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TaKaRa xho
Identification of PHD3. ( A ) Electrophoresis of full-length target gene RT-PCR product; M: <t>DNA</t> Marker DL10,000, 1: PHD3. ( B ) Hind III and <t>Xho</t> I digestion and electrophoresis of pcDNA 3.1(+)-PHD3 eukaryotic expression vector; M: DNA Marker DL10,000, 1: PHD3, 2: pcDNA 3.1(+) plasmid digested by Hind III and Xho I, 3: pcDNA 3.1(+)-PHD3 plasmid digested by Hind III and Xho I.
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Images

1) Product Images from "Construction of a recombinant eukaryotic expression vector containing PHD3 gene and its expression in HepG2 cells"

Article Title: Construction of a recombinant eukaryotic expression vector containing PHD3 gene and its expression in HepG2 cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-31-64

Identification of PHD3. ( A ) Electrophoresis of full-length target gene RT-PCR product; M: DNA Marker DL10,000, 1: PHD3. ( B ) Hind III and Xho I digestion and electrophoresis of pcDNA 3.1(+)-PHD3 eukaryotic expression vector; M: DNA Marker DL10,000, 1: PHD3, 2: pcDNA 3.1(+) plasmid digested by Hind III and Xho I, 3: pcDNA 3.1(+)-PHD3 plasmid digested by Hind III and Xho I.
Figure Legend Snippet: Identification of PHD3. ( A ) Electrophoresis of full-length target gene RT-PCR product; M: DNA Marker DL10,000, 1: PHD3. ( B ) Hind III and Xho I digestion and electrophoresis of pcDNA 3.1(+)-PHD3 eukaryotic expression vector; M: DNA Marker DL10,000, 1: PHD3, 2: pcDNA 3.1(+) plasmid digested by Hind III and Xho I, 3: pcDNA 3.1(+)-PHD3 plasmid digested by Hind III and Xho I.

Techniques Used: Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Plasmid Preparation

2) Product Images from "Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene"

Article Title: Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene

Journal: Virology Journal

doi: 10.1186/1743-422X-8-266

Cloning of DEV UL55 gene and identification of recombinant plasmid pMD18-T/UL55 by restriction enzyme digestion . A , The amplification of DEV UL55 gene by conventional PCR using primers P1, P2. Lane 1, the amplified product of DEV UL55 gene; M, DNA marker(DL2000). B , Identification of recombinant plasmid pMD18-T/UL55 by restriction enzyme digestion. Lane 1, the recombinant plasmid pMD18-T/UL55 were digested with BamH I and Xho I ; Lane 2, the PCR product of the recombinant plasmid pMD18-T/UL55; M1, DNA marker III; M2, DNA marker(DL2000).
Figure Legend Snippet: Cloning of DEV UL55 gene and identification of recombinant plasmid pMD18-T/UL55 by restriction enzyme digestion . A , The amplification of DEV UL55 gene by conventional PCR using primers P1, P2. Lane 1, the amplified product of DEV UL55 gene; M, DNA marker(DL2000). B , Identification of recombinant plasmid pMD18-T/UL55 by restriction enzyme digestion. Lane 1, the recombinant plasmid pMD18-T/UL55 were digested with BamH I and Xho I ; Lane 2, the PCR product of the recombinant plasmid pMD18-T/UL55; M1, DNA marker III; M2, DNA marker(DL2000).

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

3) Product Images from "Genome Typing of Adenovirus Type 34 Isolated in Two Cases of Conjunctivitis in Sapporo, Japan"

Article Title: Genome Typing of Adenovirus Type 34 Isolated in Two Cases of Conjunctivitis in Sapporo, Japan

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.39.11.4187-4189.2001

Restriction patterns obtained after cleavage of Ad14p, Ad34p, Ad35p, and two clinical isolates with Hin dIII (g), Hpa I (h), Pst I (i), Sal I (j), Sma I (k), Xba I (l), and Xho I (m). Lanes: M1, λ/EcoT14I; M2, 1-kb ladder; 1, Ad14p; 2, Ad34p; 3, Ad35p; 4, isolate A; 5, isolate B.
Figure Legend Snippet: Restriction patterns obtained after cleavage of Ad14p, Ad34p, Ad35p, and two clinical isolates with Hin dIII (g), Hpa I (h), Pst I (i), Sal I (j), Sma I (k), Xba I (l), and Xho I (m). Lanes: M1, λ/EcoT14I; M2, 1-kb ladder; 1, Ad14p; 2, Ad34p; 3, Ad35p; 4, isolate A; 5, isolate B.

Techniques Used:

4) Product Images from "Antiproliferative effect of double suicide gene delivery mediated by polyamidoamine dendrimers in human Tenon's capsule fibroblasts"

Article Title: Antiproliferative effect of double suicide gene delivery mediated by polyamidoamine dendrimers in human Tenon's capsule fibroblasts

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2017.5235

TK and CD gene expression in HTF cells. (A) The plasmid map showing the expression vector, pAcGFP1-Hyg, carrying the TK and CD genes. (B) The 1,617 bp fragment of the TK-CD gene was confirmed by restriction enzyme digestion of the plasmid with Xho I and Kpn I followed by 1% DNA gel electrophoresis. Lane M, DNA marker; lane 1, vector pAcGFP1-Hyg; and lane 2, pAcGFP1-Hyg-TK-CD. (C) Reverse transcription-polymerase chain reaction analysis of HFT cells transfected with pAcGFP1-Hyg-TK-CD. Lane M, DL2000 marker; lane 1, fragment of TK-CD (403 bp) and β-actin (561 bp) in the transfected cells; lane 2, untransfected; and lane 3, negative control groups. (D) TK and CD gene expression mediated either by Lipo or G5-PAMAM-D was accessed by fluorescence microscopy (magnification, ×100). TK, thymidine kinase; CD, cytosine deaminase; HTFs, human Tenon's capsule fibroblasts; G5-PAMAM-D, 5-polyamidoamine dendrimers; bp, base pairs; Lipo, Lipofectamine 2000; ctrl, control.
Figure Legend Snippet: TK and CD gene expression in HTF cells. (A) The plasmid map showing the expression vector, pAcGFP1-Hyg, carrying the TK and CD genes. (B) The 1,617 bp fragment of the TK-CD gene was confirmed by restriction enzyme digestion of the plasmid with Xho I and Kpn I followed by 1% DNA gel electrophoresis. Lane M, DNA marker; lane 1, vector pAcGFP1-Hyg; and lane 2, pAcGFP1-Hyg-TK-CD. (C) Reverse transcription-polymerase chain reaction analysis of HFT cells transfected with pAcGFP1-Hyg-TK-CD. Lane M, DL2000 marker; lane 1, fragment of TK-CD (403 bp) and β-actin (561 bp) in the transfected cells; lane 2, untransfected; and lane 3, negative control groups. (D) TK and CD gene expression mediated either by Lipo or G5-PAMAM-D was accessed by fluorescence microscopy (magnification, ×100). TK, thymidine kinase; CD, cytosine deaminase; HTFs, human Tenon's capsule fibroblasts; G5-PAMAM-D, 5-polyamidoamine dendrimers; bp, base pairs; Lipo, Lipofectamine 2000; ctrl, control.

Techniques Used: Expressing, Plasmid Preparation, DNA Gel Electrophoresis, Marker, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Fluorescence, Microscopy

5) Product Images from "Effect of stable transfection with PHD3 on growth and proliferation of HepG2 cells in vitro and in vivo"

Article Title: Effect of stable transfection with PHD3 on growth and proliferation of HepG2 cells in vitro and in vivo

Journal: International Journal of Clinical and Experimental Medicine

doi:

Verification of the PHD3 cDNA insert with restriction enzyme digestion. A: Plasmid pcDNA 3.1(+)-PHD3 was digested by Hind III and Xho I, 1: DNA Marker DL 10,000; 2: Two fragments of 721 bp 5400 bp. The small band corresponds with the size of the insert.
Figure Legend Snippet: Verification of the PHD3 cDNA insert with restriction enzyme digestion. A: Plasmid pcDNA 3.1(+)-PHD3 was digested by Hind III and Xho I, 1: DNA Marker DL 10,000; 2: Two fragments of 721 bp 5400 bp. The small band corresponds with the size of the insert.

