large construct dna purification kit  (Qiagen)


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    Name:
    QIAGEN Large Construct Kit
    Description:
    For purification of up to 50 μg BAC PAC and P1 DNA or up to 200 μg cosmid DNA free of genomic DNA Kit contents Qiagen Large Construct Kit 500mL Culture Volume BAC PAC P1 cosmid DNA Plasmid Type Anion exchange Technology Manual Processing 280 min Time Run 1 Sample Run 150g Yield For Purification of up to 50μg BAC PAC and P1 DNA or up to 200μg Cosmid DNA Free of genomic DNA Ideal for Subcloning Transfection Sequencing Optimized Gravity flow Procedure Includes 10 Qiagen tip 500 Reagents Buffers ATP Dependent Exonuclease Benefits Efficient removal of genomic DNA contamination Pure transfection grade large construct DNA Easy and simple proced
    Catalog Number:
    12462
    Price:
    433
    Category:
    QIAGEN Large Construct Kit
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    Structured Review

    Qiagen large construct dna purification kit
    QIAGEN Large Construct Kit
    For purification of up to 50 μg BAC PAC and P1 DNA or up to 200 μg cosmid DNA free of genomic DNA Kit contents Qiagen Large Construct Kit 500mL Culture Volume BAC PAC P1 cosmid DNA Plasmid Type Anion exchange Technology Manual Processing 280 min Time Run 1 Sample Run 150g Yield For Purification of up to 50μg BAC PAC and P1 DNA or up to 200μg Cosmid DNA Free of genomic DNA Ideal for Subcloning Transfection Sequencing Optimized Gravity flow Procedure Includes 10 Qiagen tip 500 Reagents Buffers ATP Dependent Exonuclease Benefits Efficient removal of genomic DNA contamination Pure transfection grade large construct DNA Easy and simple proced
    https://www.bioz.com/result/large construct dna purification kit/product/Qiagen
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    Images

    1) Product Images from "Signal peptide mutations in RANK prevent downstream activation of NF-?B"

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    Journal: Journal of Bone and Mineral Research

    doi: 10.1002/jbmr.399

    WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Figure Legend Snippet: WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.

    Techniques Used: Transfection, Staining, Confocal Microscopy

    Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.
    Figure Legend Snippet: Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.

    Techniques Used: Mutagenesis, Transduction, Staining, Confocal Microscopy

    Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .
    Figure Legend Snippet: Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Construct, Amplification, Polymerase Chain Reaction, FLAG-tag, Synthesized, Agarose Gel Electrophoresis, Transfection, Stable Transfection

    2) Product Images from "A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿"

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02388-09

    PCR amplification of DBAC-L cDNA inserts demonstrates library diversity. (A) cDNA inserts were amplified from DBAC-L genomic DNA, purified from E. coli , using primers specific for the HCMV promoter and SV40 poly(A) sequences. Extension times for PCRs varied from 1 min (lanes 1 to 4) to 5 min (lanes 6 to 9). The reaction products were separated on a 1% agarose gel, revealing cDNAs of various sizes. Lane 11, size markers; arrows indicate approximate fragment sizes in kilobases (kb). (B) Distinct cDNAs were amplified from total DNA harvested from 2 × 10 6 7b cells infected with the DBAC-L vector stock at an MOI of 0.05. Viral and cellular DNA was harvested at 24 hpi and eluted in 250 μl TE. Serial 10-fold dilutions of total DNA (lanes 1 to 5) were used as template DNA for PCR, such that one microliter (lane 1) represented approximately 400 input PFU. Lane 6, water control; lane 7, size markers. (C) Ten individual PCRs each using 10 −4 μl DBAC-L viral DNA demonstrated the presence of unique cDNA insert sizes.
    Figure Legend Snippet: PCR amplification of DBAC-L cDNA inserts demonstrates library diversity. (A) cDNA inserts were amplified from DBAC-L genomic DNA, purified from E. coli , using primers specific for the HCMV promoter and SV40 poly(A) sequences. Extension times for PCRs varied from 1 min (lanes 1 to 4) to 5 min (lanes 6 to 9). The reaction products were separated on a 1% agarose gel, revealing cDNAs of various sizes. Lane 11, size markers; arrows indicate approximate fragment sizes in kilobases (kb). (B) Distinct cDNAs were amplified from total DNA harvested from 2 × 10 6 7b cells infected with the DBAC-L vector stock at an MOI of 0.05. Viral and cellular DNA was harvested at 24 hpi and eluted in 250 μl TE. Serial 10-fold dilutions of total DNA (lanes 1 to 5) were used as template DNA for PCR, such that one microliter (lane 1) represented approximately 400 input PFU. Lane 6, water control; lane 7, size markers. (C) Ten individual PCRs each using 10 −4 μl DBAC-L viral DNA demonstrated the presence of unique cDNA insert sizes.

    Techniques Used: Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Infection, Plasmid Preparation

    Related Articles

    Clone Assay:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion. .. First, the EcoRV fragment from plasmid A was cloned into the EcoRV site of pSP72 to generate p72GateA. p72GateA was modified to replace the Cm resistance gene in the Gateway cassette with phleomycin D (Zeocin [Zeo]) resistance by isolating and end filling an XhoI-EcoRI fragment containing the Zeo coding sequence from pEM7/Zeo (Invitrogen) and subcloning between the blunted NotI and MluI sites of p72GateA.

