rapid dna ligation kit  (Thermo Fisher)


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    Name:
    Rapid DNA Ligation Kit
    Description:
    Thermo Scientific Rapid DNA Ligation Kit enables fast sticky end or blunt end DNA ligation in only 5 minutes at room temperature The kit contains T4 DNA ligase and a specially formulated 5X rapid ligation buffer optimized for fast and efficient DNA ligation Fast ligation efficiency is equal to that obtained with T4 DNA ligase in a standard 1 hour ligation The ligation reaction mixture can be used directly for bacterial transformation with the TransformAid Bacterial Transformation Kit or with other conventional transformation procedures Highlights• Fast ligation of either sticky or blunt end DNA in only 5 minutes• Convenient reaction mixture can be used directly for bacterial transformationApplications• Routine cloning experiments• Blunt end cloning• Library construction• TA cloningIncludes• T4 DNA Ligase• 5X Rapid Ligation Buffer• Water nuclease free• Detailed ProtocolNotePrior to electroporation it is necessary to inactivate T4 DNA ligase by chloroform extraction or spin column purification e g with Thermo Scientific GeneJET PCR Purification Kit K0701 Related ProductsRapid DNA Ligation Kit
    Catalog Number:
    k1422
    Price:
    None
    Applications:
    Cloning|PCR Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher rapid dna ligation kit
    SCIPay design permits CRISPR-guided integration of specific donor <t>DNA</t> sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a <t>BFP</t> transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    Thermo Scientific Rapid DNA Ligation Kit enables fast sticky end or blunt end DNA ligation in only 5 minutes at room temperature The kit contains T4 DNA ligase and a specially formulated 5X rapid ligation buffer optimized for fast and efficient DNA ligation Fast ligation efficiency is equal to that obtained with T4 DNA ligase in a standard 1 hour ligation The ligation reaction mixture can be used directly for bacterial transformation with the TransformAid Bacterial Transformation Kit or with other conventional transformation procedures Highlights• Fast ligation of either sticky or blunt end DNA in only 5 minutes• Convenient reaction mixture can be used directly for bacterial transformationApplications• Routine cloning experiments• Blunt end cloning• Library construction• TA cloningIncludes• T4 DNA Ligase• 5X Rapid Ligation Buffer• Water nuclease free• Detailed ProtocolNotePrior to electroporation it is necessary to inactivate T4 DNA ligase by chloroform extraction or spin column purification e g with Thermo Scientific GeneJET PCR Purification Kit K0701 Related ProductsRapid DNA Ligation Kit
    https://www.bioz.com/result/rapid dna ligation kit/product/Thermo Fisher
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    rapid dna ligation kit - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering"

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering

    Journal: bioRxiv

    doi: 10.1101/2020.03.25.008276

    SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    Figure Legend Snippet: SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p

    Techniques Used: CRISPR, Sequencing, Knock-Out, Plasmid Preparation, Flow Cytometry

    2) Product Images from "Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients"

