Products

GeneArt™ Seamless Cloning and Assembly Kit Invitrogen™

The GeneArt® Seamless Cloning and Assembly Kit enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. The kit contains everything required for the assembly of DNA fragments, and their transformation into E. coli for selection and growth of recombinant vectors.

Speed and Ease — Clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector (up to 13 Kb); no restriction digestion, ligation or recombination sites required
Precision and Efficiency —Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
Vector Flexibility — Use our linear vector or a vector of your choice
Free Tools — Design DNA oligos and more with our free web-based interface that walks you step-by-step through your project
Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For cloning more than 4 DNA fragments, final molecules larger than 110 Kb, or for using pre-existing DNA elements too long to be amplified by PCR; please consider the GeneArt® High-Order Genetic Assembly System (cat# A13285).

Simple and Fast Clone Creation
GeneArt® Seamless Cloning is a simple, two step process, consisting of a tube based assembly reaction followed by transformation into One Shot® Chemically Competent TOP10 E. coli. The kit uses the properties of a proprietary enzymatic mix to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). A portion of the assembly reaction is then transformed into the provided competent TOP10 cells, yielding clones ready for analysis the next day. The required 15-base pair end-homology can be easily engineered by PCR-amplification with custom DNA oligos.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Seamless Cloning and Assembly Kit the main factors effecting cloning efficiency are the size of the DNA elements (100 bp to 5 Kb), the total size of the final molecule (≤ 13 Kb), and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.

Typical cloning efficiencies for different numbers of fragments cloned into pUC19L are the following:
• 90% for a single 5 Kb DNA element
• 70% for 4 fragments of 1 Kb each
• 40% for 4 DNA fragments of 2 Kb each

Success of the cloning is independent of the insert sequence and vector type, allowing you to design and add nearly any desired sequence, or combination of sequences, to any plasmid as long as it can be linearized by either restriction enzyme digestion or by PCR. The circularized clones obtain from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

in silico Cloning Design Support
A key step in GeneArt® Seamless Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide a free online tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos with end-homology for the PCR amplification of the different elements to clone, and presents the user with a graphical representation of the vector, as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® Seamless Cloning and Assembly Kit is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites in an existing vector, and many other techniques that require manipulation of genetic sequences.

GeneArt™ Site-Directed Mutagenesis System Invitrogen™

The GeneArt® Site-Directed Mutagenesis System provides a state-of-the-art, simple, convenient, and highly efficient means to generate mutations on a target construct in vitro in less than three hours. This system replaces the popular GeneTailor™ Site-Directed Mutagenesis System, and has been completely redesigned to be at the leading edge of commercial site-directed mutagenesis products currently available on the market. This product brings our mutagenesis technology to the next level. Note: A PCR enzyme is not included with the system and must be purchased separately. The recommended enzyme for this kit is AccuPrime™ Pfx DNA Polymerase.

Powerful – Make substitutions, deletions, or insertions of up to 12 nucleotides in plasmids up to 14 Kb
Efficient – Enables you to obtain your desired mutant the first time; over 90% correct mutants for a 3 Kb plasmid
Fast – Have your mutated plasmid DNA in less then 3 hours with the simple, minimal handling protocol
Versatile – Use plasmids of many sizes, and DNA isolated from any source, with no need for specialized vectors, host strains, or restriction sites

Simple Creation of Desired Mutants
Creating mutants with the GeneArt® Site-Directed Mutagenesis System relies on the inherent properties of DNA methylase, high fidelity DNA polymerase, recombination enzymes, and the native McrBC endonuclease of E. coli. Simply incorporate the mutation or mutations (up to 12 nucleotides), that you want into primers, and after PCR, recombination, and transformation you get vectors with only the mutations you desired with up to 90% efficiency. The template vector that you add mutations to can be isolated from any source and up to 14Kb in size. For creating complete constructs, or for joining large pieces of unrelated DNA see our GeneArt® Seamless Cloning and Assembly Kit (cat# A13288) or our GeneArt® High-Order Genetic Assembly System (cat#A13286).