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1
    Article Snippet: Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen). .. Viruses were generated from AlHV-1 strain C500 BAC clones in MacT-Cre cells, grown in BT cells, and purified from the supernatant by ultracentrifugation (100,000 × g ) through a 30% (w/v) sucrose cushion for 2 h at 4 °C.

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: .. DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit. .. BAC-DNA was prepared for in situ hybridization by nick-translation using direct fluorophores Cyanine 3-dUTP (Enzo Life Sciences, ENZ-42501) or Fluorescein-12-dUTP (Thermo Scientific, R0101) .

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: Three shh -positive BAC clones were isolated. .. Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany).

    Amplification:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK). .. Primary amplification adenoviral stocks were prepared by amplification in HEK 293 cells cultured in 175-cm2 tissue culture flasks, and these stocks were titered by endpoint dilution assay.

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: That is, all primers amplified hemoglobin gene products within these BACs, and no additional BACs that were not contained within the four BACs as determined by the Atlantic salmon physical map yielded PCR products using the hemoglobin gene primers. .. Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont.

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit.

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone. .. The 5′ homology arm, a 3.9-kb DNA fragment that contains all the 5′ untranscribed region and promoter elements, was PCR amplified using a high-fidelity Thermal Ace DNA polymerase kit (Invitrogen, Inc., Carlsbad, CA).

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR across the GW cassette to assess the diversity of cDNAs recombined into TTA BAC was performed with Taq DNA polymerase and primers CMV-F1 and bGH-R1 ( ) using 100 ng BAC DNA, 0.2 μmol/l of each primer, and 0.2 mmol/l of each dNTP (Promega, Madison, WI) at the following cycling conditions: 94°C for 3 minutes; 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 3 minutes; and 72°C for 10 minutes.

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis. .. Founders were identified by PCR amplification of tail DNA with specific primers for Tlr2 Cre transgenic and wt alleles: Tlr2F (common) 5′-AGCCATAGGCCACATCTAGT-3′, Tlr2CreR 5′-GTATGCTCAGAAAACGCCTG-3′ (470b), and Tlr2wtR 5′-AAAAAGCGATGTTACCCC-3′ (784b) and backcrossed to C57BL/6 mice.

    Construct:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: .. Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The kit includes an exonuclease digestion step to eliminate E. coli genomic DNA.

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. The library was recombined with the TTA BAC Gateway cassette by incubation of 2 μg of BAC DNA and 200 ng of library plasmid DNA with clonase enzyme for 4 hours.

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: Paragraph title: Plasmid constructs and transgenic plants ... The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region.

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone. .. A replacement-targeting vector was constructed where the bacterial β-galactosidase gene was knocked into the periostin gene locus.

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1
    Article Snippet: .. Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen). .. Viruses were generated from AlHV-1 strain C500 BAC clones in MacT-Cre cells, grown in BT cells, and purified from the supernatant by ultracentrifugation (100,000 × g ) through a 30% (w/v) sucrose cushion for 2 h at 4 °C.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: .. DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit. .. BAC-DNA was prepared for in situ hybridization by nick-translation using direct fluorophores Cyanine 3-dUTP (Enzo Life Sciences, ENZ-42501) or Fluorescein-12-dUTP (Thermo Scientific, R0101) .

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR across the GW cassette to assess the diversity of cDNAs recombined into TTA BAC was performed with Taq DNA polymerase and primers CMV-F1 and bGH-R1 ( ) using 100 ng BAC DNA, 0.2 μmol/l of each primer, and 0.2 mmol/l of each dNTP (Promega, Madison, WI) at the following cycling conditions: 94°C for 3 minutes; 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 3 minutes; and 72°C for 10 minutes.

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: .. Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany). .. A total of 20 μg of BAC clone DNA in 500 μl TE (10 mM Tris, 1 mM EDTA), pH 8.0, was fragmented by sonication (Branson Sonifier, Danbury, CT, USA) with 4 × 1-second pulses at 300 W (5-mm microtip).

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis. .. The pronuclear injection of the construct (1 ng/µl) into mouse zygotes was carried out at the Transgenic Unit of IMG.

    Electrophoresis:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: Modified BAC was tested for the absence of rearrangements using EcoRV restriction analysis and pulsed field gel electrophoresis. .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ].

    Modification:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion. .. The Gateway cassette from Gateway conversion plasmid A (Invitrogen) was modified for this work in the following manner.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: Modified BAC was tested for the absence of rearrangements using EcoRV restriction analysis and pulsed field gel electrophoresis. .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ].

    Incubation:

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. The library was recombined with the TTA BAC Gateway cassette by incubation of 2 μg of BAC DNA and 200 ng of library plasmid DNA with clonase enzyme for 4 hours.

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR across the GW cassette to assess the diversity of cDNAs recombined into TTA BAC was performed with Taq DNA polymerase and primers CMV-F1 and bGH-R1 ( ) using 100 ng BAC DNA, 0.2 μmol/l of each primer, and 0.2 mmol/l of each dNTP (Promega, Madison, WI) at the following cycling conditions: 94°C for 3 minutes; 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 3 minutes; and 72°C for 10 minutes.

