kpni  (Thermo Fisher)


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    Name:
    KpnI
    Description:
    5 G G T A C ↓C 3 3 C ↑C A T G G 5 Thermo Scientific KpnI restriction enzyme recognizes GGTAC C sites and cuts best at 37°C in its own unique buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Isoschizomers Asp718I Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    Catalog Number:
    ER0523
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    Cloning|Restriction Enzyme Cloning
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    Structured Review

    Thermo Fisher kpni
    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major <t>LMSTI1</t> and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with <t>KpnI</t> and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).
    5 G G T A C ↓C 3 3 C ↑C A T G G 5 Thermo Scientific KpnI restriction enzyme recognizes GGTAC C sites and cuts best at 37°C in its own unique buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Isoschizomers Asp718I Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    https://www.bioz.com/result/kpni/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kpni - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi"

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).
    Figure Legend Snippet: A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Techniques Used: Construct, Expressing, Plasmid Preparation

    2) Product Images from "Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct"

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    Journal: Iranian Journal of Parasitology

    doi:

    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker
    Figure Legend Snippet: Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Techniques Used: Electrophoresis, Marker

    Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI
    Figure Legend Snippet: Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Techniques Used: Recombinant, Plasmid Preparation, Marker

    3) Product Images from "Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct"

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    Journal: Iranian Journal of Parasitology

    doi:

    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker
    Figure Legend Snippet: Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Techniques Used: Electrophoresis, Marker

    Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI
    Figure Legend Snippet: Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Techniques Used: Recombinant, Plasmid Preparation, Marker

    4) Product Images from "Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish"

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish

    Journal: Antibiotics

    doi: 10.3390/antibiotics7010016

    Restriction enzyme-digested fragments of the genomic DNA of A. hydrophila -phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with EcoRV; EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively.
    Figure Legend Snippet: Restriction enzyme-digested fragments of the genomic DNA of A. hydrophila -phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with EcoRV; EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively.

    Techniques Used: Marker

    5) Product Images from "A Versatile Vector System for the Fast Generation of Knock-in Cell Lines with CRISPR"

    Article Title: A Versatile Vector System for the Fast Generation of Knock-in Cell Lines with CRISPR

    Journal: bioRxiv

    doi: 10.1101/2020.02.06.927384

    The FAST-HDR vector system. (a) Diagram of the general components of the plasmid that facilitates the rapid construction of donor templates for homologous recombination. The plasmid is first digested with restriction enzymes to cut the segments with CddB cassettes (in this case, KpnI and BamHI). Previously designed synthetic recombination arms are cloned into the digested backbone plasmid using the Gibson assembly method. (b) Diagram of the protein tags and selection antibiotics that are included in the FAST-HDR system.
    Figure Legend Snippet: The FAST-HDR vector system. (a) Diagram of the general components of the plasmid that facilitates the rapid construction of donor templates for homologous recombination. The plasmid is first digested with restriction enzymes to cut the segments with CddB cassettes (in this case, KpnI and BamHI). Previously designed synthetic recombination arms are cloned into the digested backbone plasmid using the Gibson assembly method. (b) Diagram of the protein tags and selection antibiotics that are included in the FAST-HDR system.

    Techniques Used: Plasmid Preparation, Homologous Recombination, Clone Assay, Selection

    6) Product Images from "Peripheral non-viral MIDGE vector-driven delivery of ?-endorphin in inflammatory pain"

    Article Title: Peripheral non-viral MIDGE vector-driven delivery of ?-endorphin in inflammatory pain

