kpn i  (TaKaRa)

 
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  • 99
    Name:
    Kpn I
    Description:
    G GTAC | CC | CATG G
    Catalog Number:
    1068b
    Price:
    None
    Size:
    25 000 Units
    Category:
    KpnI Restriction enzymes Cloning
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    Structured Review

    TaKaRa kpn i
    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with <t>Kpn</t> I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    G GTAC | CC | CATG G
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    1) Product Images from "Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells"

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E19-02-0108

    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    Figure Legend Snippet: Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Techniques Used: Transfection, Marker, Isolation, Hybridization, Construct, Agarose Gel Electrophoresis, Fluorescence In Situ Hybridization, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction, Serial Dilution

    2) Product Images from "Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells"

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E19-02-0108

    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    Figure Legend Snippet: Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Techniques Used: Transfection, Marker, Isolation, Hybridization, Construct, Agarose Gel Electrophoresis, Fluorescence In Situ Hybridization, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction, Serial Dilution

    3) Product Images from "Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter"

    Article Title: Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17010119

    Verification of mouse Omi/HtrA2 promoter luciferase expression plasmids. Plasmids were verified by double enzyme digestion with Kpn I and Hind III, which releases a vector fragment and a variable-sized promoter insert. M: DL2000 Marker; 1 : pGL3-239; 2 : pGL3-472; 3 : pGL3-742; 4 : pGL3-931; 5 : pGL3-1298. Luc: luciferase expression plasmids.
    Figure Legend Snippet: Verification of mouse Omi/HtrA2 promoter luciferase expression plasmids. Plasmids were verified by double enzyme digestion with Kpn I and Hind III, which releases a vector fragment and a variable-sized promoter insert. M: DL2000 Marker; 1 : pGL3-239; 2 : pGL3-472; 3 : pGL3-742; 4 : pGL3-931; 5 : pGL3-1298. Luc: luciferase expression plasmids.

    Techniques Used: Luciferase, Expressing, Plasmid Preparation, Marker

    4) Product Images from "Antimicrobial peptide melittin against Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen in rice"

    Article Title: Antimicrobial peptide melittin against Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen in rice

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-016-7400-4

    The effect of melittin on the genomic DNA of X. oryzae pv. oryzae . A gel retardation assay was conducted after various amounts of peptides were incubated with 600 ng of DNA for 1 h. The weight ratios (peptide:DNA) were 0.05, 0.5, 1, 2, and 3 ( a ). Effect of melittin on DNA restriction enzyme digestion by Hin dIII (E1), Kpn I (E2), Sac I (E3), and Eco RI - HF (E4). M marker, P peptide melittin ( b )
    Figure Legend Snippet: The effect of melittin on the genomic DNA of X. oryzae pv. oryzae . A gel retardation assay was conducted after various amounts of peptides were incubated with 600 ng of DNA for 1 h. The weight ratios (peptide:DNA) were 0.05, 0.5, 1, 2, and 3 ( a ). Effect of melittin on DNA restriction enzyme digestion by Hin dIII (E1), Kpn I (E2), Sac I (E3), and Eco RI - HF (E4). M marker, P peptide melittin ( b )

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Marker

    5) Product Images from "Comparison of the Complete DNA Sequences of Human Herpesvirus 6 Variants A and B"

    Article Title: Comparison of the Complete DNA Sequences of Human Herpesvirus 6 Variants A and B

    Journal: Journal of Virology

    doi:

    Restriction fragments used for the sequencing of HHV-6 strain HST. The HHV-6 genome is represented diagrammatically, together with the positions of Bam HI, Hin dIII, Kpn I, and Pst I sites. Various plasmid-cloned restriction fragments are prefixed with Ba, H, Kp, or pSTY (indicating their terminal restriction sites). Sequenced restriction fragments are indicated by shading.
    Figure Legend Snippet: Restriction fragments used for the sequencing of HHV-6 strain HST. The HHV-6 genome is represented diagrammatically, together with the positions of Bam HI, Hin dIII, Kpn I, and Pst I sites. Various plasmid-cloned restriction fragments are prefixed with Ba, H, Kp, or pSTY (indicating their terminal restriction sites). Sequenced restriction fragments are indicated by shading.