    Article Title: Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107363

    Generation of pUbC-FLuc-ROSA αPIG-iPSC. A. Genomic DNA was isolated from indicated cell lines, digested with Eco RI and hybridized with a probe for ROSA26 locus in a Southern blot analysis. Expected size of the wild type allele is 15630 bp and of successfully targeted allele 4066 bp because the integration of FLuc cassette results in an additional Eco RI restriction site. Presence of both bands in transgenic clones indicates heterozygous integration. B. PCR amplification of genomic DNA isolated from indicated cell lines and donor plasmid to verify the site specific integration of FLuc-expression cassette into the ROSA26 locus with an expected PCR product size of 950 kb. C. Representative brightfield and fluorescent images of puromycin-purified day 16 pUbC-FLuc-ROSA αPIG-iPSC-derived EGFP positive cardiac clusters. Scale bar = 500 µm. D. Relative luminescence units (RLU) per µg protein in the two clones (C3 and C5) of undifferentiated pUbC-FLuc-ROSA αPIG-iPSC and CM derived from them on day 16 of differentiation. Values are from triplicate measurements of three independent differentiations. Statistics: two-tailed paired Student's t-test. **p
    Figure Legend Snippet: Generation of pUbC-FLuc-ROSA αPIG-iPSC. A. Genomic DNA was isolated from indicated cell lines, digested with Eco RI and hybridized with a probe for ROSA26 locus in a Southern blot analysis. Expected size of the wild type allele is 15630 bp and of successfully targeted allele 4066 bp because the integration of FLuc cassette results in an additional Eco RI restriction site. Presence of both bands in transgenic clones indicates heterozygous integration. B. PCR amplification of genomic DNA isolated from indicated cell lines and donor plasmid to verify the site specific integration of FLuc-expression cassette into the ROSA26 locus with an expected PCR product size of 950 kb. C. Representative brightfield and fluorescent images of puromycin-purified day 16 pUbC-FLuc-ROSA αPIG-iPSC-derived EGFP positive cardiac clusters. Scale bar = 500 µm. D. Relative luminescence units (RLU) per µg protein in the two clones (C3 and C5) of undifferentiated pUbC-FLuc-ROSA αPIG-iPSC and CM derived from them on day 16 of differentiation. Values are from triplicate measurements of three independent differentiations. Statistics: two-tailed paired Student's t-test. **p

    Techniques Used: Isolation, Southern Blot, Transgenic Assay, Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Expressing, Purification, Derivative Assay, Two Tailed Test

    3) Product Images from "A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors"

    Article Title: A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-9-81

    Identification of the Tol2 construct genomic insertion sites on the T2EC clone #1 . A : Splinkerette PCR products on 1.5% agar gel. 1: Fermentas DNA Ladder Mix; 2: genomic DNA from non-transfected T2EC; 3: pT2.CMV-hKO plasmid; 4: genomic DNA from T2EC clone #1, cotransfected with the pT2.CMV-hKO plasmid and the transposase plasmid, pCAGGS-T2TP (molecular ratio: 5/1). B : Alignment of the splinkerette PCR product with the Gallus gallus genome. The sequence corresponding to the Tol2 construct is shown in bold . C : Localization of the Tol2 construct insertion site into chromosome 4 of the Gallus gallus genome.
    Figure Legend Snippet: Identification of the Tol2 construct genomic insertion sites on the T2EC clone #1 . A : Splinkerette PCR products on 1.5% agar gel. 1: Fermentas DNA Ladder Mix; 2: genomic DNA from non-transfected T2EC; 3: pT2.CMV-hKO plasmid; 4: genomic DNA from T2EC clone #1, cotransfected with the pT2.CMV-hKO plasmid and the transposase plasmid, pCAGGS-T2TP (molecular ratio: 5/1). B : Alignment of the splinkerette PCR product with the Gallus gallus genome. The sequence corresponding to the Tol2 construct is shown in bold . C : Localization of the Tol2 construct insertion site into chromosome 4 of the Gallus gallus genome.

    Techniques Used: Construct, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Sequencing

    4) Product Images from "FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System"

    Article Title: FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059034

    Transcription of the master operon flhDC . ( A ) To verify the transcription of the plasmid-encoded master operon flhDC Reverse Transcriptase PCR Reaction of the isolated RNA was performed. It was assumed that within the overnight culture cells exhibiting flagellar assembly were found and therefore this culture was used as a positive control. Cells before addition of the inducer IPTG were used as reference compared to cells after induction of the plasmid-encoded flhDC . To evaluate genomic DNA contamination the isolated RNA samples were also subjected to subsequent PCR. Plasmid-encoded flhDC was used as a positive control for the PCR reaction. Fermentas 1 KB GeneRuler, ON overnight culture sample, -I whole cell sample without induction of plasmid-encoded flhDC , +I whole cell samples with induction of plasmid-encoded flhDC , RT Reverse Transcriptase Reaction, + plasmid-encoded flhDC ; ( B ) Scheme of the plasmid-encoded master operon under the control of the lacUV5 promoter
    Figure Legend Snippet: Transcription of the master operon flhDC . ( A ) To verify the transcription of the plasmid-encoded master operon flhDC Reverse Transcriptase PCR Reaction of the isolated RNA was performed. It was assumed that within the overnight culture cells exhibiting flagellar assembly were found and therefore this culture was used as a positive control. Cells before addition of the inducer IPTG were used as reference compared to cells after induction of the plasmid-encoded flhDC . To evaluate genomic DNA contamination the isolated RNA samples were also subjected to subsequent PCR. Plasmid-encoded flhDC was used as a positive control for the PCR reaction. Fermentas 1 KB GeneRuler, ON overnight culture sample, -I whole cell sample without induction of plasmid-encoded flhDC , +I whole cell samples with induction of plasmid-encoded flhDC , RT Reverse Transcriptase Reaction, + plasmid-encoded flhDC ; ( B ) Scheme of the plasmid-encoded master operon under the control of the lacUV5 promoter