Techniques Used: Plasmid Preparation, Marker

6) Product Images from "Assembly of Infectious Herpes Simplex Virus Type 1 Virions in the Absence of Full-Length VP22"

Article Title: Assembly of Infectious Herpes Simplex Virus Type 1 Virions in the Absence of Full-Length VP22

Journal: Journal of Virology

doi:

Schematic representations of the HSV-1 genome and viruses used in this study. The wild-type HSV-1 genome is shown in line 1. The unique long (U L ), unique short (U s ), and terminal repeat segments (a, b, c, a′, b′, and c′) are indicated. The coordinates of the Bam Q and Bam F fragments in the viral genome are indicated, and these regions are expanded in line 2. Line 3 shows the predicted open reading frames in the Bam Q and Bam F regions of wild-type virus. Solid arrowheads mark translation stop sites and show the direction of transcription; arrow length indicates approximate transcript size. The U L 22 transcript designated with an open arrow possesses a translational stop site beyond the boundaries of the Bam Q fragment. R2507 (line 4) is a derivative of the parental strain Δ305. Thus, R2507 possesses a deletion in the Bam Q fragment which removes 500 bp, including a portion of the U L 23 gene encoding the viral tk and a portion of the U L 24 gene. These disrupted transcripts are denoted by an X at the locations of their stop sites in the wild-type genome. In R2507, a 1.8-kb tk expression cassette containing the viral tk gene under control of the ICP27 promoter (27p) has been inserted approximately 200 bp upstream of the U L ). RD177 (line 5) and RF177 (line 6) were generated for the present study as described in Materials and Methods. Both viruses express the GFP under control of a cytomegalovirus promoter (Cp). The small solid rectangles in lines 5 and 6 indicate that the GFP cassette was inserted 45 bp 5′ of the U L 49 stop site. The deletion in the Bam Q locus present in R2507 and RD177 was repaired in RF177. Restriction sites: Bam, Bam HI; E, Eco RV; X, Xho I; N, Nsi I; Bg, Bgl II.
Figure Legend Snippet: Schematic representations of the HSV-1 genome and viruses used in this study. The wild-type HSV-1 genome is shown in line 1. The unique long (U L ), unique short (U s ), and terminal repeat segments (a, b, c, a′, b′, and c′) are indicated. The coordinates of the Bam Q and Bam F fragments in the viral genome are indicated, and these regions are expanded in line 2. Line 3 shows the predicted open reading frames in the Bam Q and Bam F regions of wild-type virus. Solid arrowheads mark translation stop sites and show the direction of transcription; arrow length indicates approximate transcript size. The U L 22 transcript designated with an open arrow possesses a translational stop site beyond the boundaries of the Bam Q fragment. R2507 (line 4) is a derivative of the parental strain Δ305. Thus, R2507 possesses a deletion in the Bam Q fragment which removes 500 bp, including a portion of the U L 23 gene encoding the viral tk and a portion of the U L 24 gene. These disrupted transcripts are denoted by an X at the locations of their stop sites in the wild-type genome. In R2507, a 1.8-kb tk expression cassette containing the viral tk gene under control of the ICP27 promoter (27p) has been inserted approximately 200 bp upstream of the U L ). RD177 (line 5) and RF177 (line 6) were generated for the present study as described in Materials and Methods. Both viruses express the GFP under control of a cytomegalovirus promoter (Cp). The small solid rectangles in lines 5 and 6 indicate that the GFP cassette was inserted 45 bp 5′ of the U L 49 stop site. The deletion in the Bam Q locus present in R2507 and RD177 was repaired in RF177. Restriction sites: Bam, Bam HI; E, Eco RV; X, Xho I; N, Nsi I; Bg, Bgl II.

Techniques Used: Expressing, Generated

7) Product Images from "Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells"

Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells

Journal: Chinese Journal of Cancer Research

doi: 10.3978/j.issn.1000-9604.2013.10.06

Agarose gel electrophoresis of pEGFP-N1 and PCR product of human syncytin cDNA and the recombinant clones with correctly sized inserts, using Xho I/ Eco R I double digestion. Lane M, DNA marker; lane 1, pEGFP-N1; lane 2, PCR product of syncytin cDNA; lanes
Figure Legend Snippet: Agarose gel electrophoresis of pEGFP-N1 and PCR product of human syncytin cDNA and the recombinant clones with correctly sized inserts, using Xho I/ Eco R I double digestion. Lane M, DNA marker; lane 1, pEGFP-N1; lane 2, PCR product of syncytin cDNA; lanes

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Recombinant, Clone Assay, Marker

Related Articles

Clone Assay:

Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region
Article Snippet: Construction, Expression, and Purification of IL23R-CHR Human IL23R-CHR (amino acid residues 124–313) was cloned by Q-PCR from human spleen cDNA library (Biomics, China) using the following primers: 5′-GCCCATGGCTCCGCCAGATATTCCTGATG-3′ and 5′-CAGCTCGAGTTAATGAAAAAACGGTGAGCTCCA-3′ . .. After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany).

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara). .. During experiment of gene cloning the ligation mixture was used to transform into chemically competent E. coli Top 10 cells.

Article Title: Molecular Characterization of a Nucleopolyhedrovirus Newly Isolated from Ophiusa disjungens in China
Article Snippet: Virus DNA was digested with Bam HI, Eco RI, Hind III, Pst I, Xho I, Xba I (TaKaRa) at 37 °C for 2–3 h, then electrophoresed on 0.7 % agarose gels at 50 V for 15–22 h to separate the fragments. .. The pst I-G fragment of OpdiNPV DNA was cloned into pUC18 (TAKARA) plasmid vector according to the methods of Sambrook [ ].

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: PCR products were cloned into pGEM T-easy vector (Promega) and used as template for DIG (digoxygenin)-labeled probe synthesis according to the manufacture’s instructions in the PCR DIG labeling kit (Roche Diagnostics). .. One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight.

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: .. After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA. ..

Centrifugation:

Article Title: Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis
Article Snippet: Briefly, the PCR product (pMD-18T/VCAM-1) was digested with Eco RI and Xho I and inserted into the His and GST fusion protein sites of the prokaryotic expression vectors pGEX-4T-1 and pET-28a (Takara) respectively, to create the recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1. .. In order to achieve fusion protein expression, the recombinant plasmids were transformed into E. coli BL21 (DE3) and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h. The recombinant E. coli cells were harvested by high speed centrifugation after IPTG induction.

Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region
Article Snippet: After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany). .. The selected positive transformants were grown in 500 ml Luria-Bertani medium containing 100 ug/ml ampicillin at 16°C for about 12 h until the absorbance at 600 nm reached 0.6.Subsequently, the transformants thus obtained were induced with 0.2 mM isopropyl-β, D-thiogalactoside (IPTG, Bio Basic, CA) for 4 h, harvested by centrifugation (10,000 g, 10 min, 4°C), and resuspended in 50 ml equilibration buffer (20 mM PBS,500 mM NaCl, 10 mM imidazole, PH7.4).

Amplification:

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: The D6E (GLELO) gene was isolated by PCR amplification from the genome of Mortierella alpina ATCC 32222 with corresponding primers GLELO-F/R (Additional file : Table S1). .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara).

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: Southern hybridation probes targeting the left and right ends of the L. oceanica mtDNA were amplified using the following primers: LoceMito_Left_spec_F (5′-AAGCAAAACGCAAGCAGAGG-3′), LoceMito_Left_spec_R (5′-GTGCGTTTTTGTAGGCCGTT-3′), LoceMito_Right_spec_F (5′-CGGATTCACCAACTCGTCAA-3′), and LoceMito_Right_spec_R (5′-TTTAGATGCTTCATCGCGCT-3′). .. One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight.

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: To construct the endogenous recA deletion cassette, the flanking sequences of the endogenous recA , designated cinA (1.4 kb) and pbpX (1.4 kb), were amplified by PCR (PrimeSTAR HS DNA polymerase, TaKaRa) using the genomic DNA of B. subtilis 168 as the template. .. After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA.

DNA Synthesis:

Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading
Article Snippet: Second-strand DNA synthesis was performed by using T7 DNA polymerase (New England Biolabs, MA, USA) and T4 DNA ligase (Nippongene, Tokyo, Japan). .. The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase.

Synthesized:

Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading
Article Snippet: The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase. .. In vitro synthesized pMM1 was doubly nicked with Nt.Bbv CI (for A-strand gap) or Nb.Bbv CI (for C-strand gap) at 37°C for 1 hr, purified, and incubated at 70°C for 20 min to dissociate the 15-nt fragment flanked by two Bbv CI sites from parental DNA.

Selection:

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. E. coli DH5α (Novagen, Germany) was transformed with this recombinant plasmid and the following selection was conducted on Luria-Bertani (LB) broth supplemented with 0.1 mg/mL ampicillin (Sigma, USA).