    Techniques Used: Sequencing, Plasmid Preparation, Clone Assay

    6) Product Images from "Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit"

    Article Title: Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit

    Journal: Scientific Reports

    doi: 10.1038/srep41851

    Detection and quantification of replicating HBV genome. Cells were infected with Ax-CM103G-kS (kS) or Ax-CM103G-dP (dP) by the indicated MOIs, or transfected with plasmids possessing the same mutant HBV expression units. ( a ) Detection of the replicating HBV genome. Total DNA either from infected or transfected HepG2 cells were analysed using Southern blot analysis. AdV (◆), Kpn I-digested genome of adenovirus vector; rc, relaxed circular DNA genome of HBV; dsL, double-stranded linear DNA genome of HBV; ss, single stranded DNA of HBV; plasmid ( ¢ ), Hind III and Dpn I-digested plasmid fragments; mock, mock infection. Overexposure of blots and full-length blots are presented in Supplementary Figs S5 and S9 , respectively. ( b ) Replicating HBV genomes were quantified using qPCR. Total DNA from infected HepG2 or PXB cells were used to performed qPCR. n = 3. Error bars represent ± s.d.; mock, mock infection of the indicated cells; ** P
    Figure Legend Snippet: Detection and quantification of replicating HBV genome. Cells were infected with Ax-CM103G-kS (kS) or Ax-CM103G-dP (dP) by the indicated MOIs, or transfected with plasmids possessing the same mutant HBV expression units. ( a ) Detection of the replicating HBV genome. Total DNA either from infected or transfected HepG2 cells were analysed using Southern blot analysis. AdV (◆), Kpn I-digested genome of adenovirus vector; rc, relaxed circular DNA genome of HBV; dsL, double-stranded linear DNA genome of HBV; ss, single stranded DNA of HBV; plasmid ( ¢ ), Hind III and Dpn I-digested plasmid fragments; mock, mock infection. Overexposure of blots and full-length blots are presented in Supplementary Figs S5 and S9 , respectively. ( b ) Replicating HBV genomes were quantified using qPCR. Total DNA from infected HepG2 or PXB cells were used to performed qPCR. n = 3. Error bars represent ± s.d.; mock, mock infection of the indicated cells; ** P

    Techniques Used: Infection, Transfection, Mutagenesis, Expressing, Southern Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

    7) Product Images from "Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries"

    Article Title: Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2009.152

    AGE and HPLC analysis of purified pGJA-P/VAX(G). (A) AGE after restriction endonuclease digestion. pGJA-P/VAX(G) digested by Kpn I (Lane 1); Xho I (Lane 2); both Xho I and Nhe I (Lane 3); Nhe I (Lane 4). Lane 5 represents the DNA marker λ Hind
    Figure Legend Snippet: AGE and HPLC analysis of purified pGJA-P/VAX(G). (A) AGE after restriction endonuclease digestion. pGJA-P/VAX(G) digested by Kpn I (Lane 1); Xho I (Lane 2); both Xho I and Nhe I (Lane 3); Nhe I (Lane 4). Lane 5 represents the DNA marker λ Hind

    Techniques Used: High Performance Liquid Chromatography, Purification, Marker

    8) Product Images from "Human endostatin gene transfer, either naked or with liposome, has the same inhibitory effect on growth of mouse liver tumor cells in vivo"

    Article Title: Human endostatin gene transfer, either naked or with liposome, has the same inhibitory effect on growth of mouse liver tumor cells in vivo

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v10.i19.2874

    Detection of the recombinant plasmid pVAX-sEN using Eco R I and Kpn I digestion. A: Marker15000; B: pVAX1 digested by Eco R I; C: Recombinant plasmid pVAX-sEN digested by Eco R I; D: Recombinant plasmid pVAX-sEN digested by Eco R I and Kpn I.
    Figure Legend Snippet: Detection of the recombinant plasmid pVAX-sEN using Eco R I and Kpn I digestion. A: Marker15000; B: pVAX1 digested by Eco R I; C: Recombinant plasmid pVAX-sEN digested by Eco R I; D: Recombinant plasmid pVAX-sEN digested by Eco R I and Kpn I.