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Isolation, Positive Control

    5) Product Images from "Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)"

    Article Title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0254-0

    High fGly conversion yields can be obtained from stably transduced cell lines under high titer cell culture conditions. Stable transduction of DNA encoding FGE, antibody light chain, and antibody heavy chain bearing one (CT only) or two (C H 1 and CT) aldehyde tags was performed with viral retrovector transduction into CHO cells (GPEx® technology). The resulting stable pools were cultured in three types of media +/- supplementation with copper(II) sulfate ( n = 3). Titers ( a ) and fGly conversion ( b ) of CT-tagged antibodies were assessed. Then, the stable pools were cloned by limiting dilution and clone performance was assessed in fed batch cultures ( c and d ; shake flask, n = 5 clones; bioreactor, n = 3 clones single tag, n = 1 clone double tag). The top performing stable single-tagged clonal cell line produced antibody in very high titer (5.2 g/L) with 98 % conversion of Cys to fGly ( c and d ). The specific productivity (75 pg/cell/d) of this top clone demonstrates the capabilities of the GPEx® technology for efficient production of highly converted antibody. The generation of fGly in single- and doubly-tagged antibodies scaled successfully to bioreactors (2 L cultures; c and d ). Error bars indicate standard deviation
    Figure Legend Snippet: High fGly conversion yields can be obtained from stably transduced cell lines under high titer cell culture conditions. Stable transduction of DNA encoding FGE, antibody light chain, and antibody heavy chain bearing one (CT only) or two (C H 1 and CT) aldehyde tags was performed with viral retrovector transduction into CHO cells (GPEx® technology). The resulting stable pools were cultured in three types of media +/- supplementation with copper(II) sulfate ( n = 3). Titers ( a ) and fGly conversion ( b ) of CT-tagged antibodies were assessed. Then, the stable pools were cloned by limiting dilution and clone performance was assessed in fed batch cultures ( c and d ; shake flask, n = 5 clones; bioreactor, n = 3 clones single tag, n = 1 clone double tag). The top performing stable single-tagged clonal cell line produced antibody in very high titer (5.2 g/L) with 98 % conversion of Cys to fGly ( c and d ). The specific productivity (75 pg/cell/d) of this top clone demonstrates the capabilities of the GPEx® technology for efficient production of highly converted antibody. The generation of fGly in single- and doubly-tagged antibodies scaled successfully to bioreactors (2 L cultures; c and d ). Error bars indicate standard deviation

    Techniques Used: Stable Transfection, Cell Culture, Transduction, Clone Assay, Produced, Standard Deviation

    Related Articles

    Clone Assay:

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    Article Snippet: .. The TOPO PCR cloning vector and DNA ligation kit were obtained from Invitrogen (Carlsbad, CA). .. The normal goat serum, the streptavidin-peroxidase, the streptavidin-phosphatase, and the diaminobenzidine reagent set were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