    Activity Assay:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    Infection:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The genome of a purified D2/BAC recombinant was circularized by infecting U2OS cells at a multiplicity of infection (MOI) of 5 for 3 h, and DNA was isolated by proteinase K digestion, phenol-chloroform extraction using PhaseLock gel (5Prime, Gaithersburg, MD), and isopropanol precipitation. .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Expressing:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    Knock-In:

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: To generate peri lacZ knock-in mice, we screened a 129SvJ mouse embryonic stem (ES) cell genomic DNA library (Genome System Inc., St. Louis, MO) by PCR and identified a bacterial artificial chromosome (BAC) clone that contains the genomic DNA that codes for the periostin gene. .. BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone.

    Derivative Assay:

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: BAC library production The pENTR-based cDNA library derived from a mixture of NGF-differentiated and undifferentiated PC12 cells was previously described. .. TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen).

    Hybridization:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: BAC shotgun library generation and sequencing Using a combination of hybridization probing and PCR (see above) to screen all BACs within the suspected hemoglobin gene containing regions, we identified two overlapping BACs from each of fps1046 (BACs S0055H05 and S0014B03) and fps135 (BACs S0155C07 and S0079J05) spanning the entire Atlantic salmon hemoglobin gene repertoire. .. Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont.

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit. .. Hybridization of BAC clones was performed with suppression of unspecific binding, by pre-hybridizing the probe with an unlabeled Cot2 fraction of repetitive axolotl DNA .

    Electroporation:

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. Following electroporation into DH10B-T1 bacteria, recombinants were titered by serial dilution on LB plates containing 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, demonstrating a total yield of ~200,000 colony forming units.

    Transfection:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    RAST Test:

    Article Title: Characterization of Escherichia coli Carrying mcr-1-Plasmids Recovered From Food Animals From Argentina
    Article Snippet: Plasmids were extracted from mcr- transconjugants strains using the Qiagen Large-Construct kit (Qiagen) and sequenced using Illumina's MiSeq system. .. The obtained reads were assembled using CLC Genomics Workbench software (CLCbio, Qiagen), annotated using RAST server ( http://rast.nmpdr.org/rast.cgi ) and the sequences (gaps were not filled) compared in a pairwise fashion using BRIG (Alikhan et al., ).

    Southern Blot:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: D2 was identified based on its plaquing dependence on both ICP4 and ICP27 complementation and was confirmed by Southern blot analysis (not shown). .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Ligation:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: Construction of recombinant adenoviruses Recombinant adenoviruses containing WT-RANK-FLAG, FEO-RANK-FLAG, and PDB-RANK-FLAG were generated by ligation using the Adeno-X-TetOff expression system (Takara Clontech, Mountain View, CA, USA) following the manufacturer's instructions. .. The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Library Screening:

    Article Title: Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group
    Article Snippet: Paragraph title: Marker Development and BAC Library Screening and Sequencing. ... DNAs were extracted from selected BACs using a large-construct kit (Qiagen) and sequenced with Illumina at the University of California (UC) Davis Genomic Center.

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: Paragraph title: BAC library Screening and BAC-clone sequencing ... Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany).

    Serial Dilution:

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. Following electroporation into DH10B-T1 bacteria, recombinants were titered by serial dilution on LB plates containing 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, demonstrating a total yield of ~200,000 colony forming units.

    Nick Translation:

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit. .. BAC-DNA was prepared for in situ hybridization by nick-translation using direct fluorophores Cyanine 3-dUTP (Enzo Life Sciences, ENZ-42501) or Fluorescein-12-dUTP (Thermo Scientific, R0101) .

    Cell Culture:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK). .. Primary amplification adenoviral stocks were prepared by amplification in HEK 293 cells cultured in 175-cm2 tissue culture flasks, and these stocks were titered by endpoint dilution assay.

    Generated:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: Construction of recombinant adenoviruses Recombinant adenoviruses containing WT-RANK-FLAG, FEO-RANK-FLAG, and PDB-RANK-FLAG were generated by ligation using the Adeno-X-TetOff expression system (Takara Clontech, Mountain View, CA, USA) following the manufacturer's instructions. .. The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The targeting plasmid for recombination was generated by insertion of the bacterial origin of replication and Cm resistance gene from pBeloBACII (New England Biolabs, Ipswich, MA) into the TK coding sequence of a UL 23 plasmid. .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1
    Article Snippet: Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen). .. Viruses were generated from AlHV-1 strain C500 BAC clones in MacT-Cre cells, grown in BT cells, and purified from the supernatant by ultracentrifugation (100,000 × g ) through a 30% (w/v) sucrose cushion for 2 h at 4 °C.

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: Tlr2 Cre and Tlr2 CreERT2 transgenic mice were generated by inserting Cre and Cre ERT2 cassettes, respectively, under the control of the Tlr2 promoter by BAC recombineering . .. BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis.

    Endpoint Dilution Assay:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK). .. Primary amplification adenoviral stocks were prepared by amplification in HEK 293 cells cultured in 175-cm2 tissue culture flasks, and these stocks were titered by endpoint dilution assay.

    Polymerase Chain Reaction:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: That is, all primers amplified hemoglobin gene products within these BACs, and no additional BACs that were not contained within the four BACs as determined by the Atlantic salmon physical map yielded PCR products using the hemoglobin gene primers. .. Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont.

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region. .. The PCR products were purified using the GenepHlow Gel/PCR Kit, and were then subcloned into the binary vector pMDC107 between the Pme I and Asc I sites to obtain pMDC107‐gCLF and pMDC107‐gSWN .