    Techniques Used: Recombinant, Plasmid Preparation

    9) Product Images from "Comprehensive molecular analysis of arginase-encoding genes in common wheat and its progenitor species"

    Article Title: Comprehensive molecular analysis of arginase-encoding genes in common wheat and its progenitor species

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07084-0

    Southern blot analysis to determine TaARG copy number in the common wheat genome. ( A ) Bam HI-digested genomic DNA of CB037; ( B ) Kpn I-digested genomic DNA of CB037. acquisition tools and image processing software package. The photo was acquired by Tanon 5200 software (YPH-bio, Co. Ltd. Beijing, China).
    Figure Legend Snippet: Southern blot analysis to determine TaARG copy number in the common wheat genome. ( A ) Bam HI-digested genomic DNA of CB037; ( B ) Kpn I-digested genomic DNA of CB037. acquisition tools and image processing software package. The photo was acquired by Tanon 5200 software (YPH-bio, Co. Ltd. Beijing, China).

    Techniques Used: Southern Blot, Software

    10) Product Images from "Expression of IPT in Asakura-sanshoo (Zanthoxylum piperitum (L.) DC. f. inerme Makino) Alters Tree Architecture, Delays Leaf Senescence, and Changes Leaf Essential Oil Composition"

    Article Title: Expression of IPT in Asakura-sanshoo (Zanthoxylum piperitum (L.) DC. f. inerme Makino) Alters Tree Architecture, Delays Leaf Senescence, and Changes Leaf Essential Oil Composition

    Journal: Plant Molecular Biology Reporter / Ispmb

    doi: 10.1007/s11105-015-0948-9

    Kanamycin-resistant shoots, GUS histochemical detection, and PCR analysis. a WT shoots. b Kanamycin-resistant shoots. Black arrows show epiphyllous shoots. c WT shoots. d GUS-positive shoots and leaves. e PCR analysis of transgenic Asakura-sanshoo plants. M DL2000 DNA marker, WT DNA from non-transgenic plant, P plasmid, 1–5 transgenic lines. f Southern blotting analysis. P pBin-Ex-Hipt plasmid; WT genomic DNA from non-transgenic plant; Y1 , Y17 , Y5 , and Y16 genomic DNA from transgenic lines. Genomic DNA from all lines and plasmid DNA were digested with Eco RI and Kpn I
    Figure Legend Snippet: Kanamycin-resistant shoots, GUS histochemical detection, and PCR analysis. a WT shoots. b Kanamycin-resistant shoots. Black arrows show epiphyllous shoots. c WT shoots. d GUS-positive shoots and leaves. e PCR analysis of transgenic Asakura-sanshoo plants. M DL2000 DNA marker, WT DNA from non-transgenic plant, P plasmid, 1–5 transgenic lines. f Southern blotting analysis. P pBin-Ex-Hipt plasmid; WT genomic DNA from non-transgenic plant; Y1 , Y17 , Y5 , and Y16 genomic DNA from transgenic lines. Genomic DNA from all lines and plasmid DNA were digested with Eco RI and Kpn I

    Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Marker, Plasmid Preparation, Southern Blot

    11) Product Images from "Development and Evaluation of a Novel and Rapid Detection Assay for Botrytis cinerea Based on Loop-Mediated Isothermal Amplification"

    Article Title: Development and Evaluation of a Novel and Rapid Detection Assay for Botrytis cinerea Based on Loop-Mediated Isothermal Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111094

    LAMP detection of the Bcos5 gene in B. cinerea and digestion of positive LAMP products. (a) LAMP for detection of B. cinerea using HNB as a visual indicator. The reaction becomes sky blue if the Bcos5 gene is present but remains violet if the gene is absent; (b) Agarose gel electrophoresis of LAMP products. The positive reaction is manifested as a ladder-like pattern on the 3.0% agarose gel. In (a) and (b), the positive reaction (with target DNA) is labeled “1″, and the negative reaction (without target DNA) is labeled “2″; (c), LAMP products were digested with Kpn I, and two fragments (153 bp, 49 bp) were observed by 3.0% agarose gel. M = 100-bp ladder, 1, LAMP products without digestion; 2, LAMP products digested by Kpn I.
    Figure Legend Snippet: LAMP detection of the Bcos5 gene in B. cinerea and digestion of positive LAMP products. (a) LAMP for detection of B. cinerea using HNB as a visual indicator. The reaction becomes sky blue if the Bcos5 gene is present but remains violet if the gene is absent; (b) Agarose gel electrophoresis of LAMP products. The positive reaction is manifested as a ladder-like pattern on the 3.0% agarose gel. In (a) and (b), the positive reaction (with target DNA) is labeled “1″, and the negative reaction (without target DNA) is labeled “2″; (c), LAMP products were digested with Kpn I, and two fragments (153 bp, 49 bp) were observed by 3.0% agarose gel. M = 100-bp ladder, 1, LAMP products without digestion; 2, LAMP products digested by Kpn I.