Optimized Mutagenesis Protocol
The GeneArt® Site-Directed Mutagenesis System has been optimized for efficiency and simplicity. The DNA methylation and amplification steps are combined into a single reaction with no need for an in vitro DpnI digestion step. After methylation and amplification, a 10 minute in vitro recombination reaction of the amplified PCR products increases the colony output by 3 to 10 fold; resulting in a higher mutagenesis efficiency. A final transformation of the mutated DNA into DH5α™ E.coli cells digests any methylated parental DNA, leaving behind only the intact unmethylated mutagenesis reaction product. No purification steps or additional digestions are needed. Individual colonies can be selected the following day to verify mutations.

GeneArt™ High-Order Genetic Assembly System Invitrogen™

The GeneArt® High-Order Genetic Assembly System is a highly efficient kit for the simultaneous and seamless assembly of up to 10 DNA fragments, totaling up to 110 Kbp in length, into any vector. The system relies on yeast’s ability to take up and recombine DNA fragments with high efficiency. This greatly reduces the in vitro handling of DNA and eliminates the need for enzymatic treatments, such as restriction and ligation, while allowing for precise fusions of DNA sequences. The kit contains materials for the transformation and purification from yeast, including yeast selective media, and competent E. coli for plasmid amplification of correct clones.

Easy and Powerful — Clone up to 10 DNA fragments, with the sequence of your choice, simultaneously in a single vector (up to 110 Kbp); no restriction digestion, ligation or recombination sites required
Precision and Efficiency — Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
Flexibility — Use our linear vector, a vector of your choice, or clone pre-existing DNA fragments that have no end-homology without further modifications
Free Tools — Design DNA oligos and more with our free web-based interface that walks you through your project step-by-step
Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For the cloning of 1 to 4 DNA fragments of limited size, and if you prefer an in vitro approach, consider using the GeneArt® Seamless Cloning and Assembly Kit (cat # A13288).

Easily Create New Specific Constructs from Diverse DNA Fragments
The GeneArt® High-Order Genetic Assembly System takes advantage of transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisae to join pre-existing DNA fragments, or chemically synthesized oligonucleotides, into a single recombinant molecule. DNA fragments and linearized vector are joined based on shared end-terminal homology. If no such end homology exists between pieces, they can be “stitched" together with recombination linkers, synthetic DNA oligonucleotides that provide end-terminal homology between two unrelated DNA fragments. The process is very efficient and seamless, leaving no extra sequences after the assembly. Even though it has been shown to work for up to 0.5 Mb and 50 DNA fragments, this product has been optimized for up to 110 Kb and 10 DNA fragments in a vector.

Simple Clone Verification
In order to minimize the work that is done in yeast, recombinant yeast clones are subjected to a simplified 10-minute DNA extraction protocol. The extraction yields assembled molecule in enough quantity to do colony PCR verification of the junctions, as well as direct transformation of E.coli cells for downstream analysis. The proprietary lysis buffer and glass beads needed for the extraction are included in the kit.

Considerations for Choosing a Cloning Vector
The GeneArt® High-Order Genetic Assembly System requires shuttle vectors that have high capacity and are compatible with yeast and E.coli (i.e. BAC-YAC shuttle vectors). There is no cloning vector included in this product, but we offer a ready-to-use linear cloning vector separately called the GeneArt® pYES1L Vector with Sapphire Technology™ (cat# A13287). You can also use your own vector, but it must be compatible with the High-Order Genetic Assembly System. This compatibility can be easily accomplished with our GeneArt® High-Order Vector Conversion Cassette (cat#A13291).

Cloning Efficiency
In GeneArt® High-Order Assembly the main factors affecting cloning efficiency are the size of the DNA elements, the number of those without end-homology, the total size of the final molecule, and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.

Typical cloning efficiencies for different numbers of fragments with end-homology assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ are the following:
• >90% for 5 DNA fragments of 10 Kb each
• >90% for 10 DNA fragments of 5 Kb each
• >50% for 10 DNA fragments of 10 Kb each

Common cloning efficiencies for pre-existing fragments, without end-homology, assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ using 'stitching’ DNA oligonucleotides are:
• >90% for 1 fragment of 10 Kb
• >75% for 2 DNA fragments of 10 Kb each

Design Your Cloning in silico
A key step in GeneArt® High-Order Genetic Assembly is the correct design of fragments and oligos with the appropriate homology and spacing to ensure successful assembly of your clone. To simplify and speed the design process we provide a free online design tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos for either PCR amplification or for stitching of the different elements to clone, and presents the user with a graphical representation of the vector as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® High-Order Genetic Assembly System is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites, clone large pre-existing DNA fragments without end-homology, and many other techniques that require manipulation of genetic sequences.