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: To generate peri lacZ knock-in mice, we screened a 129SvJ mouse embryonic stem (ES) cell genomic DNA library (Genome System Inc., St. Louis, MO) by PCR and identified a bacterial artificial chromosome (BAC) clone that contains the genomic DNA that codes for the periostin gene. .. BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone.

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR across the GW cassette to assess the diversity of cDNAs recombined into TTA BAC was performed with Taq DNA polymerase and primers CMV-F1 and bGH-R1 ( ) using 100 ng BAC DNA, 0.2 μmol/l of each primer, and 0.2 mmol/l of each dNTP (Promega, Madison, WI) at the following cycling conditions: 94°C for 3 minutes; 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 3 minutes; and 72°C for 10 minutes.

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis. .. Founders were identified by PCR amplification of tail DNA with specific primers for Tlr2 Cre transgenic and wt alleles: Tlr2F (common) 5′-AGCCATAGGCCACATCTAGT-3′, Tlr2CreR 5′-GTATGCTCAGAAAACGCCTG-3′ (470b), and Tlr2wtR 5′-AAAAAGCGATGTTACCCC-3′ (784b) and backcrossed to C57BL/6 mice.

    Sonication:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The purified BAC DNA was sheared by sonication and blunt-end repaired.

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany). .. Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany).

    Injection:

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: .. BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis. .. The pronuclear injection of the construct (1 ng/µl) into mouse zygotes was carried out at the Transgenic Unit of IMG.

    Binding Assay:

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit. .. Hybridization of BAC clones was performed with suppression of unspecific binding, by pre-hybridizing the probe with an unlabeled Cot2 fraction of repetitive axolotl DNA .

    Pulsed-Field Gel:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: Modified BAC was tested for the absence of rearrangements using EcoRV restriction analysis and pulsed field gel electrophoresis. .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ].

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: .. BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis. .. The pronuclear injection of the construct (1 ng/µl) into mouse zygotes was carried out at the Transgenic Unit of IMG.

    Nucleic Acid Electrophoresis:

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany). .. The 2- to 3-kb fraction was isolated from the sheared DNA by preparative gel electrophoresis.

    E. coli Genomic Assay:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The kit includes an exonuclease digestion step to eliminate E. coli genomic DNA.

    Fluorescence:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: Paragraph title: DNA probe preparation and fluorescence in situ hybridization (FISH) ... DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit.

    Conjugation Assay:

    Article Title: Characterization of Escherichia coli Carrying mcr-1-Plasmids Recovered From Food Animals From Argentina
    Article Snippet: Sodium azide-resistant E. coli J53 was used as a recipient strain in conjugation experiments to study the transferability of the resistance genes. .. Plasmids were extracted from mcr- transconjugants strains using the Qiagen Large-Construct kit (Qiagen) and sequenced using Illumina's MiSeq system.

    Mutagenesis:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The HSV vector D2 was created by genetic cross between the ICP4 deletion mutant virus d120 ( ) and an ICP27-deleted virus, 5dl1.2 ( ). .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Isolation:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: .. Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The kit includes an exonuclease digestion step to eliminate E. coli genomic DNA.

    Article Title: The Genomic Architecture of Novel Simulium damnosum Wolbachia Prophage Sequence Elements and Implications for Onchocerciasis Epidemiology
    Article Snippet: .. Thick mini-culture preparations from each BAC colony were pooled and their BAC DNA was isolated in single preparations using a QIAGEN large-construct kit and protocol ( https://www.qiagen.com/kr/resources/resourcedetail?id=8f67b644-6d21-4ef3-b33e-a60f32623785 & lang=en ). .. A 10-mg sample of purified BAC DNA was shot-gun sequenced commercially using a Roche 454 FLX system sequencer at the Cambridge University Biochemistry Department.

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: .. The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region. .. The PCR products were purified using the GenepHlow Gel/PCR Kit, and were then subcloned into the binary vector pMDC107 between the Pme I and Asc I sites to obtain pMDC107‐gCLF and pMDC107‐gSWN .

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: .. BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone. .. A replacement-targeting vector was constructed where the bacterial β-galactosidase gene was knocked into the periostin gene locus.

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The genome of a purified D2/BAC recombinant was circularized by infecting U2OS cells at a multiplicity of infection (MOI) of 5 for 3 h, and DNA was isolated by proteinase K digestion, phenol-chloroform extraction using PhaseLock gel (5Prime, Gaithersburg, MD), and isopropanol precipitation. .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: .. DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit. .. BAC-DNA was prepared for in situ hybridization by nick-translation using direct fluorophores Cyanine 3-dUTP (Enzo Life Sciences, ENZ-42501) or Fluorescein-12-dUTP (Thermo Scientific, R0101) .

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: Three shh -positive BAC clones were isolated. .. Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany).

    Subcloning:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion. .. First, the EcoRV fragment from plasmid A was cloned into the EcoRV site of pSP72 to generate p72GateA. p72GateA was modified to replace the Cm resistance gene in the Gateway cassette with phleomycin D (Zeocin [Zeo]) resistance by isolating and end filling an XhoI-EcoRI fragment containing the Zeo coding sequence from pEM7/Zeo (Invitrogen) and subcloning between the blunted NotI and MluI sites of p72GateA.

    Microscopy:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    Purification:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: .. The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK). .. Following PacI digestion to linearize the adenoviral DNA, recombinant adenoviruses then were produced by transfecting DNA into HEK293 cells by calcium phosphate precipitation.