    Techniques Used: Agarose Gel Electrophoresis, Labeling

    12) Product Images from "Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries"

    Article Title: Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2009.152

    AGE and HPLC analysis of purified pGJA-P/VAX(G). (A) AGE after restriction endonuclease digestion. pGJA-P/VAX(G) digested by Kpn I (Lane 1); Xho I (Lane 2); both Xho I and Nhe I (Lane 3); Nhe I (Lane 4). Lane 5 represents the DNA marker λ Hind III. (B)HPLC analysis of purified pGJA-P/VAX(G). Peak 1: open circular topology; Peak 2: supercoiled topology.
    Figure Legend Snippet: AGE and HPLC analysis of purified pGJA-P/VAX(G). (A) AGE after restriction endonuclease digestion. pGJA-P/VAX(G) digested by Kpn I (Lane 1); Xho I (Lane 2); both Xho I and Nhe I (Lane 3); Nhe I (Lane 4). Lane 5 represents the DNA marker λ Hind III. (B)HPLC analysis of purified pGJA-P/VAX(G). Peak 1: open circular topology; Peak 2: supercoiled topology.

    Techniques Used: High Performance Liquid Chromatography, Purification, Marker

    13) Product Images from "An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant"

    Article Title: An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-016-0897-z

    Plaques of mini-F recombinant PRV AH02LA strain and RFLP of BAC PRV-G . a Images of mini-F recombinant PRV AH02LA and the parental AH02LA plaques under UV excitation (upper) and phase contrast (lower) are shown. Arrowhead shows a plaque of parental PRV AH02LA virus and arrow shows a plaque formed by mini-F recombinant PRV AH02LA. Each panel represents a view of 200 × 200 μm in size. b RFLP of BAC PRV-G , DNA from PRV AH02LA BAC clone BAC PRV-G was prepared by mini-prep and digested with BamH I, Kpn I, Pst I and Sph I (lanes 1–4). The digests were separated by 0.8% agarose gel electrophoresis for 16 h under 40 V (Left). Predicted RFLP patterns of BAC PRV-G with BamH I, Kpn I, Pst I and Sph I digestion respectively. Predictions of these digestions were performed with the whole genome sequence of PRV ZJ01 strain (GenBank: KM061380.1) as reference. M: DL 15,000 DNA Marker (Takara)
    Figure Legend Snippet: Plaques of mini-F recombinant PRV AH02LA strain and RFLP of BAC PRV-G . a Images of mini-F recombinant PRV AH02LA and the parental AH02LA plaques under UV excitation (upper) and phase contrast (lower) are shown. Arrowhead shows a plaque of parental PRV AH02LA virus and arrow shows a plaque formed by mini-F recombinant PRV AH02LA. Each panel represents a view of 200 × 200 μm in size. b RFLP of BAC PRV-G , DNA from PRV AH02LA BAC clone BAC PRV-G was prepared by mini-prep and digested with BamH I, Kpn I, Pst I and Sph I (lanes 1–4). The digests were separated by 0.8% agarose gel electrophoresis for 16 h under 40 V (Left). Predicted RFLP patterns of BAC PRV-G with BamH I, Kpn I, Pst I and Sph I digestion respectively. Predictions of these digestions were performed with the whole genome sequence of PRV ZJ01 strain (GenBank: KM061380.1) as reference. M: DL 15,000 DNA Marker (Takara)

    Techniques Used: Recombinant, BAC Assay, Agarose Gel Electrophoresis, Sequencing, Marker