    Journal: Molecular Pain

    doi: 10.1186/1744-8069-5-72

    Construction of MIDGE vectors . (A) POMC exon 2-3 cDNA, encoding the signal peptide, adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH) and β-endorphin (END), was spliced into the pMOK plasmid between the SacI and KpnI restriction sites. The pMOK plasmid contains a kanamycin resistance gene, a cytomegalovirus promoter (PCMV), a chimeric intron and simian virus (SV) 40 polyadenylation (pA) site. The POMC-MIDGE-NLS was derived from the pMOK-POMC plasmid by cutting it with Eco31I, ligation with hairpin ODNs at both ends, and coupling of nuclear localization sequence (NLS) to one of the hairpin ODN. The other vectors were prepared analogously by modifying POMC gene as follows: (B) END sequence was removed to obtain control MIDGE-NLS; (C) ACTH and MSH fragments were removed while END sequence was preserved in its original place to obtain 1xEND-MIDGE-NLS encoding 1 copy of END; (D) ACTH fragment was removed, original END sequence was preserved and MSH fragment was replaced with an additional sequence of END to obtain 2xEND-MIDGE-NLS encoding 2 copies of END; (E) original END sequence was preserved and the ACTH and MSH fragments were replaced with additional END sequences to obtain 3xEND-MIDGE-NLS encoding 3 copies of END.
    Figure Legend Snippet: Construction of MIDGE vectors . (A) POMC exon 2-3 cDNA, encoding the signal peptide, adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH) and β-endorphin (END), was spliced into the pMOK plasmid between the SacI and KpnI restriction sites. The pMOK plasmid contains a kanamycin resistance gene, a cytomegalovirus promoter (PCMV), a chimeric intron and simian virus (SV) 40 polyadenylation (pA) site. The POMC-MIDGE-NLS was derived from the pMOK-POMC plasmid by cutting it with Eco31I, ligation with hairpin ODNs at both ends, and coupling of nuclear localization sequence (NLS) to one of the hairpin ODN. The other vectors were prepared analogously by modifying POMC gene as follows: (B) END sequence was removed to obtain control MIDGE-NLS; (C) ACTH and MSH fragments were removed while END sequence was preserved in its original place to obtain 1xEND-MIDGE-NLS encoding 1 copy of END; (D) ACTH fragment was removed, original END sequence was preserved and MSH fragment was replaced with an additional sequence of END to obtain 2xEND-MIDGE-NLS encoding 2 copies of END; (E) original END sequence was preserved and the ACTH and MSH fragments were replaced with additional END sequences to obtain 3xEND-MIDGE-NLS encoding 3 copies of END.

    Techniques Used: Plasmid Preparation, Derivative Assay, Ligation, Sequencing

    7) Product Images from "Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line"

    Article Title: Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker ( bp ). Asterisks (*) point the desired band.
    Figure Legend Snippet: Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker ( bp ). Asterisks (*) point the desired band.

    Techniques Used: Clone Assay, Subcloning, Amplification, Polymerase Chain Reaction, Selection, Construct, Marker

    8) Product Images from "Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion"

    Article Title: Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20060964

    Quantification of SHM in human B cell subsets. The IgκREHMA assay was modified from Andersen et al. ( 37 ) for use on genomice DNA. (A) Schematic representation of a Vκ3-20 to Jκ rearrangement on genomic DNA. The Vκ3-20 gene segment contains two Fnu4HI restriction sites in a SHM hot-spot in the CDR1 region. In addition, a KpnI site is located downstream of the Fnu4HI sites in the FR2 region. Vκ3-20–Jκ rearrangements were PCR amplified with one HEX-labeled Vκ3-20–intron forward and two Jκ reverse primers that recognize all five Jκ gene segments ( 61 ). Digestion of these PCR products with both Fnu4HI and KpnI resulted in unmutated fragments of 244 and 247 bp and mutated fragments of 262 bp. (B and C) Spectratyping of Vκ3-20–Jκ rearrangements of CD5 + B lymphocytes and memory B lymphocytes after double digest with Fnu4HI and KpnI. CD5 + B lymphocytes did not have a replication history and showed no SHM, whereas memory B lymphocytes in adult blood had undergone 11 cell cycles in the periphery and showed 30% mutated Vκ3-20–Jκ rearrangements. (D and E) The percentage of mutated Vκ3-20–Jκ rearrangements was measured in the same DNA samples that were previously used for determining the replication history (data represent means ± SD).
    Figure Legend Snippet: Quantification of SHM in human B cell subsets. The IgκREHMA assay was modified from Andersen et al. ( 37 ) for use on genomice DNA. (A) Schematic representation of a Vκ3-20 to Jκ rearrangement on genomic DNA. The Vκ3-20 gene segment contains two Fnu4HI restriction sites in a SHM hot-spot in the CDR1 region. In addition, a KpnI site is located downstream of the Fnu4HI sites in the FR2 region. Vκ3-20–Jκ rearrangements were PCR amplified with one HEX-labeled Vκ3-20–intron forward and two Jκ reverse primers that recognize all five Jκ gene segments ( 61 ). Digestion of these PCR products with both Fnu4HI and KpnI resulted in unmutated fragments of 244 and 247 bp and mutated fragments of 262 bp. (B and C) Spectratyping of Vκ3-20–Jκ rearrangements of CD5 + B lymphocytes and memory B lymphocytes after double digest with Fnu4HI and KpnI. CD5 + B lymphocytes did not have a replication history and showed no SHM, whereas memory B lymphocytes in adult blood had undergone 11 cell cycles in the periphery and showed 30% mutated Vκ3-20–Jκ rearrangements. (D and E) The percentage of mutated Vκ3-20–Jκ rearrangements was measured in the same DNA samples that were previously used for determining the replication history (data represent means ± SD).