GeneArt™ Gibson Assembly HiFi Cloning Kit, electrocompetent cells Invitrogen™

The GeneArt Gibson Assembly HiFi Cloning Kit enables DNA assembly and cloning via a technique that allows multiple overlapping DNA fragments to be seamlessly linked in a one-step, 15–60 minute isothermal reaction. DNA fragments of different lengths are uniformly assembled using complementary overlaps between fragments. The inherent flexibility of this approach is suitable for small and large DNA constructs and includes both single and multiple inserts. The resulting products can be used for a variety of downstream applications including transformation, PCR and rolling circle amplification (RCA). The GeneArt Gibson Assembly HiFi Cloning Kit, electrocompetent cells, is a complete kit that includes master mix, positive control, water, and ElectroMAX DH10B electrocompetent E. coli.

Features of the GeneArt Gibson Assembly HiFi Cloning Kit include:
Simple—seamlessly assemble and clone up to six DNA fragments in a single reaction
Efficient—high fidelity provides more correct clones than other methods
Flexible—design guidelines allow assembly into any vector of your choice
Convenient—available as master mix kits and cloning kits complete with chemical or electrocompetent cells
Trusted—over 4000 citations in the scientific literature highlighting great success

Seamless assembly of multiple fragments
GeneArt Gibson Assembly HiFi cloning is a simple, one-step process whereby up to six fragments are combined in a proprietary enzymatic mix in order to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). After a 15–60 minute incubation, a portion of the assembly reaction is then transformed into chemically competent or electrocompetent cells, yielding clones that are ready for analysis the next day. The required 20- to 40-base pair end homology is designed into the de novo fragment for synthesis or can be easily engineered by PCR amplification with custom DNA oligos. This special enzyme mix creates a seamless and covalently bound DNA construct providing high efficiency.

Robust method for maximum efficiency
Due to the covalently bound final product, the GeneArt Gibson Assembly HiFi method allows the utilization of chemically competent cells or electrocompetent cells for the highest transformation efficiency, improving the success of finding the right clone, particularly for more challenging constructs. There is no need for restriction enzymes, ligation, or recombination sites, and the method provides a perfectly seamless construct without unwanted extra bases.

Provides great versatility
The GeneArt Gibson Assembly HiFi method provides versatility, can streamline many techniques through the rapid combination, addition, deletion, or exchange of DNA segments, and eliminates the need to subclone, saving time and effort in the cloning workflow.

Convenient formats
GeneArt Gibson Assembly HiFi kits are available in multiple formats, including:
• GeneArt Gibson Assembly HiFi Cloning Kit, electrocompetent cells (with ElectroMAX DH10B electrocompetent cells) (this page)
GeneArt Gibson Assembly HiFi Cloning Kit, chemically competent cells (with One Shot TOP10 chemically competent cells)
GeneArt Gibson Assembly HiFi Master Mix kits (cloning kit without the cells)

GeneArt Prime Clone, Gene synthesis service, up to 3 kb

Exceptionally accurate, custom made, double stranded linear DNA fragments that provide high cloning success rate, especially for large DNA assemblies. These fragments up to 1.2kb in length offer unparalleled accuracy and optimum quality through our new gene synthesis platform

GeneArt™ Gibson Assembly HiFi Master Mix Invitrogen™

GeneArt Gibson Assembly HiFi Master Mix enables DNA assembly and cloning via a technique that allows multiple overlapping DNA fragments to be seamlessly linked in a one-step, 15–60 minute isothermal reaction. DNA fragments of different lengths are uniformly assembled using complementary overlaps between fragments. The inherent flexibility of this approach is suitable for small and large DNA constructs and includes both single and multiple inserts. The resulting products can be used for a variety of downstream applications including transformation, PCR and rolling circle amplification (RCA). The GeneArt Gibson Assembly HiFi Master Mix kit includes master mix, positive control, and water and accommodates the use of your own competent cells.