    14) Product Images from "Characterization and Regulation of the gbuA Gene, Encoding Guanidinobutyrase in the Arginine Dehydrogenase Pathway of Pseudomonas aeruginosa PAO1"

    Article Title: Characterization and Regulation of the gbuA Gene, Encoding Guanidinobutyrase in the Arginine Dehydrogenase Pathway of Pseudomonas aeruginosa PAO1

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.184.12.3377-3384.2002

    Northern blot of gbuA transcript. RNA was extracted from PAO1 cells cultured in MMP plus 20 mM glutamate (lane 1), MMP plus 20 mM glutamate and 4-GB (lane 2), and MMP plus 20 mM 4-GB (lane 3), blotted onto Hybond N+ nylon membranes, and then probed with the gbuA sequence between Kpn I and Sma I (nucleotides [nt] 166 to 939). Numbers to the left of the gel indicate sizes of RNA markers.
    Figure Legend Snippet: Northern blot of gbuA transcript. RNA was extracted from PAO1 cells cultured in MMP plus 20 mM glutamate (lane 1), MMP plus 20 mM glutamate and 4-GB (lane 2), and MMP plus 20 mM 4-GB (lane 3), blotted onto Hybond N+ nylon membranes, and then probed with the gbuA sequence between Kpn I and Sma I (nucleotides [nt] 166 to 939). Numbers to the left of the gel indicate sizes of RNA markers.

    Techniques Used: Northern Blot, Cell Culture, Sequencing

    15) Product Images from "Direct Regulation of Histidine Ammonia-Lyase 2 Gene by Thyroid Hormone in the Developing Adult Intestinal Stem Cells"

    Article Title: Direct Regulation of Histidine Ammonia-Lyase 2 Gene by Thyroid Hormone in the Developing Adult Intestinal Stem Cells

    Journal: Endocrinology

    doi: 10.1210/en.2016-1558

    The transgenic HAL2 promoter, driving EGFP in transgenic tadpoles, respond to T3 similarly as the endogenous HAL2 gene in the intestine. (a) The double promoter transgenic construct used to study the HAL2 promoter in vivo . The HAL2 promoter fragment flanked by the Kpn I and Age I restriction sites [with the numbers indicating the position relative to the transcription start site in Fig. 1(a) ] was cloned to drive the expression of EGFP in a double promoter transgenic construct, which also contains an eye-specific promoter, the γ -crystallin promoter, driving the expression of the GFP3 ( 36, 37 ). The HAL2 TRE in the promoter is shown as an open box. (b and c) Stage 54 premetamorphic wild-type or transgenic tadpoles were treated with 10 nM T3 for 0 to 5 days and total intestinal RNA was isolated for expression analysis of the endogenous HAL2 mRNA (b) or transgenic EGFP mRNA (c). Note that the endogenous HAL2 expression was upregulated significantly after 3 to 5 days of treatment in both wild-type and transgenic tadpoles. The expression of the transgenic EGFP mimicked that of the endogenous HAL2 in the transgenic tadpoles but was, as expected, absent in the wild-type tadpoles.
    Figure Legend Snippet: The transgenic HAL2 promoter, driving EGFP in transgenic tadpoles, respond to T3 similarly as the endogenous HAL2 gene in the intestine. (a) The double promoter transgenic construct used to study the HAL2 promoter in vivo . The HAL2 promoter fragment flanked by the Kpn I and Age I restriction sites [with the numbers indicating the position relative to the transcription start site in Fig. 1(a) ] was cloned to drive the expression of EGFP in a double promoter transgenic construct, which also contains an eye-specific promoter, the γ -crystallin promoter, driving the expression of the GFP3 ( 36, 37 ). The HAL2 TRE in the promoter is shown as an open box. (b and c) Stage 54 premetamorphic wild-type or transgenic tadpoles were treated with 10 nM T3 for 0 to 5 days and total intestinal RNA was isolated for expression analysis of the endogenous HAL2 mRNA (b) or transgenic EGFP mRNA (c). Note that the endogenous HAL2 expression was upregulated significantly after 3 to 5 days of treatment in both wild-type and transgenic tadpoles. The expression of the transgenic EGFP mimicked that of the endogenous HAL2 in the transgenic tadpoles but was, as expected, absent in the wild-type tadpoles.