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The purified BAC DNA was sheared by sonication and blunt-end repaired.

    Article Title: The Genomic Architecture of Novel Simulium damnosum Wolbachia Prophage Sequence Elements and Implications for Onchocerciasis Epidemiology
    Article Snippet: Thick mini-culture preparations from each BAC colony were pooled and their BAC DNA was isolated in single preparations using a QIAGEN large-construct kit and protocol ( https://www.qiagen.com/kr/resources/resourcedetail?id=8f67b644-6d21-4ef3-b33e-a60f32623785 & lang=en ). .. A 10-mg sample of purified BAC DNA was shot-gun sequenced commercially using a Roche 454 FLX system sequencer at the Cambridge University Biochemistry Department.

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. The library was recombined with the TTA BAC Gateway cassette by incubation of 2 μg of BAC DNA and 200 ng of library plasmid DNA with clonase enzyme for 4 hours.

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region. .. The PCR products were purified using the GenepHlow Gel/PCR Kit, and were then subcloned into the binary vector pMDC107 between the Pme I and Asc I sites to obtain pMDC107‐gCLF and pMDC107‐gSWN .

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: .. BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone. .. A replacement-targeting vector was constructed where the bacterial β-galactosidase gene was knocked into the periostin gene locus.

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion. .. The Gateway cassette from Gateway conversion plasmid A (Invitrogen) was modified for this work in the following manner.

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1
    Article Snippet: .. Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen). .. Viruses were generated from AlHV-1 strain C500 BAC clones in MacT-Cre cells, grown in BT cells, and purified from the supernatant by ultracentrifugation (100,000 × g ) through a 30% (w/v) sucrose cushion for 2 h at 4 °C.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR across the GW cassette to assess the diversity of cDNAs recombined into TTA BAC was performed with Taq DNA polymerase and primers CMV-F1 and bGH-R1 ( ) using 100 ng BAC DNA, 0.2 μmol/l of each primer, and 0.2 mmol/l of each dNTP (Promega, Madison, WI) at the following cycling conditions: 94°C for 3 minutes; 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 3 minutes; and 72°C for 10 minutes.

    Sequencing:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: Paragraph title: BAC shotgun library generation and sequencing ... Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont.

    Article Title: The Genomic Architecture of Novel Simulium damnosum Wolbachia Prophage Sequence Elements and Implications for Onchocerciasis Epidemiology
    Article Snippet: Thick mini-culture preparations from each BAC colony were pooled and their BAC DNA was isolated in single preparations using a QIAGEN large-construct kit and protocol ( https://www.qiagen.com/kr/resources/resourcedetail?id=8f67b644-6d21-4ef3-b33e-a60f32623785 & lang=en ). .. Sequence reads were quality-checked using Phred Software ( http://www.phrap.org/phredphrapconsed.html ) and assembled into 8,238 contigs using Phrap (Ewing and Green, ; Ewing et al., ).

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: .. The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region. .. The PCR products were purified using the GenepHlow Gel/PCR Kit, and were then subcloned into the binary vector pMDC107 between the Pme I and Asc I sites to obtain pMDC107‐gCLF and pMDC107‐gSWN .

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The targeting plasmid for recombination was generated by insertion of the bacterial origin of replication and Cm resistance gene from pBeloBACII (New England Biolabs, Ipswich, MA) into the TK coding sequence of a UL 23 plasmid. .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Article Title: Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group
    Article Snippet: Paragraph title: Marker Development and BAC Library Screening and Sequencing. ... DNAs were extracted from selected BACs using a large-construct kit (Qiagen) and sequenced with Illumina at the University of California (UC) Davis Genomic Center.

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1
    Article Snippet: Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen). .. Paired-ended, 150 nucleotide sequence reads were generated for the strain C500 BAC clone and the reconstituted virus by using a MiSeq DNA sequencer running version 2 chemistry (Illumina) and assembled by using methods described previously .

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: Integrity of the hBDNF-EGFP reading frame was confirmed by sequencing. .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ].

    Article Title: Characterization of Escherichia coli Carrying mcr-1-Plasmids Recovered From Food Animals From Argentina
    Article Snippet: Isolates were also genotyped by multilocus sequence typing (MLST). .. Plasmids were extracted from mcr- transconjugants strains using the Qiagen Large-Construct kit (Qiagen) and sequenced using Illumina's MiSeq system.

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: Paragraph title: BAC library Screening and BAC-clone sequencing ... Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany).

    Selection:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: BAC elements were engineered into the D2 genome at the thymidine kinase (TK) (UL 23) locus by homologous recombination in 7b cells and selection for ganciclovir resistance. .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Gel Extraction:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The sonicated DNA was size fractioned by agarose gel electrophoresis and 2-5 kb fragments were purified using the QIAquick Gel Extraction Kit (Qiagen, Mississauga, Ont.

    cDNA Library Assay:

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: BAC library production The pENTR-based cDNA library derived from a mixture of NGF-differentiated and undifferentiated PC12 cells was previously described. .. TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen).