    Article Title: 5-Lipoxygenase and Leukotriene B4 Receptor Are Expressed in Human Pancreatic Cancers But Not in Pancreatic Ducts in Normal Tissue
    Article Snippet: .. The TOPO PCR cloning vector and DNA ligation kit were obtained from Invitrogen (Carlsbad, CA). .. The normal goat serum, the streptavidin-peroxidase, the streptavidin-phosphatase, the diaminobenzidine reagent set, and the Histo-Mark-Red AP-system were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering
    Article Snippet: .. This modular plasmid allows insertion of gRNA-coding DNA oligonucleotides using BpiI as outlined above and subsequent cloning of HA- and bait-coding DNA fragments engineered to be in-frame with the preceding genome sequence and following P2A-BFP sequence with BsaI using Gibson or restriction/ligation cloning. .. SCIPay-human-CD69-tdTomato and SCIPay-human-TRAC-CAR were generated by first cloning gRNA-coding DNA oligonucleotides as described above and subsequently inserting DNA fragments (Twist Bioscience) designed in silico with gRNA-specific bait sequences into pQCi using Gibson assembly.

    Amplification:

    Article Title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)
    Article Snippet: .. Both the FGE vector and the EF1α PCR product were digested with NruI and NheI and ligated with the Rapid DNA Ligation Kit (Thermo/Life Technologies) to generate WT pEF-FGE-hygro, which was amplified in XL-10 gold cells (Agilent) and purified according to the manufacturer’s instructions. .. Quikchange Mutagenesis (Agilent) was used to mutate the furin cleavage site (HRYSRE to HRYSGE) and/or add the KDEL retention signal to the C-terminus of FGE to generate Δfurin, +KDEL, and Δfurin + KDEL versions of FGE.

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    Article Snippet: .. UbC promoter (pUbC) was amplified from plasmid pLenti6/UbC/V5-DEST (Invitrogen, Life Technologies, Carlsbad, CA) with Dream Taq PCR Master Mix (Fermentas, Thermo Scientific, Waltham, MA) using primers flanked by restriction sites for Xho I and Hind III (Fermentas). pGL4.14 [luc2 /Hygro] plasmid and the PCR-product were digested with both enzymes, purified with NucleoSpin Gel and PCR Clean-up-Kit (Macherey-Nagel, Düren, Germany) and ligated overnight at 4°C with Rapid DNA Ligation Kit (Fermentas) to yield the pGL4.14-pUbC [luc2 /Hygro] vector. .. The PGK promoter was removed from the pPPGKIP vector by digestion with Sal I and Hind III and subcloned between the XhoI and HindIII cutting sites of the pGL4.14 [luc2 /Hygro] plasmid to obtain pGL4.14-pPGK [luc2 /Hygro] vector.

    DNA Ligation:

    Article Title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)
    Article Snippet: .. Both the FGE vector and the EF1α PCR product were digested with NruI and NheI and ligated with the Rapid DNA Ligation Kit (Thermo/Life Technologies) to generate WT pEF-FGE-hygro, which was amplified in XL-10 gold cells (Agilent) and purified according to the manufacturer’s instructions. .. Quikchange Mutagenesis (Agilent) was used to mutate the furin cleavage site (HRYSRE to HRYSGE) and/or add the KDEL retention signal to the C-terminus of FGE to generate Δfurin, +KDEL, and Δfurin + KDEL versions of FGE.

    Article Title: Differential Expression of Thromboxane Synthase in Prostate Carcinoma
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    Article Title: A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors
    Article Snippet: .. Considering that a 4 bp recognition site will occur roughly every 256 bp, and therefore that 200 ng of Tai I-digested gDNA correspond to 1.28 pmol DNA, 200 ng of digested DNA were ligated to a 3× molar excess of the annealed splinkerette adaptor (Splinklong-ACGT+Splinkshort-hairpin) with the T4 DNA ligase (4 U), for 1 hour at 22°C, using the Fermentas Rapid DNA Ligation kit. .. In order to improve the ligation yield, digested DNA was heated for 5 min at 60°C before being ligated.