    Techniques Used: Transgenic Assay, Construct, In Vivo, Clone Assay, Expressing, Isolation

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    Clone Assay:

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells
    Article Snippet: .. Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ). ..

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells
    Article Snippet: .. Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ). ..

    Amplification:

    Article Title: A new location to split Cre recombinase for protein fragment complementation
    Article Snippet: .. Finally, 2NLZ and 3NLZ fragments were retrieved by cleavage with Kpn I and Sph I and inserted between P 35S (Kpn I) and T nos (Sph I) of pMM23. pMM23‐CLZ series constructs: 1C‐LZ‐cre and 3C‐LZ‐cre fragments were PCR amplified from pMM23 using primers C‐R2, 1CLZ‐F or 3CLZ‐F for insertion between the Eco RI and Sph I sites of pMD18‐T (Takara). ..

    Article Title: A new location to split Cre recombinase for protein fragment complementation
    Article Snippet: .. Finally, 1C‐LZ‐cre and 3C‐LZ‐cre fragments were retrieved by cleavage with Kpn I and Sph I and inserted between P 35S (Kpn I) and T nos (Sph I) of pMM23. pMR1: An Xho I‐gus‐Sph I fragment was PCR amplified from pZH36 (ZG Han, Ow lab, unpublished) using primers Gus‐F and Gus‐R for insertion into a T‐vector (Takara) between the corresponding sites. .. An hpt fragment (with nos terminator) was also PCR amplified from pZH36, using primers lhl‐F and lhl‐R with overhanging lox sites to create a Kpn I‐lox‐hpt‐lox‐Xho I fragment for insertion upstream of the gus gene between .

    Southern Blot:

    Article Title: Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit
    Article Snippet: .. The pellet was dissolved with TE containing 20 mg/ml RNase A. Twenty μg of DNA from the AdV-infected cells were digested with 100 U Kpn I (Takara Bio, Inc., Shiga, Japan) at 37 °C for 4 h, while DNA from plasmid-transfected cells was treated with both 100 U Hind III (New England Biolabs, Inc., MA, USA) and 100 U Dpn I (New England Biolabs, Inc., MA, USA) at 37 °C for 4 h. Southern blot analysis was then perfomed as described in Maekawa et al . .. The HBV103-AdV system in microplate format Huh-7 cells were infected with Ax-CM103G-ΔpreS or Ax-CM103G-dP at MOI 10, and seeded in a 96-well microplate at 3 × 104 cells per well in 100 μl of high-glucose DMEM supplemented with 5% FCS.

    Construct:

    Article Title: A new location to split Cre recombinase for protein fragment complementation
    Article Snippet: .. Finally, 2NLZ and 3NLZ fragments were retrieved by cleavage with Kpn I and Sph I and inserted between P 35S (Kpn I) and T nos (Sph I) of pMM23. pMM23‐CLZ series constructs: 1C‐LZ‐cre and 3C‐LZ‐cre fragments were PCR amplified from pMM23 using primers C‐R2, 1CLZ‐F or 3CLZ‐F for insertion between the Eco RI and Sph I sites of pMD18‐T (Takara). ..

    Article Title: Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter
    Article Snippet: .. Recombinant constructs pMD18-T-239 to pMD18-T-1298 and pGL3 luciferase reporter vector (#E1751; Promega, Madison, WI, USA) were digested for 4h at 37 °C with Kpn I (#1068A; Takara) and Hind III (#1060A; Takara), and the fragments of the recombinant constructs were subcloned into pGL3. .. The ligation mixtures, including 0.3 μg of recombinant fragments (Kpn I/Hind III), 0.1 μg of pGL3-basic plasmid (Kpn I/Hind III), 1 μL of 10× buffer M, and 1U of T4 ligase in a final volume of 10 μL, were incubated at 16 °C overnight and then 65 °C for 5 min.

    Purification:

    Article Title: Comparison of the Complete DNA Sequences of Human Herpesvirus 6 Variants A and B
    Article Snippet: .. Purified viral genomic DNA was digested with restriction enzymes Pst I, Bam HI, Hin dIII, or Kpn I (Takara Shuzo). .. The resultant fragments were cloned into the corresponding restriction enzyme sites of pUC19.