Construct:

Article Title: Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells
Article Snippet: The 2.2-kb KDR-TK fragment was extracted from pBluescript II KDR-TK by a double digestion with Xho I and Sal I (Takara Biotechnology Co.,Ltd, Dalian, China) and identified by electrophoresis and DNA sequencing. .. The KDR-TK was inserted into the same clone location of the pEGFP vector with T4 ligase (Takara) to construct a plasmid termed pEGFP-KDR-TK.

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: .. The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. E. coli DH5α (Novagen, Germany) was transformed with this recombinant plasmid and the following selection was conducted on Luria-Bertani (LB) broth supplemented with 0.1 mg/mL ampicillin (Sigma, USA).

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: The PCR fragments were then cloned into the SalI-EcoRI site and the BamHI-SacII site of pBluescript II SK(+) to construct pCP. .. After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA.

Article Title: Screening and identification of proteins interacting with nucleostemin
Article Snippet: The full length of NS cDNA was cut by Bam HI and Xho I restriction endonucleases from pCDNA3-NS, then inserted into the downstream of the Gal4 DNA-binding domain of the bait vector pGBKT7 (Clontech Laboratories) with T4 DNA ligase. .. To construct the eukaryotic vector expressing the protein of the positive clone, DNA of the positive clone was digested by restriction endonucleases Bam HI and Xho I, and the DNA fragment was inserted into the pCDNA-myc vector.

Electrophoresis:

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight. .. After electrophoresis, digested DNA fragments were transferred to a positively charged nylon membrane by capillary transfer.

Article Title: Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells
Article Snippet: .. The 2.2-kb KDR-TK fragment was extracted from pBluescript II KDR-TK by a double digestion with Xho I and Sal I (Takara Biotechnology Co.,Ltd, Dalian, China) and identified by electrophoresis and DNA sequencing. .. The pEGFP 4.7 kb carrier was released from EGFP plasmid DNA (donated by Doctor Zhang Ge, Laboratory of Molecular Medicine, Sun Yat-Sen University, China) containing the restriction sites for Xho I and Sal I using the same technique.

Incubation:

Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading
Article Snippet: The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase. .. In vitro synthesized pMM1 was doubly nicked with Nt.Bbv CI (for A-strand gap) or Nb.Bbv CI (for C-strand gap) at 37°C for 1 hr, purified, and incubated at 70°C for 20 min to dissociate the 15-nt fragment flanked by two Bbv CI sites from parental DNA.

In Silico:

Article Title: Comparative genomic analysis of two emergent human adenovirus type 14 respiratory pathogen isolates in China reveals similar yet divergent genomes
Article Snippet: .. Genome type identification In silico restriction maps of available genome sequences from all HAdV-14 strains CHN/GZ01/2010, 303600, CHN/BJ430, CHN2012, and prototype de Wit were generated using the Vector NTI 10.3.0 software (Invitrogen Corp.; San Diego, CA, USA) as described in earlier studies., , These included profiles from restriction enzymes Bam HI, Bcl I, Bgl l, Bgl II, Bst EII, Eco RI, Hin dIII, Hpa I, Sal l, Sma I, Xba I, and Xho I (TaKaRa Corp.; China), all of which were chosen in order to be consistent with the original nomenclature system devised by Li and colleagues. .. Genome typing of these strains was determined by comparing its in silico RE profiles with other HAdV-14 genome types.,

Expressing:

Article Title: Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis
Article Snippet: .. Briefly, the PCR product (pMD-18T/VCAM-1) was digested with Eco RI and Xho I and inserted into the His and GST fusion protein sites of the prokaryotic expression vectors pGEX-4T-1 and pET-28a (Takara) respectively, to create the recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1. .. In order to achieve fusion protein expression, the recombinant plasmids were transformed into E. coli BL21 (DE3) and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h. The recombinant E. coli cells were harvested by high speed centrifugation after IPTG induction.

Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region
Article Snippet: Paragraph title: Construction, Expression, and Purification of IL23R-CHR ... After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany).

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: .. The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. E. coli DH5α (Novagen, Germany) was transformed with this recombinant plasmid and the following selection was conducted on Luria-Bertani (LB) broth supplemented with 0.1 mg/mL ampicillin (Sigma, USA).

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: Paragraph title: Construction of the inducible recA expression BGM vector (iREX) ... After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA.

Article Title: Screening and identification of proteins interacting with nucleostemin
Article Snippet: The full length of NS cDNA was cut by Bam HI and Xho I restriction endonucleases from pCDNA3-NS, then inserted into the downstream of the Gal4 DNA-binding domain of the bait vector pGBKT7 (Clontech Laboratories) with T4 DNA ligase. .. The expression of NS fusion protein with yeast Gal4 DNA-binding domain was checked by Western blot with antibody against NS.

Modification:

Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading
Article Snippet: To introduce a site-specific biotin modification, an additional oligonucleotide carrying a site-specific biotin-dT modification (5’-CGCCTTGATCGT[Biotin-dT]GGGAACCGGAGCTGAATGAAGC-3’) was also added. .. The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase.

Western Blot:

Article Title: Screening and identification of proteins interacting with nucleostemin
Article Snippet: The full length of NS cDNA was cut by Bam HI and Xho I restriction endonucleases from pCDNA3-NS, then inserted into the downstream of the Gal4 DNA-binding domain of the bait vector pGBKT7 (Clontech Laboratories) with T4 DNA ligase. .. The expression of NS fusion protein with yeast Gal4 DNA-binding domain was checked by Western blot with antibody against NS.

Transformation Assay:

Article Title: Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis
Article Snippet: Briefly, the PCR product (pMD-18T/VCAM-1) was digested with Eco RI and Xho I and inserted into the His and GST fusion protein sites of the prokaryotic expression vectors pGEX-4T-1 and pET-28a (Takara) respectively, to create the recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1. .. In order to achieve fusion protein expression, the recombinant plasmids were transformed into E. coli BL21 (DE3) and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h. The recombinant E. coli cells were harvested by high speed centrifugation after IPTG induction.

Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region
Article Snippet: After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany). .. E.coli BL21 (DE3) cells were transformed with the pET32a/IL23R-CHR and the positive clones were selected by ampicillin resistance and confirmed by DNA sequencing.

Article Title: Molecular Characterization of a Nucleopolyhedrovirus Newly Isolated from Ophiusa disjungens in China
Article Snippet: Virus DNA was digested with Bam HI, Eco RI, Hind III, Pst I, Xho I, Xba I (TaKaRa) at 37 °C for 2–3 h, then electrophoresed on 0.7 % agarose gels at 50 V for 15–22 h to separate the fragments. .. The recombinant plasmids were transformed into E. coli DH 5α cells (TIANGEN), screened for lacZ complementation, recovered by using DNA Product Purification Kit (TIANGEN) from agarose gels and purified by precipitation with glass milk.

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. E. coli DH5α (Novagen, Germany) was transformed with this recombinant plasmid and the following selection was conducted on Luria-Bertani (LB) broth supplemented with 0.1 mg/mL ampicillin (Sigma, USA).

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: .. After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA. ..

Hybridization:

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: Paragraph title: Southern hybridization analysis ... One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight.

Transfection:

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. After verification of correct sequences by restriction digestion and DNA sequencing (Invitrogen, USA), the plasmids for cell transfection were prepared using EndoFree Maxi Plasmid Kit (Tiangen, China) and then linearized with Pvu I (Takara, China).

Southern Blot:

Article Title: Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa)
Article Snippet: .. Southern blot Genomic DNA from the transgenic plants was digested with the Xho I restriction enzyme (2,000 U, Takara). .. The resulting fragments were fractionated on a 1% agarose gel, denatured, and transferred from the gel onto a Hybond H+ nylon membrane (Amersham Pharmacia Biotech) using the capillary transfer method (Sambrook and Russell ).

Article Title: Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae
Article Snippet: .. Native and denatured southern blotting Genomic DNA from non-synchronised saturated cell cultures was digested overnight with XhoI (Takara, 1094A) and then separated on 1% agarose gel (15 cm in length) for 18 hours at 25 volts. .. E. coli exonuclease I digestion (New England biolabs, M0293S) was performed prior to XhoI digestion of the genomic DNA.

Ligation:

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara). .. During experiment of gene cloning the ligation mixture was used to transform into chemically competent E. coli Top 10 cells.

Introduce:

Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading
Article Snippet: To introduce a site-specific biotin modification, an additional oligonucleotide carrying a site-specific biotin-dT modification (5’-CGCCTTGATCGT[Biotin-dT]GGGAACCGGAGCTGAATGAAGC-3’) was also added. .. The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase.