    Article Title: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress
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    Article Title: Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients
    Article Snippet: .. UbC promoter (pUbC) was amplified from plasmid pLenti6/UbC/V5-DEST (Invitrogen, Life Technologies, Carlsbad, CA) with Dream Taq PCR Master Mix (Fermentas, Thermo Scientific, Waltham, MA) using primers flanked by restriction sites for Xho I and Hind III (Fermentas). pGL4.14 [luc2 /Hygro] plasmid and the PCR-product were digested with both enzymes, purified with NucleoSpin Gel and PCR Clean-up-Kit (Macherey-Nagel, Düren, Germany) and ligated overnight at 4°C with Rapid DNA Ligation Kit (Fermentas) to yield the pGL4.14-pUbC [luc2 /Hygro] vector. .. The PGK promoter was removed from the pPPGKIP vector by digestion with Sal I and Hind III and subcloned between the XhoI and HindIII cutting sites of the pGL4.14 [luc2 /Hygro] plasmid to obtain pGL4.14-pPGK [luc2 /Hygro] vector.

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    Article Title: FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System
    Article Snippet: .. Rapid DNA ligation kit, Pfu DNA polymerase and 10× MgSO4 -PCR buffer were obtained from Fermentas. .. Tris-Glycine gels were purchased from Invitrogen and Biorad.

    Purification:

    Article Title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)
    Article Snippet: .. Both the FGE vector and the EF1α PCR product were digested with NruI and NheI and ligated with the Rapid DNA Ligation Kit (Thermo/Life Technologies) to generate WT pEF-FGE-hygro, which was amplified in XL-10 gold cells (Agilent) and purified according to the manufacturer’s instructions. .. Quikchange Mutagenesis (Agilent) was used to mutate the furin cleavage site (HRYSRE to HRYSGE) and/or add the KDEL retention signal to the C-terminus of FGE to generate Δfurin, +KDEL, and Δfurin + KDEL versions of FGE.

    Article Title: Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients
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    Polymerase Chain Reaction:

    Article Title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)
    Article Snippet: .. Both the FGE vector and the EF1α PCR product were digested with NruI and NheI and ligated with the Rapid DNA Ligation Kit (Thermo/Life Technologies) to generate WT pEF-FGE-hygro, which was amplified in XL-10 gold cells (Agilent) and purified according to the manufacturer’s instructions. .. Quikchange Mutagenesis (Agilent) was used to mutate the furin cleavage site (HRYSRE to HRYSGE) and/or add the KDEL retention signal to the C-terminus of FGE to generate Δfurin, +KDEL, and Δfurin + KDEL versions of FGE.

    Article Title: Differential Expression of Thromboxane Synthase in Prostate Carcinoma
    Article Snippet: .. The TOPO PCR cloning vector and DNA ligation kit were obtained from Invitrogen (Carlsbad, CA). .. The normal goat serum, the streptavidin-peroxidase, the streptavidin-phosphatase, and the diaminobenzidine reagent set were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

    Article Title: Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients
    Article Snippet: .. UbC promoter (pUbC) was amplified from plasmid pLenti6/UbC/V5-DEST (Invitrogen, Life Technologies, Carlsbad, CA) with Dream Taq PCR Master Mix (Fermentas, Thermo Scientific, Waltham, MA) using primers flanked by restriction sites for Xho I and Hind III (Fermentas). pGL4.14 [luc2 /Hygro] plasmid and the PCR-product were digested with both enzymes, purified with NucleoSpin Gel and PCR Clean-up-Kit (Macherey-Nagel, Düren, Germany) and ligated overnight at 4°C with Rapid DNA Ligation Kit (Fermentas) to yield the pGL4.14-pUbC [luc2 /Hygro] vector. .. The PGK promoter was removed from the pPPGKIP vector by digestion with Sal I and Hind III and subcloned between the XhoI and HindIII cutting sites of the pGL4.14 [luc2 /Hygro] plasmid to obtain pGL4.14-pPGK [luc2 /Hygro] vector.