    Luciferase:

    Article Title: Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter
    Article Snippet: .. Recombinant constructs pMD18-T-239 to pMD18-T-1298 and pGL3 luciferase reporter vector (#E1751; Promega, Madison, WI, USA) were digested for 4h at 37 °C with Kpn I (#1068A; Takara) and Hind III (#1060A; Takara), and the fragments of the recombinant constructs were subcloned into pGL3. .. The ligation mixtures, including 0.3 μg of recombinant fragments (Kpn I/Hind III), 0.1 μg of pGL3-basic plasmid (Kpn I/Hind III), 1 μL of 10× buffer M, and 1U of T4 ligase in a final volume of 10 μL, were incubated at 16 °C overnight and then 65 °C for 5 min.

    Expressing:

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells
    Article Snippet: .. Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ). ..

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells
    Article Snippet: .. Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ). ..

    Polymerase Chain Reaction:

    Article Title: A new location to split Cre recombinase for protein fragment complementation
    Article Snippet: .. Finally, 2NLZ and 3NLZ fragments were retrieved by cleavage with Kpn I and Sph I and inserted between P 35S (Kpn I) and T nos (Sph I) of pMM23. pMM23‐CLZ series constructs: 1C‐LZ‐cre and 3C‐LZ‐cre fragments were PCR amplified from pMM23 using primers C‐R2, 1CLZ‐F or 3CLZ‐F for insertion between the Eco RI and Sph I sites of pMD18‐T (Takara). ..

    Article Title: A new location to split Cre recombinase for protein fragment complementation
    Article Snippet: .. Finally, 1C‐LZ‐cre and 3C‐LZ‐cre fragments were retrieved by cleavage with Kpn I and Sph I and inserted between P 35S (Kpn I) and T nos (Sph I) of pMM23. pMR1: An Xho I‐gus‐Sph I fragment was PCR amplified from pZH36 (ZG Han, Ow lab, unpublished) using primers Gus‐F and Gus‐R for insertion into a T‐vector (Takara) between the corresponding sites. .. An hpt fragment (with nos terminator) was also PCR amplified from pZH36, using primers lhl‐F and lhl‐R with overhanging lox sites to create a Kpn I‐lox‐hpt‐lox‐Xho I fragment for insertion upstream of the gus gene between .

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells
    Article Snippet: .. Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ). ..

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells
    Article Snippet: .. Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ). ..

    Recombinant:

    Article Title: Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter
    Article Snippet: .. Recombinant constructs pMD18-T-239 to pMD18-T-1298 and pGL3 luciferase reporter vector (#E1751; Promega, Madison, WI, USA) were digested for 4h at 37 °C with Kpn I (#1068A; Takara) and Hind III (#1060A; Takara), and the fragments of the recombinant constructs were subcloned into pGL3. .. The ligation mixtures, including 0.3 μg of recombinant fragments (Kpn I/Hind III), 0.1 μg of pGL3-basic plasmid (Kpn I/Hind III), 1 μL of 10× buffer M, and 1U of T4 ligase in a final volume of 10 μL, were incubated at 16 °C overnight and then 65 °C for 5 min.

    Plasmid Preparation:

    Article Title: Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter
    Article Snippet: .. Recombinant constructs pMD18-T-239 to pMD18-T-1298 and pGL3 luciferase reporter vector (#E1751; Promega, Madison, WI, USA) were digested for 4h at 37 °C with Kpn I (#1068A; Takara) and Hind III (#1060A; Takara), and the fragments of the recombinant constructs were subcloned into pGL3. .. The ligation mixtures, including 0.3 μg of recombinant fragments (Kpn I/Hind III), 0.1 μg of pGL3-basic plasmid (Kpn I/Hind III), 1 μL of 10× buffer M, and 1U of T4 ligase in a final volume of 10 μL, were incubated at 16 °C overnight and then 65 °C for 5 min.