    Techniques Used: Modification, Polymerase Chain Reaction, Amplification, Labeling

    9) Product Images from "pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110"

    Article Title: pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110

    Journal: Microbiology Resource Announcements

    doi: 10.1128/MRA.00596-20

    (A) Novel integrative pSETT4 cloning system. The lacZ′ cassette is flanked by the recognition sites of the restriction enzyme BsaI. BsaI enables exchange of lacZ by the gene of interest using Gibson assembly, restriction/ligation cloning, or Golden Gate cloning. As strong expression needs strong termination, T4 terminators were introduced before and after the cloning site. Behind the cloning site, two antiparallel-oriented T4 terminators prevent read-through from both directions. For exchange of the promoter sequence, NdeI and KpnI restriction sites were introduced. Furthermore, the vector contains the integrase gene int and the attachment site attP of the phage φC31, the origin of transfer ( incP ) and relaxosome gene traJ , the high-copy-number ColE1 origin of replication, and the apramycin resistance gene aac(3)-IV . (B) Growth and acarbose formation of Actinoplanes sp. SE50/110 (pSETT4 tip ), Actinoplanes sp. SE50/110 (pSET152), and the wild-type Actinoplanes sp. SE50/110 took place in a shake flask in maltose minimal medium. Numbers of replicates are indicated by the n values shown in parentheses for both the cell dry weight (cdw) and the acarbose concentration (acb).
    Figure Legend Snippet: (A) Novel integrative pSETT4 cloning system. The lacZ′ cassette is flanked by the recognition sites of the restriction enzyme BsaI. BsaI enables exchange of lacZ by the gene of interest using Gibson assembly, restriction/ligation cloning, or Golden Gate cloning. As strong expression needs strong termination, T4 terminators were introduced before and after the cloning site. Behind the cloning site, two antiparallel-oriented T4 terminators prevent read-through from both directions. For exchange of the promoter sequence, NdeI and KpnI restriction sites were introduced. Furthermore, the vector contains the integrase gene int and the attachment site attP of the phage φC31, the origin of transfer ( incP ) and relaxosome gene traJ , the high-copy-number ColE1 origin of replication, and the apramycin resistance gene aac(3)-IV . (B) Growth and acarbose formation of Actinoplanes sp. SE50/110 (pSETT4 tip ), Actinoplanes sp. SE50/110 (pSET152), and the wild-type Actinoplanes sp. SE50/110 took place in a shake flask in maltose minimal medium. Numbers of replicates are indicated by the n values shown in parentheses for both the cell dry weight (cdw) and the acarbose concentration (acb).

    Techniques Used: Clone Assay, Ligation, Expressing, Sequencing, Plasmid Preparation, Concentration Assay

    Related Articles

    Generated:

    Article Title: Unusual organization of a developmentally regulated mitochondrial RNA polymerase (TBMTRNAP) gene in Trypanosoma brucei
    Article Snippet: Poly(A + ) RNA (4.5 µg) and 30 µg of total RNA were electrophoresed on a 0.66 M formaldehyde 1% agarose gel. .. The resolved RNA was transferred to a Hybond Nytran membrane using downward capillary transfer and hybridized with an internal 1.8 kb riboprobe generated by digestion of the Bam HI clone with Kpn I and transcription from the T3 promoter using the Ambion T3 Maxiscript kit. .. The templates for the 5′ probe and 3′ UTR antisense riboprobes were created via PCR to incorporate a T7 promoter.

    Expressing:

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi
    Article Snippet: The integrities of the pT-LmSTI1 and pT-PpSP42 plasmids were verified by nucleotide sequencing using M13F and M13R universal primers from Gen Fanavaran Co. (Iran). .. Construction of eukaryotic expression vector for the fusion protein LmSTI1 was cloned into pcNDA3.1 (+) to produce pCLmSTI1 as follows: the TA-cloned pT-LmSTI1 plasmid and pcDNA3.1 (+) were double-digested separately with KpnI and EcoRI (Fermentas, Germany) in 1X Tango buffer. ..