    Shotgun Sequencing:

    Article Title: The Genomic Architecture of Novel Simulium damnosum Wolbachia Prophage Sequence Elements and Implications for Onchocerciasis Epidemiology
    Article Snippet: Paragraph title: Shotgun sequencing of the genomic DNA regions flanking the w Dam cell-division protein FtsZ ... Thick mini-culture preparations from each BAC colony were pooled and their BAC DNA was isolated in single preparations using a QIAGEN large-construct kit and protocol ( https://www.qiagen.com/kr/resources/resourcedetail?id=8f67b644-6d21-4ef3-b33e-a60f32623785 & lang=en ).

    Mouse Assay:

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: To generate peri lacZ knock-in mice, we screened a 129SvJ mouse embryonic stem (ES) cell genomic DNA library (Genome System Inc., St. Louis, MO) by PCR and identified a bacterial artificial chromosome (BAC) clone that contains the genomic DNA that codes for the periostin gene. .. BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: Paragraph title: Generation of transgenic mice ... In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ].

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: Tlr2 Cre and Tlr2 CreERT2 transgenic mice were generated by inserting Cre and Cre ERT2 cassettes, respectively, under the control of the Tlr2 promoter by BAC recombineering . .. BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis.

    In Situ Hybridization:

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: Paragraph title: DNA probe preparation and fluorescence in situ hybridization (FISH) ... DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit.

    Plasmid Preparation:

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. The library was recombined with the TTA BAC Gateway cassette by incubation of 2 μg of BAC DNA and 200 ng of library plasmid DNA with clonase enzyme for 4 hours.

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: Paragraph title: Plasmid constructs and transgenic plants ... The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region.

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone. .. A replacement-targeting vector was constructed where the bacterial β-galactosidase gene was knocked into the periostin gene locus.

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: Paragraph title: Plasmids and vector construction. ... The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Article Title: Characterization of Escherichia coli Carrying mcr-1-Plasmids Recovered From Food Animals From Argentina
    Article Snippet: Plasmid profile of the isolates was analyzed by S1-PFGE. .. Plasmids were extracted from mcr- transconjugants strains using the Qiagen Large-Construct kit (Qiagen) and sequenced using Illumina's MiSeq system.

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany). .. DNA fragments were end-polished with Klenow enzyme (Roche, Basel, Switzerland) and blunt-end ligated into pUC18 vector (Roche).

    Software:

    Article Title: The Genomic Architecture of Novel Simulium damnosum Wolbachia Prophage Sequence Elements and Implications for Onchocerciasis Epidemiology
    Article Snippet: Thick mini-culture preparations from each BAC colony were pooled and their BAC DNA was isolated in single preparations using a QIAGEN large-construct kit and protocol ( https://www.qiagen.com/kr/resources/resourcedetail?id=8f67b644-6d21-4ef3-b33e-a60f32623785 & lang=en ). .. Sequence reads were quality-checked using Phred Software ( http://www.phrap.org/phredphrapconsed.html ) and assembled into 8,238 contigs using Phrap (Ewing and Green, ; Ewing et al., ).

    Article Title: Characterization of Escherichia coli Carrying mcr-1-Plasmids Recovered From Food Animals From Argentina
    Article Snippet: Plasmids were extracted from mcr- transconjugants strains using the Qiagen Large-Construct kit (Qiagen) and sequenced using Illumina's MiSeq system. .. The obtained reads were assembled using CLC Genomics Workbench software (CLCbio, Qiagen), annotated using RAST server ( http://rast.nmpdr.org/rast.cgi ) and the sequences (gaps were not filled) compared in a pairwise fashion using BRIG (Alikhan et al., ).

    Recombinant:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: Paragraph title: Construction of recombinant adenoviruses ... The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: The genome of a purified D2/BAC recombinant was circularized by infecting U2OS cells at a multiplicity of infection (MOI) of 5 for 3 h, and DNA was isolated by proteinase K digestion, phenol-chloroform extraction using PhaseLock gel (5Prime, Gaithersburg, MD), and isopropanol precipitation. .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Agarose Gel Electrophoresis:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The sonicated DNA was size fractioned by agarose gel electrophoresis and 2-5 kb fragments were purified using the QIAquick Gel Extraction Kit (Qiagen, Mississauga, Ont.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Restriction solution was separated in low-melt agarose gel (Fermentas, Lithuania) using CHEF-DR II Pulsed Field Electrophoresis System (Bio-Rad, USA).

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining.

    Transgenic Assay:

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: Paragraph title: Plasmid constructs and transgenic plants ... The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: Paragraph title: Generation of transgenic mice ... In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ].

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: Tlr2 Cre and Tlr2 CreERT2 transgenic mice were generated by inserting Cre and Cre ERT2 cassettes, respectively, under the control of the Tlr2 promoter by BAC recombineering . .. BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis.

    Next-Generation Sequencing:

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1
    Article Snippet: .. Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen). .. Viruses were generated from AlHV-1 strain C500 BAC clones in MacT-Cre cells, grown in BT cells, and purified from the supernatant by ultracentrifugation (100,000 × g ) through a 30% (w/v) sucrose cushion for 2 h at 4 °C.

    Chromosome Walking:

    Article Title: Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group
    Article Snippet: The physical map was assembled by chromosome walking, starting from the closest proximal marker CJ641478 and from the completely linked marker EX24785 in both directions. .. DNAs were extracted from selected BACs using a large-construct kit (Qiagen) and sequenced with Illumina at the University of California (UC) Davis Genomic Center.

    Produced:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK). .. Following PacI digestion to linearize the adenoviral DNA, recombinant adenoviruses then were produced by transfecting DNA into HEK293 cells by calcium phosphate precipitation.