Features of the GeneArt Gibson Assembly HiFi Master Mix include:
Simple—seamlessly assemble and clone up to six DNA fragments in a single reaction
Efficient—high fidelity provides more correct clones than other methods
Flexible—design guidelines allow assembly into any vector of your choice
Convenient—available as master mix kits and cloning kits complete with chemical or electrocompetent cells
Trusted—over 4000 citations in the scientific literature highlighting great success

Seamless assembly of multiple fragments
GeneArt Gibson Assembly HiFi cloning is a simple, one-step process whereby up to six fragments are combined in a proprietary enzymatic mix in order to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). After a 15–60 minute incubation, a portion of the assembly reaction is then transformed into chemically competent or electrocompetent cells, yielding clones that are ready for analysis the next day. The required 20- to 40-base pair end homology is designed into the de novo fragment for synthesis or can be easily engineered by PCR amplification with custom DNA oligos. This special enzyme mix creates a seamless and covalently bound DNA construct providing high efficiency.

Robust method for maximum efficiency
Due to the covalently bound final product, the GeneArt Gibson Assembly HiFi method allows the utilization of chemically competent cells or electrocompetent cells for the highest transformation efficiency, improving the success of finding the right clone, particularly for more challenging constructs. There is no need for restriction enzymes, ligation, or recombination sites, and the method provides a perfectly seamless construct without unwanted extra bases.

Provides great versatility
The GeneArt Gibson Assembly HiFi method provides versatility, can streamline many techniques through the rapid combination, addition, deletion, or exchange of DNA segments, and eliminates the need to subclone, saving time and effort in the cloning workflow.

Convenient formats
GeneArt Gibson Assembly HiFi kits are available in multiple formats, including:
GeneArt Gibson Assembly HiFi Cloning Kit, chemically competent cells (with One Shot TOP10 chemically competent cells)
GeneArt Gibson Assembly HiFi Cloning Kit, electrocompetent cells (with ElectroMAX DH10B electrocompetent cells)
• GeneArt Gibson Assembly HiFi Master Mix kits (cloning kit without the cells) (this page)

GeneArt™ Gibson Assembly EX Cloning Kit, electrocompetent cells Invitrogen™

The GeneArt Gibson Assembly EX Cloning Kit enables DNA assembly via a technique that allows up to 15 overlapping DNA fragments to be seamlessly linked in a single-tube, two-step optimized reaction. DNA fragments of different lengths are uniformly assembled using complementary overlaps between fragments. The inherent flexibility and success of this approach is suitable for small and large DNA constructs, includes both single and multiple inserts, and has been used to build entire genomes. The resulting products can be used for a variety of downstream applications, including transformation, PCR, and rolling circle amplification (RCA). The GeneArt Gibson Assembly EX Cloning Kit, electrocompetent cells, is a complete kit that includes master mix, positive control, water, and ElectroMAX DH10B electrocompetent E. coli.

Features of the GeneArt Gibson Assembly EX Cloning Kit include:
Simple—seamlessly assemble and clone up to 15 DNA fragments in a single reaction
Efficient—robust efficiency provides successful clones for both simple and challenging constructs
Flexible—design guidelines allow assembly into any vector of your choice
Convenient—available as master mix kits and cloning kits complete with chemical or electrocompetent cells
Trusted—over 4000 citations in the scientific literature highlighting great success

Seamless assembly of up to 15 fragments
GeneArt Gibson Assembly EX cloning is a robust, single-tube, two-step process whereby up to 15 inserts and vector are combined in a proprietary enzymatic mix in order to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). After this dually optimized reaction is complete, a portion of the assembly reaction is then transformed into chemically competent or electrocompetent cells, yielding clones ready for analysis the next day. The required 20- to 40-base pair end homology is designed into the de novo fragment for synthesis or can be easily engineered by PCR amplification with custom DNA oligos. This special enzyme mix creates a seamless and covalently bound DNA construct providing high efficiency.

Robust method for maximum efficiency
Due to the covalently bound final product, the GeneArt Gibson Assembly EX method allows the utilization of chemically competent cells or electrocompetent cells for the highest transformation efficiency, especially important for challenging constructs. There is no need for restriction enzymes, ligation, or recombination sites, and the method provides a perfectly seamless construct without unwanted extra bases.

Provides great versatility
The GeneArt Gibson Assembly EX method provides versatility, can streamline many techniques through the rapid combination, addition, deletion, or exchange of DNA segments, and eliminates the need to subclone, saving time and effort in the cloning workflow.