    Plasmid Preparation:

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi
    Article Snippet: The integrities of the pT-LmSTI1 and pT-PpSP42 plasmids were verified by nucleotide sequencing using M13F and M13R universal primers from Gen Fanavaran Co. (Iran). .. Construction of eukaryotic expression vector for the fusion protein LmSTI1 was cloned into pcNDA3.1 (+) to produce pCLmSTI1 as follows: the TA-cloned pT-LmSTI1 plasmid and pcDNA3.1 (+) were double-digested separately with KpnI and EcoRI (Fermentas, Germany) in 1X Tango buffer. ..

    Article Title: Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells
    Article Snippet: The PCR product was purified using Qiaquick Gel extraction kit (Qiagen,USA). .. Both PCR product and pGL3-basic vector were digested with Kpn-I and Xho-I restriction enzymes (Fermentas). .. The digested fragment was then ligated to restriction enzyme digested pGL3-basic vector using DNA ligase and designated as pGL3-1217.

    Clone Assay:

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi
    Article Snippet: The integrities of the pT-LmSTI1 and pT-PpSP42 plasmids were verified by nucleotide sequencing using M13F and M13R universal primers from Gen Fanavaran Co. (Iran). .. Construction of eukaryotic expression vector for the fusion protein LmSTI1 was cloned into pcNDA3.1 (+) to produce pCLmSTI1 as follows: the TA-cloned pT-LmSTI1 plasmid and pcDNA3.1 (+) were double-digested separately with KpnI and EcoRI (Fermentas, Germany) in 1X Tango buffer. ..

    Sequencing:

    Article Title: PepGMV Rep-Protein Expression in Mammalian Cells
    Article Snippet: Insert and vector purification was performed using the Silica Bead DNA Gel Extraction kit (Fermentas) and ligation reaction was done using the ligase T4 (Invitrogen) and a 4:1 insert/vector molar ratio. .. The insert presence was confirmed by double digestion with SpeI and KpnI enzymes (Fermentas).Gene correct orientation was confirmed by sequencing (Genetic Analyzer 3130, Applied Biosystems). .. Cell Culture and Transfection 3T3L1 mice fibroblast cells (ATCC No. CL-173) were grown in Dubelco’s Modified Eagle Medium (DMEM, Gibco) supplemented with penicillin (62.1 mg/L; Sigma), streptomycin (100 mg/L; Sigma), anphotericin (250 µg/L; Gibco), and 5% of calf serum (Biowest).

    Recombinant:

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct
    Article Snippet: The ptr1 coding region was amplified from genomic DNA and the PCR product was ligated to a 3′ T-tailed, EcoRV-digested pBluescript and sequenced. .. Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI. .. The recombinant plasmid was transformed into E. coli TOP10 strain.

    Purification:

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct
    Article Snippet: The ptr1 coding region was amplified from genomic DNA and the PCR product was ligated to a 3′ T-tailed, EcoRV-digested pBluescript and sequenced. .. Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI. .. The recombinant plasmid was transformed into E. coli TOP10 strain.

    DNA Purification:

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct
    Article Snippet: The ptr1 coding region was amplified from genomic DNA and the PCR product was ligated to a 3′ T-tailed, EcoRV-digested pBluescript and sequenced. .. Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI. .. The recombinant plasmid was transformed into E. coli TOP10 strain.

    Polymerase Chain Reaction:

    Article Title: Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells
    Article Snippet: The PCR product was purified using Qiaquick Gel extraction kit (Qiagen,USA). .. Both PCR product and pGL3-basic vector were digested with Kpn-I and Xho-I restriction enzymes (Fermentas). .. The digested fragment was then ligated to restriction enzyme digested pGL3-basic vector using DNA ligase and designated as pGL3-1217.

