title: Characterization of Helicobacter pylori Bacteriophage KHP30
Article Snippet: The phage DNAs inside the H. pylori cells were extracted using a FastGene plasmid minikit (Nippon Genetics Co., Tokyo, Japan). .. The phage DNAs (1 μg) were digested with EcoRI (TaKaRa Bio) and then resolved electrophoretically in a 0.8% agarose gel.

Article Title: Tay1 Protein, a Novel Telomere Binding Factor from Yarrowia lipolytica *
Article Snippet: .. The plasmid pMH25 carrying ten Y. lipolytica telomeric repeats was constructed by annealing oligonucleotides YlTEL-sense and YlTEL-antisense followed by the digestion of the resulting dsDNA with EcoRI and ligation into the EcoRI-digested and -dephosphorylated plasmid pHIS2 (Clontech). .. We obtained the desired recombinant plasmid carrying 2 × 5 telomeric repeats separated by an EcoRI site and named it pMH25.

Software:

Article Title: Analysis of DNA Methylation in Various Swine Tissues
Article Snippet: DNA methylation polymorphism profiles were subsequently obtained using GeneScan 3.1 software. .. The digestion-ligation reaction was performed in a volume of 25 uL containing 500 ng DNA template, 3 U EcoRI, 3 U HpaII (or MspI), 1.5 U T4 DNA ligase (TaKaRa, Dalian, China), 5 pmol EcoRI adapter, 50 pmol HpaII/Msp I adapter and 2.5 uL 10× T4 ligase buffer.

Real-time Polymerase Chain Reaction:

Article Title: Cloning, expression and characterization of gE protein of Duck plague virus
Article Snippet: Restriction enzymes, EcoRI and XhoI, pMD18-T vector, the Total RNA Isolation System and RNase-free DNase I were purchased from TaKaRa Biotechnology Co. Ltd. .. The Gel extraction kit purification, and the real-time PCR Master Mix SYBR Green I were purchased from Tiangen Biotechnology Co. Ltd. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, the fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit) and DAB (3'3'-Diaminobenzidine tetrahydrochloride peroxidase) were from Beijing Zhongshan Co. Ltd.

Recombinant:

Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells
Article Snippet: .. The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn. .. Cell culture and transfection Chinese hamster ovary (CHO) cells (Shanghai Institute of Cell Bank, China) were cultured in DMEM/F12K medium containing 10% FBS supplemented with penicillin (50 U/mL, Gibco) and streptomycin (50 U/mL, Gibco) at 37℃ in a humidified atmosphere with 5% CO2 .

Article Title: Tay1 Protein, a Novel Telomere Binding Factor from Yarrowia lipolytica *
Article Snippet: The plasmid pMH25 carrying ten Y. lipolytica telomeric repeats was constructed by annealing oligonucleotides YlTEL-sense and YlTEL-antisense followed by the digestion of the resulting dsDNA with EcoRI and ligation into the EcoRI-digested and -dephosphorylated plasmid pHIS2 (Clontech). .. We obtained the desired recombinant plasmid carrying 2 × 5 telomeric repeats separated by an EcoRI site and named it pMH25.

Agarose Gel Electrophoresis:

Article Title: Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy
Article Snippet: .. Preparation of D4Z4 repeats from the BAC clone RP11-242C23 was digested using EcoRI and treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 20 min. DNA was subjected to electrophoresis on a 0.5% agarose gel. .. Bands larger than the 10-kb marker (GeneRuler 1 kb DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA) were excised using a razor under ultraviolet light.

Article Title: Characterization of Helicobacter pylori Bacteriophage KHP30
Article Snippet: .. The phage DNAs (1 μg) were digested with EcoRI (TaKaRa Bio) and then resolved electrophoretically in a 0.8% agarose gel. .. After staining with ethidium bromide, the gels were visualized under UV transillumination.

Article Title: New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients
Article Snippet: .. Five micrograms of DNA were digested with EcoRI, EcoRI/BlnI, or HindIII (Takara, Japan), and separated by pulsed-field gel electrophoresis on a 1.2% agarose gel (Sangon, China) in × 0.5 TBE for 39 h. Nytran + membranes (GE Healthcare, USA) for allele sizing were hybridized with probe p13E-11, and those for allele typing were hybridized with probe 4qA and 4qB as previously described. ..