Convenient formats
GeneArt Gibson Assembly EX kits are available in multiple formats, including:
• GeneArt Gibson Assembly EX Cloning Kit, electrocompetent cells (with ElectroMAX DH10B electrocompetent cells) (this page)
GeneArt Gibson Assembly EX Cloning Kit, chemically competent cells (with One Shot TOP10 chemically competent cells)
GeneArt Gibson Assembly EX Master Mix kits (cloning kit without the cells)

GeneArt™ Data Storage Material Documentation

The GeneArt® Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence.

GeneArt Prime Clone, gene synthesis service, 3 to 5 kb

Exceptionally accurate, custom made, double stranded linear DNA fragments that provide high cloning success rate, especially for large DNA assemblies. These fragments up to 1.2kb in length offer unparalleled accuracy and optimum quality through our new gene synthesis platform

GeneArt™ Precision TAL Construction Service, Native TAL vp16 Activator

The GeneArt® Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence.

GeneArt™ Gibson Assembly EX Master Mix Invitrogen™

GeneArt Gibson Assembly EX Master Mix enables DNA assembly via a technique that allows multiple overlapping DNA fragments to be seamlessly linked in a single-tube, two-step optimized reaction. DNA fragments of different lengths are uniformly assembled using complementary overlaps between fragments. The inherent flexibility and success of this approach is suitable for small and large DNA constructs, includes both single and multiple inserts, and has been used to build entire genomes. The resulting products can be used for a variety of downstream applications, including transformation, PCR, and rolling circle amplification (RCA). The GeneArt Gibson Assembly EX Master Mix kit includes master mix, positive control, and water, and accommodates the use of your own competent cells.

Features of the GeneArt Gibson Assembly EX Master Mix include:
Simple—seamlessly assemble and clone up to 15 DNA fragments in a single reaction
Efficient—robust efficiency provides successful clones for both simple and challenging constructs
Flexible—design guidelines allow assembly into any vector of your choice
Convenient—available as master mix kits and cloning kits complete with chemical or electrocompetent cells
Trusted—over 4000 citations in the scientific literature highlighting great success

Seamless assembly of up to 15 fragments
GeneArt Gibson Assembly EX cloning is a robust, single-tube, two-step process whereby up to 15 inserts and vector are combined in a proprietary enzymatic mix in order to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). After this dually optimized reaction is complete, a portion of the assembly reaction is then transformed into chemically competent or electrocompetent cells, yielding clones ready for analysis the next day. The required 20- to 40-base pair end homology is designed into the de novo fragment for synthesis or can be easily engineered by PCR amplification with custom DNA oligos. This special enzyme mix creates a seamless and covalently bound DNA construct providing high efficiency.

Robust method for maximum efficiency
Due to the covalently bound final product, the GeneArt Gibson Assembly EX method allows the utilization of chemically competent cells or electrocompetent cells for the highest transformation efficiency, especially important for challenging constructs. There is no need for restriction enzymes, ligation, or recombination sites, and the method provides a perfectly seamless construct without unwanted extra bases.

Provides great versatility
The GeneArt Gibson Assembly EX method provides versatility, can streamline many techniques through the rapid combination, addition, deletion, or exchange of DNA segments, and eliminates the need to subclone, saving time and effort in the cloning workflow.

Convenient formats
GeneArt Gibson Assembly EX kits are available in multiple formats, including:
GeneArt Gibson Assembly EX Cloning Kit, electrocompetent cells (with ElectroMAX DH10B electrocompetent cells)
GeneArt Gibson Assembly EX Cloning Kit, chemically competent cells (with One Shot TOP10 chemically competent cells)
• GeneArt Gibson Assembly EX Master Mix kits (cloning kit without the cells) (this page)

GeneArt™ Data Storage GLP Source Documentation

The GeneArt® Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence.

GeneArt Prime Clone, gene synthesis service, 5 to 12 kb

Exceptionally accurate, custom made, double stranded linear DNA fragments that provide high cloning success rate, especially for large DNA assemblies. These fragments up to 1.2kb in length offer unparalleled accuracy and optimum quality through our new gene synthesis platform

GeneArt™ Precision TAL Construction Service, Native TAL vp64 Activator

The GeneArt® Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence.

GeneArt™ Precision TAL 18 Base DNA Binding Domain

The GeneArt® Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence.
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