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    Thermo Fisher kpn i
    Identification of PPA1 minimal promoter. (A) Determination of transcription start site of PPA1 gene. Total RNA was isolated from MCF7 cells and analyzed by primer extension analysis using a primer complementary to the -20 to -1 region of exon 1 of PPA1 gene. The sequencing reaction was set using same primer as in primer extension. Both the sequencing reaction products (A, T, G and C) and primer extension product (PE) were run on a 7.5% urea-polyacrylamide gel. (B) Schematic representation of 5’-deletion constructs of PPA1 promoter. All the deletion constructs were generated by <t>PCR</t> using pGL3-1217 construct as template and cloned into the <t>Kpn-I</t> and Xho-I restriction site of pGL3-Basic vector. (C) Promoter activity of all the 5’-deleted promoter constructs was determined by Dual-Luciferase assay. MCF7 cells were transfected with promoter constructs and after 24 h luciferase assay was carried out using Dual-Luciferase assay kit (Promega). Each transfection was conducted in triplicate and the results are represented as mean ± S.E.M.
    Kpn I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Identification of PPA1 minimal promoter. (A) Determination of transcription start site of PPA1 gene. Total RNA was isolated from MCF7 cells and analyzed by primer extension analysis using a primer complementary to the -20 to -1 region of exon 1 of PPA1 gene. The sequencing reaction was set using same primer as in primer extension. Both the sequencing reaction products (A, T, G and C) and primer extension product (PE) were run on a 7.5% urea-polyacrylamide gel. (B) Schematic representation of 5’-deletion constructs of PPA1 promoter. All the deletion constructs were generated by PCR using pGL3-1217 construct as template and cloned into the Kpn-I and Xho-I restriction site of pGL3-Basic vector. (C) Promoter activity of all the 5’-deleted promoter constructs was determined by Dual-Luciferase assay. MCF7 cells were transfected with promoter constructs and after 24 h luciferase assay was carried out using Dual-Luciferase assay kit (Promega). Each transfection was conducted in triplicate and the results are represented as mean ± S.E.M.

    Journal: PLoS ONE

    Article Title: Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells

    doi: 10.1371/journal.pone.0124864

    Figure Lengend Snippet: Identification of PPA1 minimal promoter. (A) Determination of transcription start site of PPA1 gene. Total RNA was isolated from MCF7 cells and analyzed by primer extension analysis using a primer complementary to the -20 to -1 region of exon 1 of PPA1 gene. The sequencing reaction was set using same primer as in primer extension. Both the sequencing reaction products (A, T, G and C) and primer extension product (PE) were run on a 7.5% urea-polyacrylamide gel. (B) Schematic representation of 5’-deletion constructs of PPA1 promoter. All the deletion constructs were generated by PCR using pGL3-1217 construct as template and cloned into the Kpn-I and Xho-I restriction site of pGL3-Basic vector. (C) Promoter activity of all the 5’-deleted promoter constructs was determined by Dual-Luciferase assay. MCF7 cells were transfected with promoter constructs and after 24 h luciferase assay was carried out using Dual-Luciferase assay kit (Promega). Each transfection was conducted in triplicate and the results are represented as mean ± S.E.M.

    Article Snippet: Both PCR product and pGL3-basic vector were digested with Kpn-I and Xho-I restriction enzymes (Fermentas).

    Techniques: Isolation, Sequencing, Construct, Generated, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Activity Assay, Luciferase, Transfection

    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Journal: Reports of Biochemistry & Molecular Biology

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi

    doi:

    Figure Lengend Snippet: A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Article Snippet: Construction of eukaryotic expression vector for the fusion protein LmSTI1 was cloned into pcNDA3.1 (+) to produce pCLmSTI1 as follows: the TA-cloned pT-LmSTI1 plasmid and pcDNA3.1 (+) were double-digested separately with KpnI and EcoRI (Fermentas, Germany) in 1X Tango buffer.

    Techniques: Construct, Expressing, Plasmid Preparation

    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Electrophoresis, Marker

    Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Recombinant, Plasmid Preparation, Marker

    Agarose gel electrophoresis of Rep gene PCR amplification and construction double digestion.( a ) PCR amplification of Rep gene. Lanes: 1, size molecular marker; 2, Rep; 3, RepATG − ; 4, negative control. ( b ) Construction digestion with SpeI and KpnI . Lanes: 1, size molecular marker; 2, pTracerSV40:Rep; 3, pTracerSV40:RepATG − . The samples were loaded in a 1.2% agarose gel stained with EtBr.

    Journal: Viruses

    Article Title: PepGMV Rep-Protein Expression in Mammalian Cells

    doi: 10.3390/v4091792

    Figure Lengend Snippet: Agarose gel electrophoresis of Rep gene PCR amplification and construction double digestion.( a ) PCR amplification of Rep gene. Lanes: 1, size molecular marker; 2, Rep; 3, RepATG − ; 4, negative control. ( b ) Construction digestion with SpeI and KpnI . Lanes: 1, size molecular marker; 2, pTracerSV40:Rep; 3, pTracerSV40:RepATG − . The samples were loaded in a 1.2% agarose gel stained with EtBr.

    Article Snippet: The insert presence was confirmed by double digestion with SpeI and KpnI enzymes (Fermentas).Gene correct orientation was confirmed by sequencing (Genetic Analyzer 3130, Applied Biosystems).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Negative Control, Staining