    Alkaline Lysis:

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    DNA Purification:

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
    Article Snippet: .. The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK). .. Following PacI digestion to linearize the adenoviral DNA, recombinant adenoviruses then were produced by transfecting DNA into HEK293 cells by calcium phosphate precipitation.

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion. .. The Gateway cassette from Gateway conversion plasmid A (Invitrogen) was modified for this work in the following manner.

    Marker:

    Article Title: Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group
    Article Snippet: Paragraph title: Marker Development and BAC Library Screening and Sequencing. ... DNAs were extracted from selected BACs using a large-construct kit (Qiagen) and sequenced with Illumina at the University of California (UC) Davis Genomic Center.

    Staining:

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining.

    Fluorescence In Situ Hybridization:

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: Paragraph title: DNA probe preparation and fluorescence in situ hybridization (FISH) ... DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit.

    BAC Assay:

    Article Title: Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire
    Article Snippet: .. Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. .. The kit includes an exonuclease digestion step to eliminate E. coli genomic DNA.

    Article Title: The Genomic Architecture of Novel Simulium damnosum Wolbachia Prophage Sequence Elements and Implications for Onchocerciasis Epidemiology
    Article Snippet: .. Thick mini-culture preparations from each BAC colony were pooled and their BAC DNA was isolated in single preparations using a QIAGEN large-construct kit and protocol ( https://www.qiagen.com/kr/resources/resourcedetail?id=8f67b644-6d21-4ef3-b33e-a60f32623785 & lang=en ). .. A 10-mg sample of purified BAC DNA was shot-gun sequenced commercially using a Roche 454 FLX system sequencer at the Cambridge University Biochemistry Department.

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. TTA BAC DNA was purified using the Qiagen Large Construct Kit (Qiagen). .. The library was recombined with the TTA BAC Gateway cassette by incubation of 2 μg of BAC DNA and 200 ng of library plasmid DNA with clonase enzyme for 4 hours.

    Article Title: Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings, et al. Genome‐wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings
    Article Snippet: .. The BAC plasmids were isolated using QIAGEN Large‐Construct Kit, and then used as the templates to amplify the CLF and SWN genomic sequence including the promoter/regulatory region. .. The PCR products were purified using the GenepHlow Gel/PCR Kit, and were then subcloned into the binary vector pMDC107 between the Pme I and Asc I sites to obtain pMDC107‐gCLF and pMDC107‐gSWN .

    Article Title: periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype
    Article Snippet: .. BAC DNA was isolated using a large-construct purification kit (QIAGEN Inc., Valencia, CA), and Southern blots were used to confirm the identity and integrity of the clone. .. A replacement-targeting vector was constructed where the bacterial β-galactosidase gene was knocked into the periostin gene locus.

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion. .. The Gateway cassette from Gateway conversion plasmid A (Invitrogen) was modified for this work in the following manner.

    Article Title: Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group
    Article Snippet: Paragraph title: Marker Development and BAC Library Screening and Sequencing. ... DNAs were extracted from selected BACs using a large-construct kit (Qiagen) and sequenced with Illumina at the University of California (UC) Davis Genomic Center.

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1
    Article Snippet: .. Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen). .. Viruses were generated from AlHV-1 strain C500 BAC clones in MacT-Cre cells, grown in BT cells, and purified from the supernatant by ultracentrifugation (100,000 × g ) through a 30% (w/v) sucrose cushion for 2 h at 4 °C.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ]. .. Five days after transfection EGFP expression and distribution in COS-7 cells was visualized using fluorescence microscopy (Eclipse 80i upright microscope, Nikon). hEGFP-BDNF BAC DNA was purified for microinjection by alkaline lysis and linearized with PI-SceI enzyme (NEB, USA).

    Article Title: Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum
    Article Snippet: .. DNA was isolated from tyr BAC clones (AMMCBa_160B14, AMMCBa_31L9) using a Qiagen Large Construct kit. .. BAC-DNA was prepared for in situ hybridization by nick-translation using direct fluorophores Cyanine 3-dUTP (Enzo Life Sciences, ENZ-42501) or Fluorescein-12-dUTP (Thermo Scientific, R0101) .

    Article Title: An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain
    Article Snippet: .. En masse bacterial amplification was performed by overnight incubation at 30°C under shaking in 500 ml LB supplemented with 15 μg/ml of chloramphenicol and 30 μg/ml of ampicillin, with BAC DNA purified using the Qiagen Large Construct Kit. .. PCR across the GW cassette to assess the diversity of cDNAs recombined into TTA BAC was performed with Taq DNA polymerase and primers CMV-F1 and bGH-R1 ( ) using 100 ng BAC DNA, 0.2 μmol/l of each primer, and 0.2 mmol/l of each dNTP (Promega, Madison, WI) at the following cycling conditions: 94°C for 3 minutes; 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 3 minutes; and 72°C for 10 minutes.

    Article Title: Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position
    Article Snippet: .. Large-scale BAC clone preparation was obtained with the Large Construct Kit (Qiagen, Hilden, Germany). .. A total of 20 μg of BAC clone DNA in 500 μl TE (10 mM Tris, 1 mM EDTA), pH 8.0, was fragmented by sonication (Branson Sonifier, Danbury, CT, USA) with 4 × 1-second pulses at 300 W (5-mm microtip).

    Homologous Recombination:

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿
    Article Snippet: BAC elements were engineered into the D2 genome at the thymidine kinase (TK) (UL 23) locus by homologous recombination in 7b cells and selection for ganciclovir resistance. .. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Article Title: Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice
    Article Snippet: Red® /ET® homologous recombination in E. coli (Counter-Selection BAC Modification Kit, Gene Bridges GmbH, Germany) was used to delete BDNF stop codon and to insert EGFP reporter gene with the linker sequence (CGG GAT CCA CCG GTC GCC ACC) into the 3' end of BDNF. .. In order to validate the reporter activity, BAC DNA was purified using the Large Construct Purification Kit (Qiagen, USA) and transfected into COS-7 cells using DEAE-dextran mediated transfection system [ ].

    Article Title: Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors
    Article Snippet: By homologous recombination, the open-reading frame of Tlr2 exon 3 was substituted with Cre and Cre ERT2 cDNA, respectively. .. BAC DNA for pronuclear injection was prepared using the QIAGEN Large-Construct Kit and analyzed by pulsed-field gel electrophoresis.

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    Qiagen large construct dna purification kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Large Construct Dna Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Transfection, Staining, Confocal Microscopy

    Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Mutagenesis, Transduction, Staining, Confocal Microscopy

    Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Construct, Amplification, Polymerase Chain Reaction, FLAG-tag, Synthesized, Agarose Gel Electrophoresis, Transfection, Stable Transfection

    Presence of a duplicated region in parental strain C500 and the BAC clone. ( A ) Diagram of C500DT showing the duplicated region from LUR containing a part of ORF48, ORF50 and A6 in their entirety and a part of A7, inserted into a copy of TR. Nucleotide positions are indicated. ( B ) Southern blot of SacI-digested DNA showing the presence of the duplicated region in the BAC clone and the parental virus. The ORF50 probe was produced by PCR using primers C500-1 and C500-2 24 . EtBr indicates ethidium bromide-stained lanes prior to blotting. The entire lanes analysed by Southern blotting are shown.

    Journal: Scientific Reports

    Article Title: Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1

    doi: 10.1038/srep38607

    Figure Lengend Snippet: Presence of a duplicated region in parental strain C500 and the BAC clone. ( A ) Diagram of C500DT showing the duplicated region from LUR containing a part of ORF48, ORF50 and A6 in their entirety and a part of A7, inserted into a copy of TR. Nucleotide positions are indicated. ( B ) Southern blot of SacI-digested DNA showing the presence of the duplicated region in the BAC clone and the parental virus. The ORF50 probe was produced by PCR using primers C500-1 and C500-2 24 . EtBr indicates ethidium bromide-stained lanes prior to blotting. The entire lanes analysed by Southern blotting are shown.

    Article Snippet: Next-generation sequencing BAC clone DNA was purified by using the Large Construct DNA prep kit (Qiagen).

    Techniques: BAC Assay, Southern Blot, Produced, Polymerase Chain Reaction, Staining

    PCR amplification of DBAC-L cDNA inserts demonstrates library diversity. (A) cDNA inserts were amplified from DBAC-L genomic DNA, purified from E. coli , using primers specific for the HCMV promoter and SV40 poly(A) sequences. Extension times for PCRs varied from 1 min (lanes 1 to 4) to 5 min (lanes 6 to 9). The reaction products were separated on a 1% agarose gel, revealing cDNAs of various sizes. Lane 11, size markers; arrows indicate approximate fragment sizes in kilobases (kb). (B) Distinct cDNAs were amplified from total DNA harvested from 2 × 10 6 7b cells infected with the DBAC-L vector stock at an MOI of 0.05. Viral and cellular DNA was harvested at 24 hpi and eluted in 250 μl TE. Serial 10-fold dilutions of total DNA (lanes 1 to 5) were used as template DNA for PCR, such that one microliter (lane 1) represented approximately 400 input PFU. Lane 6, water control; lane 7, size markers. (C) Ten individual PCRs each using 10 −4 μl DBAC-L viral DNA demonstrated the presence of unique cDNA insert sizes.

    Journal: Journal of Virology

    Article Title: A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries ▿

    doi: 10.1128/JVI.02388-09

    Figure Lengend Snippet: PCR amplification of DBAC-L cDNA inserts demonstrates library diversity. (A) cDNA inserts were amplified from DBAC-L genomic DNA, purified from E. coli , using primers specific for the HCMV promoter and SV40 poly(A) sequences. Extension times for PCRs varied from 1 min (lanes 1 to 4) to 5 min (lanes 6 to 9). The reaction products were separated on a 1% agarose gel, revealing cDNAs of various sizes. Lane 11, size markers; arrows indicate approximate fragment sizes in kilobases (kb). (B) Distinct cDNAs were amplified from total DNA harvested from 2 × 10 6 7b cells infected with the DBAC-L vector stock at an MOI of 0.05. Viral and cellular DNA was harvested at 24 hpi and eluted in 250 μl TE. Serial 10-fold dilutions of total DNA (lanes 1 to 5) were used as template DNA for PCR, such that one microliter (lane 1) represented approximately 400 input PFU. Lane 6, water control; lane 7, size markers. (C) Ten individual PCRs each using 10 −4 μl DBAC-L viral DNA demonstrated the presence of unique cDNA insert sizes.

    Article Snippet: The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.

    Techniques: Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Infection, Plasmid Preparation