Generated:

Article Title: Comparative genomic analysis of two emergent human adenovirus type 14 respiratory pathogen isolates in China reveals similar yet divergent genomes
Article Snippet: .. Genome type identification In silico restriction maps of available genome sequences from all HAdV-14 strains CHN/GZ01/2010, 303600, CHN/BJ430, CHN2012, and prototype de Wit were generated using the Vector NTI 10.3.0 software (Invitrogen Corp.; San Diego, CA, USA) as described in earlier studies., , These included profiles from restriction enzymes Bam HI, Bcl I, Bgl l, Bgl II, Bst EII, Eco RI, Hin dIII, Hpa I, Sal l, Sma I, Xba I, and Xho I (TaKaRa Corp.; China), all of which were chosen in order to be consistent with the original nomenclature system devised by Li and colleagues. .. Genome typing of these strains was determined by comparing its in silico RE profiles with other HAdV-14 genome types.,

Polymerase Chain Reaction:

Article Title: Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis
Article Snippet: .. Briefly, the PCR product (pMD-18T/VCAM-1) was digested with Eco RI and Xho I and inserted into the His and GST fusion protein sites of the prokaryotic expression vectors pGEX-4T-1 and pET-28a (Takara) respectively, to create the recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1. .. In order to achieve fusion protein expression, the recombinant plasmids were transformed into E. coli BL21 (DE3) and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h. The recombinant E. coli cells were harvested by high speed centrifugation after IPTG induction.

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara). .. During experiment of gene cloning the ligation mixture was used to transform into chemically competent E. coli Top 10 cells.

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: PCR products were cloned into pGEM T-easy vector (Promega) and used as template for DIG (digoxygenin)-labeled probe synthesis according to the manufacture’s instructions in the PCR DIG labeling kit (Roche Diagnostics). .. One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight.

Article Title: Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa)
Article Snippet: Southern blot Genomic DNA from the transgenic plants was digested with the Xho I restriction enzyme (2,000 U, Takara). .. The probe for the LTB gene was prepared by PCR from pMJ103-LTB-CTB.

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: After purification with Tian-quick midi purification kit (Tiangen, China), the tIL12rβ1 was ligated with Fc gene using overlap PCR extension with the primers F1 and F4. .. The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA).

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: The PCR fragments were then cloned into the SalI-EcoRI site and the BamHI-SacII site of pBluescript II SK(+) to construct pCP. .. After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA.

DNA Sequencing:

Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region
Article Snippet: After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany). .. E.coli BL21 (DE3) cells were transformed with the pET32a/IL23R-CHR and the positive clones were selected by ampicillin resistance and confirmed by DNA sequencing.

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara). .. The plasmids isolated from these transformants were verified by doing PCR and the gene sequences were confirmed by DNA sequencing.

Article Title: Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells
Article Snippet: .. The 2.2-kb KDR-TK fragment was extracted from pBluescript II KDR-TK by a double digestion with Xho I and Sal I (Takara Biotechnology Co.,Ltd, Dalian, China) and identified by electrophoresis and DNA sequencing. .. The pEGFP 4.7 kb carrier was released from EGFP plasmid DNA (donated by Doctor Zhang Ge, Laboratory of Molecular Medicine, Sun Yat-Sen University, China) containing the restriction sites for Xho I and Sal I using the same technique.

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. After verification of correct sequences by restriction digestion and DNA sequencing (Invitrogen, USA), the plasmids for cell transfection were prepared using EndoFree Maxi Plasmid Kit (Tiangen, China) and then linearized with Pvu I (Takara, China).

Reverse Transcription Polymerase Chain Reaction:

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: Construction, expression and purification of tIL12rβ1/Fc Based on the amino acid sequence of human IL12rβ1 receptor (Genbank NP_005526) and human IgG1 Fc (Genbank AEV43323.1), the human truncated IL12rβ1 receptor (tIL12rβ1) gene containing a signal peptide sequence (708 bp) and the human IgG1 Fc fragment (699 bp) was obtained by RT-PCR from human spleen cDNA library (Biomics, China) using the primers F1, F2 and F3, F4 (Table ), respectively. .. The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA).

Recombinant:

Article Title: Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis
Article Snippet: .. Briefly, the PCR product (pMD-18T/VCAM-1) was digested with Eco RI and Xho I and inserted into the His and GST fusion protein sites of the prokaryotic expression vectors pGEX-4T-1 and pET-28a (Takara) respectively, to create the recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1. .. In order to achieve fusion protein expression, the recombinant plasmids were transformed into E. coli BL21 (DE3) and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h. The recombinant E. coli cells were harvested by high speed centrifugation after IPTG induction.

Article Title: Molecular Characterization of a Nucleopolyhedrovirus Newly Isolated from Ophiusa disjungens in China
Article Snippet: Virus DNA was digested with Bam HI, Eco RI, Hind III, Pst I, Xho I, Xba I (TaKaRa) at 37 °C for 2–3 h, then electrophoresed on 0.7 % agarose gels at 50 V for 15–22 h to separate the fragments. .. The recombinant plasmids were transformed into E. coli DH 5α cells (TIANGEN), screened for lacZ complementation, recovered by using DNA Product Purification Kit (TIANGEN) from agarose gels and purified by precipitation with glass milk.

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. E. coli DH5α (Novagen, Germany) was transformed with this recombinant plasmid and the following selection was conducted on Luria-Bertani (LB) broth supplemented with 0.1 mg/mL ampicillin (Sigma, USA).

Article Title: Screening and identification of proteins interacting with nucleostemin
Article Snippet: The full length of NS cDNA was cut by Bam HI and Xho I restriction endonucleases from pCDNA3-NS, then inserted into the downstream of the Gal4 DNA-binding domain of the bait vector pGBKT7 (Clontech Laboratories) with T4 DNA ligase. .. The recombinant vector pGBKT7-NS was sequenced and NS protein was in the reading frame.

Mutagenesis:

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara). .. During experiment of gene cloning the ligation mixture was used to transform into chemically competent E. coli Top 10 cells.

Isolation:

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: The D6E (GLELO) gene was isolated by PCR amplification from the genome of Mortierella alpina ATCC 32222 with corresponding primers GLELO-F/R (Additional file : Table S1). .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara).

Labeling:

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: PCR products were cloned into pGEM T-easy vector (Promega) and used as template for DIG (digoxygenin)-labeled probe synthesis according to the manufacture’s instructions in the PCR DIG labeling kit (Roche Diagnostics). .. One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight.

Purification:

Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region
Article Snippet: .. After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany). .. The IL23R-CHR protein was successfully expressed in E.coli BL21 (DE3) (Novagen Germany) cells and purified by the following procedures.

Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading
Article Snippet: The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase. .. The covalently closed products were purified with the Cesium Chloride density gradient ultracentrifugation.

Article Title: Molecular Characterization of a Nucleopolyhedrovirus Newly Isolated from Ophiusa disjungens in China
Article Snippet: Virus DNA was digested with Bam HI, Eco RI, Hind III, Pst I, Xho I, Xba I (TaKaRa) at 37 °C for 2–3 h, then electrophoresed on 0.7 % agarose gels at 50 V for 15–22 h to separate the fragments. .. The recombinant plasmids were transformed into E. coli DH 5α cells (TIANGEN), screened for lacZ complementation, recovered by using DNA Product Purification Kit (TIANGEN) from agarose gels and purified by precipitation with glass milk.

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: Paragraph title: Construction, expression and purification of tIL12rβ1/Fc ... The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA).

Sequencing:

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: Construction, expression and purification of tIL12rβ1/Fc Based on the amino acid sequence of human IL12rβ1 receptor (Genbank NP_005526) and human IgG1 Fc (Genbank AEV43323.1), the human truncated IL12rβ1 receptor (tIL12rβ1) gene containing a signal peptide sequence (708 bp) and the human IgG1 Fc fragment (699 bp) was obtained by RT-PCR from human spleen cDNA library (Biomics, China) using the primers F1, F2 and F3, F4 (Table ), respectively. .. The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA).