    Article Title: 5-Lipoxygenase and Leukotriene B4 Receptor Are Expressed in Human Pancreatic Cancers But Not in Pancreatic Ducts in Normal Tissue
    Article Snippet: .. The TOPO PCR cloning vector and DNA ligation kit were obtained from Invitrogen (Carlsbad, CA). .. The normal goat serum, the streptavidin-peroxidase, the streptavidin-phosphatase, the diaminobenzidine reagent set, and the Histo-Mark-Red AP-system were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

    Article Title: FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System
    Article Snippet: .. Rapid DNA ligation kit, Pfu DNA polymerase and 10× MgSO4 -PCR buffer were obtained from Fermentas. .. Tris-Glycine gels were purchased from Invitrogen and Biorad.

    Sequencing:

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering
    Article Snippet: .. This modular plasmid allows insertion of gRNA-coding DNA oligonucleotides using BpiI as outlined above and subsequent cloning of HA- and bait-coding DNA fragments engineered to be in-frame with the preceding genome sequence and following P2A-BFP sequence with BsaI using Gibson or restriction/ligation cloning. .. SCIPay-human-CD69-tdTomato and SCIPay-human-TRAC-CAR were generated by first cloning gRNA-coding DNA oligonucleotides as described above and subsequently inserting DNA fragments (Twist Bioscience) designed in silico with gRNA-specific bait sequences into pQCi using Gibson assembly.

    Plasmid Preparation:

    Article Title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)
    Article Snippet: .. Both the FGE vector and the EF1α PCR product were digested with NruI and NheI and ligated with the Rapid DNA Ligation Kit (Thermo/Life Technologies) to generate WT pEF-FGE-hygro, which was amplified in XL-10 gold cells (Agilent) and purified according to the manufacturer’s instructions. .. Quikchange Mutagenesis (Agilent) was used to mutate the furin cleavage site (HRYSRE to HRYSGE) and/or add the KDEL retention signal to the C-terminus of FGE to generate Δfurin, +KDEL, and Δfurin + KDEL versions of FGE.

    Article Title: Differential Expression of Thromboxane Synthase in Prostate Carcinoma
    Article Snippet: .. The TOPO PCR cloning vector and DNA ligation kit were obtained from Invitrogen (Carlsbad, CA). .. The normal goat serum, the streptavidin-peroxidase, the streptavidin-phosphatase, and the diaminobenzidine reagent set were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

    Article Title: Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients
    Article Snippet: .. UbC promoter (pUbC) was amplified from plasmid pLenti6/UbC/V5-DEST (Invitrogen, Life Technologies, Carlsbad, CA) with Dream Taq PCR Master Mix (Fermentas, Thermo Scientific, Waltham, MA) using primers flanked by restriction sites for Xho I and Hind III (Fermentas). pGL4.14 [luc2 /Hygro] plasmid and the PCR-product were digested with both enzymes, purified with NucleoSpin Gel and PCR Clean-up-Kit (Macherey-Nagel, Düren, Germany) and ligated overnight at 4°C with Rapid DNA Ligation Kit (Fermentas) to yield the pGL4.14-pUbC [luc2 /Hygro] vector. .. The PGK promoter was removed from the pPPGKIP vector by digestion with Sal I and Hind III and subcloned between the XhoI and HindIII cutting sites of the pGL4.14 [luc2 /Hygro] plasmid to obtain pGL4.14-pPGK [luc2 /Hygro] vector.

    Article Title: 5-Lipoxygenase and Leukotriene B4 Receptor Are Expressed in Human Pancreatic Cancers But Not in Pancreatic Ducts in Normal Tissue
    Article Snippet: .. The TOPO PCR cloning vector and DNA ligation kit were obtained from Invitrogen (Carlsbad, CA). .. The normal goat serum, the streptavidin-peroxidase, the streptavidin-phosphatase, the diaminobenzidine reagent set, and the Histo-Mark-Red AP-system were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering
    Article Snippet: .. This modular plasmid allows insertion of gRNA-coding DNA oligonucleotides using BpiI as outlined above and subsequent cloning of HA- and bait-coding DNA fragments engineered to be in-frame with the preceding genome sequence and following P2A-BFP sequence with BsaI using Gibson or restriction/ligation cloning. .. SCIPay-human-CD69-tdTomato and SCIPay-human-TRAC-CAR were generated by first cloning gRNA-coding DNA oligonucleotides as described above and subsequently inserting DNA fragments (Twist Bioscience) designed in silico with gRNA-specific bait sequences into pQCi using Gibson assembly.