    Article Title: Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit
    Article Snippet: .. The pellet was dissolved with TE containing 20 mg/ml RNase A. Twenty μg of DNA from the AdV-infected cells were digested with 100 U Kpn I (Takara Bio, Inc., Shiga, Japan) at 37 °C for 4 h, while DNA from plasmid-transfected cells was treated with both 100 U Hind III (New England Biolabs, Inc., MA, USA) and 100 U Dpn I (New England Biolabs, Inc., MA, USA) at 37 °C for 4 h. Southern blot analysis was then perfomed as described in Maekawa et al . .. The HBV103-AdV system in microplate format Huh-7 cells were infected with Ax-CM103G-ΔpreS or Ax-CM103G-dP at MOI 10, and seeded in a 96-well microplate at 3 × 104 cells per well in 100 μl of high-glucose DMEM supplemented with 5% FCS.

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    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with <t>Kpn</t> I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
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    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Journal: Molecular Biology of the Cell

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

    doi: 10.1091/mbc.E19-02-0108

    Figure Lengend Snippet: Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Article Snippet: Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ).

    Techniques: Transfection, Marker, Isolation, Hybridization, Construct, Agarose Gel Electrophoresis, Fluorescence In Situ Hybridization, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction, Serial Dilution

    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Journal: Molecular Biology of the Cell

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

    doi: 10.1091/mbc.E19-02-0108

    Figure Lengend Snippet: Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Article Snippet: Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ).

    Techniques: Transfection, Marker, Isolation, Hybridization, Construct, Agarose Gel Electrophoresis, Fluorescence In Situ Hybridization, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction, Serial Dilution

    Verification of mouse Omi/HtrA2 promoter luciferase expression plasmids. Plasmids were verified by double enzyme digestion with Kpn I and Hind III, which releases a vector fragment and a variable-sized promoter insert. M: DL2000 Marker; 1 : pGL3-239; 2 : pGL3-472; 3 : pGL3-742; 4 : pGL3-931; 5 : pGL3-1298. Luc: luciferase expression plasmids.

    Journal: International Journal of Molecular Sciences

    Article Title: Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter

    doi: 10.3390/ijms17010119

    Figure Lengend Snippet: Verification of mouse Omi/HtrA2 promoter luciferase expression plasmids. Plasmids were verified by double enzyme digestion with Kpn I and Hind III, which releases a vector fragment and a variable-sized promoter insert. M: DL2000 Marker; 1 : pGL3-239; 2 : pGL3-472; 3 : pGL3-742; 4 : pGL3-931; 5 : pGL3-1298. Luc: luciferase expression plasmids.

    Article Snippet: Recombinant constructs pMD18-T-239 to pMD18-T-1298 and pGL3 luciferase reporter vector (#E1751; Promega, Madison, WI, USA) were digested for 4h at 37 °C with Kpn I (#1068A; Takara) and Hind III (#1060A; Takara), and the fragments of the recombinant constructs were subcloned into pGL3.

    Techniques: Luciferase, Expressing, Plasmid Preparation, Marker

    The effect of melittin on the genomic DNA of X. oryzae pv. oryzae . A gel retardation assay was conducted after various amounts of peptides were incubated with 600 ng of DNA for 1 h. The weight ratios (peptide:DNA) were 0.05, 0.5, 1, 2, and 3 ( a ). Effect of melittin on DNA restriction enzyme digestion by Hin dIII (E1), Kpn I (E2), Sac I (E3), and Eco RI - HF (E4). M marker, P peptide melittin ( b )

    Journal: Applied Microbiology and Biotechnology

    Article Title: Antimicrobial peptide melittin against Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen in rice

    doi: 10.1007/s00253-016-7400-4

    Figure Lengend Snippet: The effect of melittin on the genomic DNA of X. oryzae pv. oryzae . A gel retardation assay was conducted after various amounts of peptides were incubated with 600 ng of DNA for 1 h. The weight ratios (peptide:DNA) were 0.05, 0.5, 1, 2, and 3 ( a ). Effect of melittin on DNA restriction enzyme digestion by Hin dIII (E1), Kpn I (E2), Sac I (E3), and Eco RI - HF (E4). M marker, P peptide melittin ( b )

    Article Snippet: In addition, samples with peptide/DNA weight ratios of 0.5 were dissolved in 0.5 μl Tris-HCl and then digested with 1 μl Hin dIII, Kpn I, SacI , or Eco RI- HF (Takara Biotech).

    Techniques: Electrophoretic Mobility Shift Assay, Incubation, Marker