Cell Culture:

Article Title: Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries
Article Snippet: After being cultured at 30 °C for 6–12 h, the upper layer of the double-decker agar plate was checked for phage plaque. .. Single identification digestion was made using only one restriction enzyme, Kpn I (Takara Bio Inc, Otsu, Japan) or Nhe I (Takara Bio Inc, Otsu, Japan), both of which produced one fragment 7349 bp in size; or Xho I (Takara Bio Inc, Otsu, Japan), which produced two fragments of 2273 bp and 5076 bp.

cDNA Library Assay:

Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region
Article Snippet: Construction, Expression, and Purification of IL23R-CHR Human IL23R-CHR (amino acid residues 124–313) was cloned by Q-PCR from human spleen cDNA library (Biomics, China) using the following primers: 5′-GCCCATGGCTCCGCCAGATATTCCTGATG-3′ and 5′-CAGCTCGAGTTAATGAAAAAACGGTGAGCTCCA-3′ . .. After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany).

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: Construction, expression and purification of tIL12rβ1/Fc Based on the amino acid sequence of human IL12rβ1 receptor (Genbank NP_005526) and human IgG1 Fc (Genbank AEV43323.1), the human truncated IL12rβ1 receptor (tIL12rβ1) gene containing a signal peptide sequence (708 bp) and the human IgG1 Fc fragment (699 bp) was obtained by RT-PCR from human spleen cDNA library (Biomics, China) using the primers F1, F2 and F3, F4 (Table ), respectively. .. The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA).

Plasmid Preparation:

Article Title: Comparative genomic analysis of two emergent human adenovirus type 14 respiratory pathogen isolates in China reveals similar yet divergent genomes
Article Snippet: .. Genome type identification In silico restriction maps of available genome sequences from all HAdV-14 strains CHN/GZ01/2010, 303600, CHN/BJ430, CHN2012, and prototype de Wit were generated using the Vector NTI 10.3.0 software (Invitrogen Corp.; San Diego, CA, USA) as described in earlier studies., , These included profiles from restriction enzymes Bam HI, Bcl I, Bgl l, Bgl II, Bst EII, Eco RI, Hin dIII, Hpa I, Sal l, Sma I, Xba I, and Xho I (TaKaRa Corp.; China), all of which were chosen in order to be consistent with the original nomenclature system devised by Li and colleagues. .. Genome typing of these strains was determined by comparing its in silico RE profiles with other HAdV-14 genome types.,

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara). .. During experiment of gene cloning the ligation mixture was used to transform into chemically competent E. coli Top 10 cells.

Article Title: Molecular Characterization of a Nucleopolyhedrovirus Newly Isolated from Ophiusa disjungens in China
Article Snippet: Virus DNA was digested with Bam HI, Eco RI, Hind III, Pst I, Xho I, Xba I (TaKaRa) at 37 °C for 2–3 h, then electrophoresed on 0.7 % agarose gels at 50 V for 15–22 h to separate the fragments. .. The pst I-G fragment of OpdiNPV DNA was cloned into pUC18 (TAKARA) plasmid vector according to the methods of Sambrook [ ].

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: PCR products were cloned into pGEM T-easy vector (Promega) and used as template for DIG (digoxygenin)-labeled probe synthesis according to the manufacture’s instructions in the PCR DIG labeling kit (Roche Diagnostics). .. One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight.

Article Title: Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries
Article Snippet: Restriction enzyme digestion and 0.8% agarose gel electrophoresis (AGE) were performed to confirm the identity of the plasmid. .. Single identification digestion was made using only one restriction enzyme, Kpn I (Takara Bio Inc, Otsu, Japan) or Nhe I (Takara Bio Inc, Otsu, Japan), both of which produced one fragment 7349 bp in size; or Xho I (Takara Bio Inc, Otsu, Japan), which produced two fragments of 2273 bp and 5076 bp.

Article Title: Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells
Article Snippet: Paragraph title: Preparation of plasmid DNA ... The 2.2-kb KDR-TK fragment was extracted from pBluescript II KDR-TK by a double digestion with Xho I and Sal I (Takara Biotechnology Co.,Ltd, Dalian, China) and identified by electrophoresis and DNA sequencing.

Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation
Article Snippet: .. The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA). .. E. coli DH5α (Novagen, Germany) was transformed with this recombinant plasmid and the following selection was conducted on Luria-Bertani (LB) broth supplemented with 0.1 mg/mL ampicillin (Sigma, USA).

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: Paragraph title: Construction of the inducible recA expression BGM vector (iREX) ... After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA.

Article Title: Screening and identification of proteins interacting with nucleostemin
Article Snippet: .. The full length of NS cDNA was cut by Bam HI and Xho I restriction endonucleases from pCDNA3-NS, then inserted into the downstream of the Gal4 DNA-binding domain of the bait vector pGBKT7 (Clontech Laboratories) with T4 DNA ligase. .. The recombinant vector pGBKT7-NS was sequenced and NS protein was in the reading frame.

Software:

Article Title: Comparative genomic analysis of two emergent human adenovirus type 14 respiratory pathogen isolates in China reveals similar yet divergent genomes
Article Snippet: .. Genome type identification In silico restriction maps of available genome sequences from all HAdV-14 strains CHN/GZ01/2010, 303600, CHN/BJ430, CHN2012, and prototype de Wit were generated using the Vector NTI 10.3.0 software (Invitrogen Corp.; San Diego, CA, USA) as described in earlier studies., , These included profiles from restriction enzymes Bam HI, Bcl I, Bgl l, Bgl II, Bst EII, Eco RI, Hin dIII, Hpa I, Sal l, Sma I, Xba I, and Xho I (TaKaRa Corp.; China), all of which were chosen in order to be consistent with the original nomenclature system devised by Li and colleagues. .. Genome typing of these strains was determined by comparing its in silico RE profiles with other HAdV-14 genome types.,

Article Title: Molecular Characterization of a Nucleopolyhedrovirus Newly Isolated from Ophiusa disjungens in China
Article Snippet: Virus DNA was digested with Bam HI, Eco RI, Hind III, Pst I, Xho I, Xba I (TaKaRa) at 37 °C for 2–3 h, then electrophoresed on 0.7 % agarose gels at 50 V for 15–22 h to separate the fragments. .. The sizes of viral DNA fragments were estimated using GelScan XL software (Pharmacia LKB).

Positron Emission Tomography:

Article Title: Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis
Article Snippet: .. Briefly, the PCR product (pMD-18T/VCAM-1) was digested with Eco RI and Xho I and inserted into the His and GST fusion protein sites of the prokaryotic expression vectors pGEX-4T-1 and pET-28a (Takara) respectively, to create the recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1. .. In order to achieve fusion protein expression, the recombinant plasmids were transformed into E. coli BL21 (DE3) and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h. The recombinant E. coli cells were harvested by high speed centrifugation after IPTG induction.

Agarose Gel Electrophoresis:

Article Title: Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries
Article Snippet: Restriction enzyme digestion and 0.8% agarose gel electrophoresis (AGE) were performed to confirm the identity of the plasmid. .. Single identification digestion was made using only one restriction enzyme, Kpn I (Takara Bio Inc, Otsu, Japan) or Nhe I (Takara Bio Inc, Otsu, Japan), both of which produced one fragment 7349 bp in size; or Xho I (Takara Bio Inc, Otsu, Japan), which produced two fragments of 2273 bp and 5076 bp.

Article Title: Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa)
Article Snippet: Southern blot Genomic DNA from the transgenic plants was digested with the Xho I restriction enzyme (2,000 U, Takara). .. The resulting fragments were fractionated on a 1% agarose gel, denatured, and transferred from the gel onto a Hybond H+ nylon membrane (Amersham Pharmacia Biotech) using the capillary transfer method (Sambrook and Russell ).

Article Title: Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae
Article Snippet: .. Native and denatured southern blotting Genomic DNA from non-synchronised saturated cell cultures was digested overnight with XhoI (Takara, 1094A) and then separated on 1% agarose gel (15 cm in length) for 18 hours at 25 volts. .. E. coli exonuclease I digestion (New England biolabs, M0293S) was performed prior to XhoI digestion of the genomic DNA.

Article Title: Genetic Analysis of Type E Botulinum Toxin-Producing Clostridium butyricum Strains †
Article Snippet: The plugs were digested with Sma I or Xho I (Takara Shuzo). .. All samples were electrophoresed with a CHEF-DR II (Bio-Rad Laboratories) apparatus through a 1% Pulsed-Field Certified Agarose gel (Bio-Rad Laboratories) in 0.5× Tris-borate-EDTA buffer at 14°C and 6 V/cm (200 V).

In Vitro:

Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading
Article Snippet: Preparation of the MMR substrates In vitro synthesis of mismatch-carrying plasmids was performed essentially as described previously ( ). .. The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase.