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    Thermo Fisher rapid dna ligation kit
    SCIPay design permits CRISPR-guided integration of specific donor <t>DNA</t> sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a <t>BFP</t> transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    Rapid Dna Ligation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p

    Journal: bioRxiv

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering

    doi: 10.1101/2020.03.25.008276

    Figure Lengend Snippet: SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p

    Article Snippet: This modular plasmid allows insertion of gRNA-coding DNA oligonucleotides using BpiI as outlined above and subsequent cloning of HA- and bait-coding DNA fragments engineered to be in-frame with the preceding genome sequence and following P2A-BFP sequence with BsaI using Gibson or restriction/ligation cloning.

    Techniques: CRISPR, Sequencing, Knock-Out, Plasmid Preparation, Flow Cytometry

    High fGly conversion yields can be obtained from stably transduced cell lines under high titer cell culture conditions. Stable transduction of DNA encoding FGE, antibody light chain, and antibody heavy chain bearing one (CT only) or two (C H 1 and CT) aldehyde tags was performed with viral retrovector transduction into CHO cells (GPEx® technology). The resulting stable pools were cultured in three types of media +/- supplementation with copper(II) sulfate ( n = 3). Titers ( a ) and fGly conversion ( b ) of CT-tagged antibodies were assessed. Then, the stable pools were cloned by limiting dilution and clone performance was assessed in fed batch cultures ( c and d ; shake flask, n = 5 clones; bioreactor, n = 3 clones single tag, n = 1 clone double tag). The top performing stable single-tagged clonal cell line produced antibody in very high titer (5.2 g/L) with 98 % conversion of Cys to fGly ( c and d ). The specific productivity (75 pg/cell/d) of this top clone demonstrates the capabilities of the GPEx® technology for efficient production of highly converted antibody. The generation of fGly in single- and doubly-tagged antibodies scaled successfully to bioreactors (2 L cultures; c and d ). Error bars indicate standard deviation

    Journal: BMC Biotechnology

    Article Title: Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)

    doi: 10.1186/s12896-016-0254-0

    Figure Lengend Snippet: High fGly conversion yields can be obtained from stably transduced cell lines under high titer cell culture conditions. Stable transduction of DNA encoding FGE, antibody light chain, and antibody heavy chain bearing one (CT only) or two (C H 1 and CT) aldehyde tags was performed with viral retrovector transduction into CHO cells (GPEx® technology). The resulting stable pools were cultured in three types of media +/- supplementation with copper(II) sulfate ( n = 3). Titers ( a ) and fGly conversion ( b ) of CT-tagged antibodies were assessed. Then, the stable pools were cloned by limiting dilution and clone performance was assessed in fed batch cultures ( c and d ; shake flask, n = 5 clones; bioreactor, n = 3 clones single tag, n = 1 clone double tag). The top performing stable single-tagged clonal cell line produced antibody in very high titer (5.2 g/L) with 98 % conversion of Cys to fGly ( c and d ). The specific productivity (75 pg/cell/d) of this top clone demonstrates the capabilities of the GPEx® technology for efficient production of highly converted antibody. The generation of fGly in single- and doubly-tagged antibodies scaled successfully to bioreactors (2 L cultures; c and d ). Error bars indicate standard deviation

    Article Snippet: Both the FGE vector and the EF1α PCR product were digested with NruI and NheI and ligated with the Rapid DNA Ligation Kit (Thermo/Life Technologies) to generate WT pEF-FGE-hygro, which was amplified in XL-10 gold cells (Agilent) and purified according to the manufacturer’s instructions.

    Techniques: Stable Transfection, Cell Culture, Transduction, Clone Assay, Produced, Standard Deviation