Electrophoresis:

Article Title: Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy
Article Snippet: .. Preparation of D4Z4 repeats from the BAC clone RP11-242C23 was digested using EcoRI and treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 20 min. DNA was subjected to electrophoresis on a 0.5% agarose gel. .. Bands larger than the 10-kb marker (GeneRuler 1 kb DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA) were excised using a razor under ultraviolet light.

DNA Methylation Assay:

Article Title: Analysis of DNA Methylation in Various Swine Tissues
Article Snippet: DNA methylation polymorphism profiles were subsequently obtained using GeneScan 3.1 software. .. The digestion-ligation reaction was performed in a volume of 25 uL containing 500 ng DNA template, 3 U EcoRI, 3 U HpaII (or MspI), 1.5 U T4 DNA ligase (TaKaRa, Dalian, China), 5 pmol EcoRI adapter, 50 pmol HpaII/Msp I adapter and 2.5 uL 10× T4 ligase buffer.

Molecular Weight:

Article Title: New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients
Article Snippet: DNA isolation, analysis of D4Z4 repeats, and 4qA/4qB variant determination High molecular weight DNA was extracted from peripheral lymphocytes derived from freshly collected blood using the standard protocols. .. Five micrograms of DNA were digested with EcoRI, EcoRI/BlnI, or HindIII (Takara, Japan), and separated by pulsed-field gel electrophoresis on a 1.2% agarose gel (Sangon, China) in × 0.5 TBE for 39 h. Nytran + membranes (GE Healthcare, USA) for allele sizing were hybridized with probe p13E-11, and those for allele typing were hybridized with probe 4qA and 4qB as previously described.

BAC Assay:

Article Title: Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy
Article Snippet: .. Preparation of D4Z4 repeats from the BAC clone RP11-242C23 was digested using EcoRI and treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 20 min. DNA was subjected to electrophoresis on a 0.5% agarose gel. .. Bands larger than the 10-kb marker (GeneRuler 1 kb DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA) were excised using a razor under ultraviolet light.

Marker:

Article Title: Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy
Article Snippet: Preparation of D4Z4 repeats from the BAC clone RP11-242C23 was digested using EcoRI and treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 20 min. DNA was subjected to electrophoresis on a 0.5% agarose gel. .. Bands larger than the 10-kb marker (GeneRuler 1 kb DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA) were excised using a razor under ultraviolet light.

Staining:

Article Title: Characterization of Helicobacter pylori Bacteriophage KHP30
Article Snippet: The phage DNAs (1 μg) were digested with EcoRI (TaKaRa Bio) and then resolved electrophoretically in a 0.8% agarose gel. .. After staining with ethidium bromide, the gels were visualized under UV transillumination.

Variant Assay:

Article Title: New Insights into Genotype-phenotype Correlations in Chinese Facioscapulohumeral Muscular Dystrophy: A Retrospective Analysis of 178 Patients
Article Snippet: Paragraph title: DNA isolation, analysis of D4Z4 repeats, and 4qA/4qB variant determination ... Five micrograms of DNA were digested with EcoRI, EcoRI/BlnI, or HindIII (Takara, Japan), and separated by pulsed-field gel electrophoresis on a 1.2% agarose gel (Sangon, China) in × 0.5 TBE for 39 h. Nytran + membranes (GE Healthcare, USA) for allele sizing were hybridized with probe p13E-11, and those for allele typing were hybridized with probe 4qA and 4qB as previously described.

Homologous Recombination:

Article Title: Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria
Article Snippet: Preparation of asp -disrupted or -introduced strain Fragments of strain 288 chromosomal DNA digested with the EcoRI were inserted into the EcoRI site of pUC119 ( ) and introduced into E. coli HB101 (TaKaRa Co., Kyoto, Japan). .. Disruption of asp and orf2 was carried out according to the homologous recombination technique ( ) using a suicide vector pXAC623 ( ; ).

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  • 99
    TaKaRa ecori
    (a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection <t>DNA</t> probe (b, horizontal bar) that had <t>EcoRI</t> digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.
    Ecori, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa 233 bp sspi ecori fragment
    Restriction map of the 17 kb genomic clone and subclones containing the 5′ region of the human α2(V) collagen gene. E, <t>EcoRI;</t> B, BamHI; K, KpnI; S, SacI; Pv, PvuII; Bg, BglII; P, PstI; X, XbaI; Sp, SphI; C, ClaI; Sc, ScaI; D, DraI; M, MluI; Ss, <t>SspI;</t> (X), XbaI site overlapping a dam methylation site. White and black boxes denote untranslated and coding regions of the first exon, respectively. The cross hatched bar represents intron sequences. Sl, ScaI-SspI fragment used as Sl probe; asterisk denotes 32 P-labeled 5′ end. Arrows indicate region and direction of sequencing.
    233 Bp Sspi Ecori Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection DNA probe (b, horizontal bar) that had EcoRI digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.

    Journal: Fems Microbiology Letters

    Article Title: Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria

    doi: 10.1111/j.1574-6968.2008.01184.x

    Figure Lengend Snippet: (a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection DNA probe (b, horizontal bar) that had EcoRI digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.

    Article Snippet: Preparation of asp -disrupted or -introduced strain Fragments of strain 288 chromosomal DNA digested with the EcoRI were inserted into the EcoRI site of pUC119 ( ) and introduced into E. coli HB101 (TaKaRa Co., Kyoto, Japan).

    Techniques: Plasmid Preparation, Homologous Recombination, Produced, Mutagenesis, Hybridization, Amplification, Labeling, DNA Labeling, Agarose Gel Electrophoresis, SDS Page, Staining

    Generation of Tg(RE:HSE:EGFP) zebrafish line. ( a ) Schematic diagram of the structure of PSGH2/RUNX1-Evi-1 recombinant plasmid. A human-RUNX1-Evi-1 fragment was cloned into the EcoRI and EcoRV sites of the PSGH2 vector. ( b ) A schematic presentation of the eight multimerized heat shock element (HSE) promoter, which is flanked by two minimal promoters in opposed orientation (black arrowhead) to bidirectionally induce EGFP and RUNX1-Evi-1 expression. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. ( c ) Transgenic verification by PCR: M: TAKARA DL2000 marker; lane 1 and 2: wild type and Tg(RE:HSE:EGFP) zebrafish larvae at 3 dpf, respectively; lane 3: PSGH2/RUNX1-Evi-1 plasmid; lane 4: double distilled water. ( d ) EGFP expression in Tg(RE:HSE:EGFP) zebrafish F2 generation at 3dpf (×4)

    Journal: BMC Cancer

    Article Title: RUNX1-Evi-1 fusion gene inhibited differentiation and apoptosis in myelopoiesis: an in vivo study

    doi: 10.1186/s12885-015-1961-y

    Figure Lengend Snippet: Generation of Tg(RE:HSE:EGFP) zebrafish line. ( a ) Schematic diagram of the structure of PSGH2/RUNX1-Evi-1 recombinant plasmid. A human-RUNX1-Evi-1 fragment was cloned into the EcoRI and EcoRV sites of the PSGH2 vector. ( b ) A schematic presentation of the eight multimerized heat shock element (HSE) promoter, which is flanked by two minimal promoters in opposed orientation (black arrowhead) to bidirectionally induce EGFP and RUNX1-Evi-1 expression. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. ( c ) Transgenic verification by PCR: M: TAKARA DL2000 marker; lane 1 and 2: wild type and Tg(RE:HSE:EGFP) zebrafish larvae at 3 dpf, respectively; lane 3: PSGH2/RUNX1-Evi-1 plasmid; lane 4: double distilled water. ( d ) EGFP expression in Tg(RE:HSE:EGFP) zebrafish F2 generation at 3dpf (×4)

    Article Snippet: It was subcloned into EcoRI and EcoRV (Takara, Japan) sites of the pSGH2 vector [ ], which contains eight HSE sequence (AGAACGTTCTAGAAC) and EGFP segment.

    Techniques: Recombinant, Plasmid Preparation, Clone Assay, Expressing, Transgenic Assay, Polymerase Chain Reaction, Marker

    (A) Location of the intracellular phage genome in phage-producing H. pylori strain NY43. The DNAs of H. pylori strains 3401 and NY43 were separated by PFGE (left) and subjected to Southern blot analysis with a phage-specific probe (right). H. pylori strain 3401 was used as the non-phage-harboring control. Black and gray arrows, phage and H. pylori DNAs, respectively. Lanes M, 1, and 2 in both panels, bacteriophage lambda ladder, strain 3401, and strain NY43, respectively. (B) Restriction digestion analysis of phage DNAs. (Left) Phage DNAs derived from the purified particles or the phage DNAs derived from H. pylori strain NY43 were digested with EcoRI and separated electrophoretically in a 0.8% agarose gel. Bacteriophage lambda DNA digested with HindIII was also separated electrophoretically as a marker. The DNA bands are indicated by arrows. (Right) In silico EcoRI digestion patterns of phage KHP30 DNA in the linear form, the circular form, a mixture of the linear and circular forms, and the concatemeric form, which were resolved in a 0.7% agarose gel.

    Journal: Applied and Environmental Microbiology

    Article Title: Characterization of Helicobacter pylori Bacteriophage KHP30

    doi: 10.1128/AEM.03530-12

    Figure Lengend Snippet: (A) Location of the intracellular phage genome in phage-producing H. pylori strain NY43. The DNAs of H. pylori strains 3401 and NY43 were separated by PFGE (left) and subjected to Southern blot analysis with a phage-specific probe (right). H. pylori strain 3401 was used as the non-phage-harboring control. Black and gray arrows, phage and H. pylori DNAs, respectively. Lanes M, 1, and 2 in both panels, bacteriophage lambda ladder, strain 3401, and strain NY43, respectively. (B) Restriction digestion analysis of phage DNAs. (Left) Phage DNAs derived from the purified particles or the phage DNAs derived from H. pylori strain NY43 were digested with EcoRI and separated electrophoretically in a 0.8% agarose gel. Bacteriophage lambda DNA digested with HindIII was also separated electrophoretically as a marker. The DNA bands are indicated by arrows. (Right) In silico EcoRI digestion patterns of phage KHP30 DNA in the linear form, the circular form, a mixture of the linear and circular forms, and the concatemeric form, which were resolved in a 0.7% agarose gel.

    Article Snippet: The phage DNAs (1 μg) were digested with EcoRI (TaKaRa Bio) and then resolved electrophoretically in a 0.8% agarose gel.

    Techniques: Southern Blot, Derivative Assay, Purification, Agarose Gel Electrophoresis, Lambda DNA Preparation, Marker, In Silico

    Restriction map of the 17 kb genomic clone and subclones containing the 5′ region of the human α2(V) collagen gene. E, EcoRI; B, BamHI; K, KpnI; S, SacI; Pv, PvuII; Bg, BglII; P, PstI; X, XbaI; Sp, SphI; C, ClaI; Sc, ScaI; D, DraI; M, MluI; Ss, SspI; (X), XbaI site overlapping a dam methylation site. White and black boxes denote untranslated and coding regions of the first exon, respectively. The cross hatched bar represents intron sequences. Sl, ScaI-SspI fragment used as Sl probe; asterisk denotes 32 P-labeled 5′ end. Arrows indicate region and direction of sequencing.

    Journal: Gene Expression

    Article Title: Homology between α2(V) and αl(lll) collagen promoters and evidence for negatively acting elements in the α2(V) first intron and 5′ flanking sequences

    doi:

    Figure Lengend Snippet: Restriction map of the 17 kb genomic clone and subclones containing the 5′ region of the human α2(V) collagen gene. E, EcoRI; B, BamHI; K, KpnI; S, SacI; Pv, PvuII; Bg, BglII; P, PstI; X, XbaI; Sp, SphI; C, ClaI; Sc, ScaI; D, DraI; M, MluI; Ss, SspI; (X), XbaI site overlapping a dam methylation site. White and black boxes denote untranslated and coding regions of the first exon, respectively. The cross hatched bar represents intron sequences. Sl, ScaI-SspI fragment used as Sl probe; asterisk denotes 32 P-labeled 5′ end. Arrows indicate region and direction of sequencing.

    Article Snippet: A 233 bp SspI-EcoRI fragment, corresponding to 5′ untranslated sequences, was isolated from a human α2(V) collagen cDNA clone , and used to screen a human leukocyte genomic library in EMBL3, purchased from Clontech.

    Techniques: Methylation, Labeling, Sequencing