Transgenic Assay:

Article Title: Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa)
Article Snippet: .. Southern blot Genomic DNA from the transgenic plants was digested with the Xho I restriction enzyme (2,000 U, Takara). .. The resulting fragments were fractionated on a 1% agarose gel, denatured, and transferred from the gel onto a Hybond H+ nylon membrane (Amersham Pharmacia Biotech) using the capillary transfer method (Sambrook and Russell ).

Produced:

Article Title: Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries
Article Snippet: .. Single identification digestion was made using only one restriction enzyme, Kpn I (Takara Bio Inc, Otsu, Japan) or Nhe I (Takara Bio Inc, Otsu, Japan), both of which produced one fragment 7349 bp in size; or Xho I (Takara Bio Inc, Otsu, Japan), which produced two fragments of 2273 bp and 5076 bp. ..

Concentration Assay:

Article Title: Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics
Article Snippet: One μg aliquots of total DNA were digested with Hin dIII, Xho I and Acc I restriction enzymes (Takara) at 37 °C overnight. .. Southern hybridization was carried out in DIG Easy Hyb buffer (Roche Diagnostics) with 2 μl/ml concentration of DIG-labeled probe at 37 °C overnight.

Marker:

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: The pyrG encodes orotidine 5’-phosphate decarboxylase which produce uridine as a selectable marker and flanking sequences corresponding to regions surrounding the carotenogenic carRP –carRP gene allows its chromosomal integration by homologous recombination [ , ]. .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara).

Staining:

Article Title: Genetic Analysis of Type E Botulinum Toxin-Producing Clostridium butyricum Strains †
Article Snippet: The plugs were digested with Sma I or Xho I (Takara Shuzo). .. The gel was stained with 1 μg of ethidium bromide per ml for 30 min and destained in distilled water for 30 min.

Homologous Recombination:

Article Title: Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides
Article Snippet: The pyrG encodes orotidine 5’-phosphate decarboxylase which produce uridine as a selectable marker and flanking sequences corresponding to regions surrounding the carotenogenic carRP –carRP gene allows its chromosomal integration by homologous recombination [ , ]. .. After digestion the plasmid with XhoI, The PCR fragment was ligated into this site to generate plasmid as pMAT1552-GLELO for mutant pleu-MU402 (One step cloning kit, Takara).

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments
Article Snippet: The inducible recA expression cassette is flanked by 5’- and 3’- amyE ; thus, the cassette can be inserted into the amyE locus of the BEST310 genome by homologous recombination. pX-recA was digested with ScaI (TaKaRa), and linearized pX-recA was introduced into the BEST310 genome via transformation. .. After these steps, an EcoRI-BamHI fragment of the tetracycline resistance gene from pBEST307 [ ] was cloned into the EcoRI-BamHI site of pCP to generate pCTP. pCTP was digested with XhoI (TaKaRa), and the linearized pCTP was used for transformation to delete the endogenous recA of BEST310/pX-recA.

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    TaKaRa xho i s1 treated mouse mtdna
    Linear 11 kb fragments of <t>mtDNA</t> are released from control mouse liver mtDNA by single-stranded nuclease treatment. The figure shows a schematic diagram of a replication intermediate of mammalian mtDNA, where fork arrest has occurred in the NCR and near O L ; nicking by S1, or other, nuclease can in theory release a linear fragment of mtDNA of ∼11 kb as illustrated. S1 nuclease treatment of Balb C mouse liver mtDNA yielded just such fragments based on a series of probes from around the mouse mitochondrial genome (Panels A – F ). (Panels A–D) represent a single gel: 0.62% agarose, 60 V, 30 h; whereas, the mtDNA of (Panel E) and (Panel F) was separated on a 0.5% agarose gel, at 65 V for 30 h. Treating mouse liver mtDNA with <t>Xho</t> I or Sac I, in addition to S1 nuclease, produced shorter fragments (Panel G , i–iii), after separation at 100 V for 4 h on a 0.8% agarose gel. Probes were assigned lowercase letters (a–f), and the numbers at the foot of the gel panels indicate the span of the probes, based on the revised mouse mtDNA reference sequence ( 17 ).
    Xho I S1 Treated Mouse Mtdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa xho i
    Detection of tracts of <t>DNA</t> repair synthesis during gap-directed MMR in NPE. Covalently closed (lanes 1–3), A-strand-gap-carrying (lanes 4–6) or C-strand-gap-carrying pMM1 AC (lanes 7–9) was incubated in NPE, and sampled at the indicated times. DNA was purified and digested with Xmn I and either Bam HI (upper, A to G repair) or <t>Xho</t> I (middle, C to T repair). To analyze the incorporation of radioactivity, DNA was digested with Drd I (bottom). α-[ 32 P]-dCTP was preferentially incorporated into the 1 kb fragment corresponding to the shorter path between the gap and the mismatch in both 5’-gap- and 3’-gap-directed MMR. DOI: http://dx.doi.org/10.7554/eLife.15155.005
    Xho I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 677 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Linear 11 kb fragments of mtDNA are released from control mouse liver mtDNA by single-stranded nuclease treatment. The figure shows a schematic diagram of a replication intermediate of mammalian mtDNA, where fork arrest has occurred in the NCR and near O L ; nicking by S1, or other, nuclease can in theory release a linear fragment of mtDNA of ∼11 kb as illustrated. S1 nuclease treatment of Balb C mouse liver mtDNA yielded just such fragments based on a series of probes from around the mouse mitochondrial genome (Panels A – F ). (Panels A–D) represent a single gel: 0.62% agarose, 60 V, 30 h; whereas, the mtDNA of (Panel E) and (Panel F) was separated on a 0.5% agarose gel, at 65 V for 30 h. Treating mouse liver mtDNA with Xho I or Sac I, in addition to S1 nuclease, produced shorter fragments (Panel G , i–iii), after separation at 100 V for 4 h on a 0.8% agarose gel. Probes were assigned lowercase letters (a–f), and the numbers at the foot of the gel panels indicate the span of the probes, based on the revised mouse mtDNA reference sequence ( 17 ).

    Journal: Nucleic Acids Research

    Article Title: Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA

    doi: 10.1093/nar/gkp091

    Figure Lengend Snippet: Linear 11 kb fragments of mtDNA are released from control mouse liver mtDNA by single-stranded nuclease treatment. The figure shows a schematic diagram of a replication intermediate of mammalian mtDNA, where fork arrest has occurred in the NCR and near O L ; nicking by S1, or other, nuclease can in theory release a linear fragment of mtDNA of ∼11 kb as illustrated. S1 nuclease treatment of Balb C mouse liver mtDNA yielded just such fragments based on a series of probes from around the mouse mitochondrial genome (Panels A – F ). (Panels A–D) represent a single gel: 0.62% agarose, 60 V, 30 h; whereas, the mtDNA of (Panel E) and (Panel F) was separated on a 0.5% agarose gel, at 65 V for 30 h. Treating mouse liver mtDNA with Xho I or Sac I, in addition to S1 nuclease, produced shorter fragments (Panel G , i–iii), after separation at 100 V for 4 h on a 0.8% agarose gel. Probes were assigned lowercase letters (a–f), and the numbers at the foot of the gel panels indicate the span of the probes, based on the revised mouse mtDNA reference sequence ( 17 ).

    Article Snippet: Xho I/S1 treated mouse mtDNA was recovered from 1D agarose gels by electrophoresis at 70 V for 2 h into Recochips™ (TaKaRa, Japan) and ligated overnight with 200 U of T4 ligase (New England Biolabs), at 22°C; the junction of the ligated DNA fragments was amplified over the course of 30 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 30 s using Biotaq (Bioline), or KOD HiFi DNA polymerase (Novagen).

    Techniques: Agarose Gel Electrophoresis, Produced, Sequencing

    The ends of the prominent linear molecules of mutator mouse mtDNA map to the NCR and in the vicinity of O L . S1 products of mutator mouse mtDNA were recovered from specific regions of agarose gels using Recochips™; repeated separation of some of the material by 1D-AGE (0.8% agarose, 4 h, 100 V) was used to confirm successful recovery (panel A , i and ii; panel D , iii). Fragments were circularized, and the junction amplified, cloned and sequenced. Panel ( B ) A partial linear map of the mouse mitochondrial genome encompassing the NCR. The ends (red diamonds) identified by direct sequencing were nt 16 033, 16 016, 15 997, 15 995, 15 978, 15 956 (Recochip 1); and 15 875, 15 751, 15 751, 15 750, 15 747, 15 529, 15 489, 15 487, 15 486, 15 467 and 15 389 (Recochip 2). Scarcer ends mapping to the NCR were also cloned and sequenced from control littermates (yellow diamonds). The NCR of mouse mtDNA includes three so-called conserved sequenced boxes (CSBs 1–3), a conserved central domain, and a predicted clover-leaf structure, TAS, which is believed to effect termination of 7S DNA (D-loop) synthesis. Three of the ends (15 751, 15 751 and 15 750) in the conserved central domain are flanked by a GC-rich sequence CCGGGCCC and GGGGG, which is extremely rare in the L-strand of mammalian mtDNAs, suggesting that this may represent a cis -element. Free 5′ ends of DNA identified previously by LM-PCR are represented as vertical blue lines; in panel B the free 5′ ends comprise two clusters, cluster I (O H ) and cluster II ( 12 , 13 , 38 ). The height of the most prominent free end (nt 16 034) was set arbitrarily and the others expressed as a fraction of its height. Panel ( C ) The sequenced Sac I/S1 products with an end mapping close to O L (nt 5160–5191) were nt 4946, 5082, 5133, 5201, 5217, 5255, 5257, 5318, 5477 (red diamonds) in mutator mouse samples and nt 5169, 5193, 5196, 5199, 5244 and 5362 (yellow diamonds) in controls. In humans, free 5′ ends are concentrated in the tRNA Cys gene (C), which is adjacent to O L ( 38 , 39 ) and free 5′ ends map to similar positions in mouse mtDNA (see Supplementary Figure 2 ); in panel (C) they are again represented as blue vertical lines. Panel (D) Sac I/S1-treated mutator mouse mtDNA analysed by 1D-AGE, iii is the material that was used to define the ends of DNA represented as red diamonds in panel (C).

    Journal: Nucleic Acids Research

    Article Title: Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA

    doi: 10.1093/nar/gkp091

    Figure Lengend Snippet: The ends of the prominent linear molecules of mutator mouse mtDNA map to the NCR and in the vicinity of O L . S1 products of mutator mouse mtDNA were recovered from specific regions of agarose gels using Recochips™; repeated separation of some of the material by 1D-AGE (0.8% agarose, 4 h, 100 V) was used to confirm successful recovery (panel A , i and ii; panel D , iii). Fragments were circularized, and the junction amplified, cloned and sequenced. Panel ( B ) A partial linear map of the mouse mitochondrial genome encompassing the NCR. The ends (red diamonds) identified by direct sequencing were nt 16 033, 16 016, 15 997, 15 995, 15 978, 15 956 (Recochip 1); and 15 875, 15 751, 15 751, 15 750, 15 747, 15 529, 15 489, 15 487, 15 486, 15 467 and 15 389 (Recochip 2). Scarcer ends mapping to the NCR were also cloned and sequenced from control littermates (yellow diamonds). The NCR of mouse mtDNA includes three so-called conserved sequenced boxes (CSBs 1–3), a conserved central domain, and a predicted clover-leaf structure, TAS, which is believed to effect termination of 7S DNA (D-loop) synthesis. Three of the ends (15 751, 15 751 and 15 750) in the conserved central domain are flanked by a GC-rich sequence CCGGGCCC and GGGGG, which is extremely rare in the L-strand of mammalian mtDNAs, suggesting that this may represent a cis -element. Free 5′ ends of DNA identified previously by LM-PCR are represented as vertical blue lines; in panel B the free 5′ ends comprise two clusters, cluster I (O H ) and cluster II ( 12 , 13 , 38 ). The height of the most prominent free end (nt 16 034) was set arbitrarily and the others expressed as a fraction of its height. Panel ( C ) The sequenced Sac I/S1 products with an end mapping close to O L (nt 5160–5191) were nt 4946, 5082, 5133, 5201, 5217, 5255, 5257, 5318, 5477 (red diamonds) in mutator mouse samples and nt 5169, 5193, 5196, 5199, 5244 and 5362 (yellow diamonds) in controls. In humans, free 5′ ends are concentrated in the tRNA Cys gene (C), which is adjacent to O L ( 38 , 39 ) and free 5′ ends map to similar positions in mouse mtDNA (see Supplementary Figure 2 ); in panel (C) they are again represented as blue vertical lines. Panel (D) Sac I/S1-treated mutator mouse mtDNA analysed by 1D-AGE, iii is the material that was used to define the ends of DNA represented as red diamonds in panel (C).

    Article Snippet: Xho I/S1 treated mouse mtDNA was recovered from 1D agarose gels by electrophoresis at 70 V for 2 h into Recochips™ (TaKaRa, Japan) and ligated overnight with 200 U of T4 ligase (New England Biolabs), at 22°C; the junction of the ligated DNA fragments was amplified over the course of 30 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 30 s using Biotaq (Bioline), or KOD HiFi DNA polymerase (Novagen).

    Techniques: Amplification, Clone Assay, Sequencing, Polymerase Chain Reaction

    Linear 11 kb fragments of mtDNA are present at high abundance in mutator mouse liver mtDNA, in the absence of single-strand nuclease treatment. Liver mtDNA samples isolated from wild-type (Wt) and mutator (M) mice were digested with restriction enzymes, Xho I, Mlu I or Sac I, or left uncut, and fractionated by 1D-AGE in TBE buffer. Separation conditions were 55 V for 20 h in 0.4% agarose (Panels A and B ) or 100 V for 4 h in 0.8% agarose (Panels C and D ). After Southern blotting, membranes were probed with PCR products corresponding to nt 14 903–15 401 (Panels A and C); and nt 8031–8625 (Panels B and D) of mouse mtDNA. The samples in panels (C) and (D) were treated additionally with single-strand specific (S1) nuclease after restriction digestion (see ‘Materials and Methods’ section). The two lanes in panel (C) are different exposures of the same gel; a longer exposure of the digest of Wt mtDNA was needed to show that S1 nuclease generated some fragments of similar size to the abundant short fragments seen in mutator mouse mtDNA digests. The species that distinguished mutator mouse mtDNA from wild-type mtDNA are interpreted as follows: 1 —replicating theta structures, or eyebrows, as illustrated at the base of the figure. Wild-type mtDNA samples also include theta structures but their low abundance means they are difficult to detect unless 2D-AGE is applied ( 12 ); 2 —linear mtDNA fragments of approximately 11 kb; 3 — Xho I digested mtDNA fragments with one end corresponding to the restriction site at nt 13 558 and the other mapping to the major non-coding region (NCR); 4 — Sac I fragments ∼7 kb, spanning nt 9047 to the NCR; 5 —fragments ∼8 kb, with one end close to O L the other nt 13 558; 6 —fragments ∼4 kb, with termini at nt 9047 and near O L . The fragments labelled 7 in panel D were gel-extracted, converted to circles, cloned and sequenced, without recourse to S1 nuclease treatment, and found to contain point mutations, which indicated that the novel fragments were the result of Sac I site gains (see Supplementary Figure 3 for details).

    Journal: Nucleic Acids Research

    Article Title: Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA

    doi: 10.1093/nar/gkp091

    Figure Lengend Snippet: Linear 11 kb fragments of mtDNA are present at high abundance in mutator mouse liver mtDNA, in the absence of single-strand nuclease treatment. Liver mtDNA samples isolated from wild-type (Wt) and mutator (M) mice were digested with restriction enzymes, Xho I, Mlu I or Sac I, or left uncut, and fractionated by 1D-AGE in TBE buffer. Separation conditions were 55 V for 20 h in 0.4% agarose (Panels A and B ) or 100 V for 4 h in 0.8% agarose (Panels C and D ). After Southern blotting, membranes were probed with PCR products corresponding to nt 14 903–15 401 (Panels A and C); and nt 8031–8625 (Panels B and D) of mouse mtDNA. The samples in panels (C) and (D) were treated additionally with single-strand specific (S1) nuclease after restriction digestion (see ‘Materials and Methods’ section). The two lanes in panel (C) are different exposures of the same gel; a longer exposure of the digest of Wt mtDNA was needed to show that S1 nuclease generated some fragments of similar size to the abundant short fragments seen in mutator mouse mtDNA digests. The species that distinguished mutator mouse mtDNA from wild-type mtDNA are interpreted as follows: 1 —replicating theta structures, or eyebrows, as illustrated at the base of the figure. Wild-type mtDNA samples also include theta structures but their low abundance means they are difficult to detect unless 2D-AGE is applied ( 12 ); 2 —linear mtDNA fragments of approximately 11 kb; 3 — Xho I digested mtDNA fragments with one end corresponding to the restriction site at nt 13 558 and the other mapping to the major non-coding region (NCR); 4 — Sac I fragments ∼7 kb, spanning nt 9047 to the NCR; 5 —fragments ∼8 kb, with one end close to O L the other nt 13 558; 6 —fragments ∼4 kb, with termini at nt 9047 and near O L . The fragments labelled 7 in panel D were gel-extracted, converted to circles, cloned and sequenced, without recourse to S1 nuclease treatment, and found to contain point mutations, which indicated that the novel fragments were the result of Sac I site gains (see Supplementary Figure 3 for details).

    Article Snippet: Xho I/S1 treated mouse mtDNA was recovered from 1D agarose gels by electrophoresis at 70 V for 2 h into Recochips™ (TaKaRa, Japan) and ligated overnight with 200 U of T4 ligase (New England Biolabs), at 22°C; the junction of the ligated DNA fragments was amplified over the course of 30 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 30 s using Biotaq (Bioline), or KOD HiFi DNA polymerase (Novagen).

    Techniques: Isolation, Mouse Assay, Southern Blot, Polymerase Chain Reaction, Generated, Clone Assay

    Detection of tracts of DNA repair synthesis during gap-directed MMR in NPE. Covalently closed (lanes 1–3), A-strand-gap-carrying (lanes 4–6) or C-strand-gap-carrying pMM1 AC (lanes 7–9) was incubated in NPE, and sampled at the indicated times. DNA was purified and digested with Xmn I and either Bam HI (upper, A to G repair) or Xho I (middle, C to T repair). To analyze the incorporation of radioactivity, DNA was digested with Drd I (bottom). α-[ 32 P]-dCTP was preferentially incorporated into the 1 kb fragment corresponding to the shorter path between the gap and the mismatch in both 5’-gap- and 3’-gap-directed MMR. DOI: http://dx.doi.org/10.7554/eLife.15155.005

    Journal: eLife

    Article Title: MutSα maintains the mismatch repair capability by inhibiting PCNA unloading

    doi: 10.7554/eLife.15155

    Figure Lengend Snippet: Detection of tracts of DNA repair synthesis during gap-directed MMR in NPE. Covalently closed (lanes 1–3), A-strand-gap-carrying (lanes 4–6) or C-strand-gap-carrying pMM1 AC (lanes 7–9) was incubated in NPE, and sampled at the indicated times. DNA was purified and digested with Xmn I and either Bam HI (upper, A to G repair) or Xho I (middle, C to T repair). To analyze the incorporation of radioactivity, DNA was digested with Drd I (bottom). α-[ 32 P]-dCTP was preferentially incorporated into the 1 kb fragment corresponding to the shorter path between the gap and the mismatch in both 5’-gap- and 3’-gap-directed MMR. DOI: http://dx.doi.org/10.7554/eLife.15155.005

    Article Snippet: The mismatch-carrying DNA was treated with Xho I (Takara Bio, Kusatsu, Japan) to digest DNA whose mismatch base was edited by T7 DNA polymerase.

    Techniques: Incubation, Purification, Radioactivity

    Generation of IL23R-CHR protein. A, Restriction sites are indicated in bold, the translated amino acid and Trx-6×His-DDDDKA tag are shown in the box; B, IL23R-CHR gene from human spleen cDNA library amplified by PCR; C, One pET32a/IL23R-CHR clone digested with NcoI and XhoI and analyzed by agrose gel electrophoresis (1%). MW = molecular weight markers, bp. D, Trx-IL23R-CHR induced by IPTG in E.Coli BL21 (DE3), SDS-PAGE (15%), protein bands were stained with coomassie brilliant blue R250 reagent. Lane 2, uninduced bacterial lysate; Lane 3, IPTG induced bacterial lysate. E, Trx-IL23R-CHR purified from BL21 (DE3) lysate and cleaved by enterokinase, Lane 5, purified Trx-IL23R-CHR; Lane 6, purified IL23R-CHR. F, Western blot using mouse mAbs against human IL23R (Lane 8). Lane 1, 4 and 7 in D, E, F: molecular weight markers, KD.

    Journal: PLoS ONE

    Article Title: Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region

    doi: 10.1371/journal.pone.0045625

    Figure Lengend Snippet: Generation of IL23R-CHR protein. A, Restriction sites are indicated in bold, the translated amino acid and Trx-6×His-DDDDKA tag are shown in the box; B, IL23R-CHR gene from human spleen cDNA library amplified by PCR; C, One pET32a/IL23R-CHR clone digested with NcoI and XhoI and analyzed by agrose gel electrophoresis (1%). MW = molecular weight markers, bp. D, Trx-IL23R-CHR induced by IPTG in E.Coli BL21 (DE3), SDS-PAGE (15%), protein bands were stained with coomassie brilliant blue R250 reagent. Lane 2, uninduced bacterial lysate; Lane 3, IPTG induced bacterial lysate. E, Trx-IL23R-CHR purified from BL21 (DE3) lysate and cleaved by enterokinase, Lane 5, purified Trx-IL23R-CHR; Lane 6, purified IL23R-CHR. F, Western blot using mouse mAbs against human IL23R (Lane 8). Lane 1, 4 and 7 in D, E, F: molecular weight markers, KD.

    Article Snippet: After purification with Tianquick midi purification kit (Tiangen, China), the IL23R-CHR gene was digested with NcoI and XhoI (takara) and inserted into pET32a following the manufacturer’s instructions (Novagen, Germany).

    Techniques: cDNA Library Assay, Amplification, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Molecular Weight, SDS Page, Staining, Purification, Western Blot

    Construction, expression, and purification of tIL12rβ1/Fc A. Plasmid map of the eukaryotic expression plasmid containing human truncated IL-12rβ1 receptor (pcDNA3.1(+)-tIL12rβ1/Fc). The gene sequence encoding tIL12rβ1/Fc was inserted into pcDNA3.1(+) vector at the corresponding restriction sites Hind III and Xho I. B. PCR products of tIL12rβ1/Fc recombinant gene. Lane 1, tIL12rβ1/Fc gene sequence containing the signal peptide. Lane 2, IgG1 Fc fragment. Lane 3, recombinant tIL12rβ1/Fc fused gene product using overlap PCR. M, DNA molecular weight markers, bp. C. Correctly constructed plasmid was verified by digestion at the Hind III and Xho I sites. Lane 1, pcDNA3.1(+)-tIL12rβ1/Fc after digestion. Lane 2, pcDNA3.1(+) -tIL12rβ1/Fc before digestion. M, DNA molecular weight markers, bp. D. Plasmids were linearized for cell transfection using Pvu I. Lane 1 and 2, linearized pcDNA3.1(+)-tIL12rβ1/Fc. M, DNA molecular weight markers, bp. E. Total RNA of the two strains were identified by RT-PCR with primers F1 and F4. F. SDS-PAGE analysis of the purified tIL12rβ1/Fc fusion protein using Protein A column. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa. G. Western blot analysis of tIL12rβ1/Fc using mAbs against human IL12rβ1. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa.

    Journal: Oncotarget

    Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation

    doi:

    Figure Lengend Snippet: Construction, expression, and purification of tIL12rβ1/Fc A. Plasmid map of the eukaryotic expression plasmid containing human truncated IL-12rβ1 receptor (pcDNA3.1(+)-tIL12rβ1/Fc). The gene sequence encoding tIL12rβ1/Fc was inserted into pcDNA3.1(+) vector at the corresponding restriction sites Hind III and Xho I. B. PCR products of tIL12rβ1/Fc recombinant gene. Lane 1, tIL12rβ1/Fc gene sequence containing the signal peptide. Lane 2, IgG1 Fc fragment. Lane 3, recombinant tIL12rβ1/Fc fused gene product using overlap PCR. M, DNA molecular weight markers, bp. C. Correctly constructed plasmid was verified by digestion at the Hind III and Xho I sites. Lane 1, pcDNA3.1(+)-tIL12rβ1/Fc after digestion. Lane 2, pcDNA3.1(+) -tIL12rβ1/Fc before digestion. M, DNA molecular weight markers, bp. D. Plasmids were linearized for cell transfection using Pvu I. Lane 1 and 2, linearized pcDNA3.1(+)-tIL12rβ1/Fc. M, DNA molecular weight markers, bp. E. Total RNA of the two strains were identified by RT-PCR with primers F1 and F4. F. SDS-PAGE analysis of the purified tIL12rβ1/Fc fusion protein using Protein A column. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa. G. Western blot analysis of tIL12rβ1/Fc using mAbs against human IL12rβ1. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa.

    Article Snippet: The constructed tIL12rβ1/Fc fragment was digested with Xho I and Hind III (Takara, China) and then inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA).

    Techniques: Expressing, Purification, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Recombinant, Molecular Weight, Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot