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    CloneMiner™ II cDNA Library Construction Kit Invitrogen™

    The CloneMiner™ II cDNA Library Construction Kit is a second generation CloneMiner™ kit that enables rapid construction of highly representative cDNA libraries without restriction enzyme cloning. This innovative library construction technology combines SuperScript® III Reverse Transcriptase with Gateway® cloning technology, resulting in the discovery of previously unobtainable, full-length clones. The kit also avoids the use of time-consuming, inefficient ligation reactions, making library construction faster and offering better representation.

    The CloneMiner™ II cDNA Library Construction Kit ensures:
    • High primary titers
    • Large average insert sizes
    • The highest percentage of full-length genes
    • Highly efficient cloning of cDNA to multiple destination vectors without the need of restriction enzyme digestion and ligation

    How the CloneMiner™ II Kit Works:

    • High yields of full-length cDNA: The CloneMiner™ II kit contains SuperScript® III for generating high cDNA yields. SuperScript® III Reverse Transcriptase (RT), a proprietary mutant of SuperScript® II RT, is active at 50°C and has a half-life of 220 minutes. It also has a point mutation that reduces RNase H activity, thereby decreasing RNA degradation during first-strand synthesis and increasing the percentage of full-length genes.

    • Gateway® cloning technology avoids restriction enzyme cloning: Library construction is mediated by Gateway® Technology, a site-specific recombination system that eliminates the use of restriction enzymes and ligase in cloning. Each cDNA insert is flanked by specific att recombination sites (added during the cDNA synthesis steps) that recombine with complementary att sites present in Gateway® donor vectors to create entry clones. Entry clones are subsequently recombined with Gateway® expression vectors to create expression clones, effectively replacing the use of restriction enzymes and ligase. The resulting clones maintain the original orientation and reading frame enabling functional analysis of full-length genes and whole libraries.

    How is This Kit Different From the Original CloneMiner™ Kit?
    • The new kit incorporates a simplified protocol.
    • To ensure high cDNA yields, SuperScript® II Reverse Transcriptase (RT) has been replaced by SuperScript® III Reverse Transcriptase.
    • To enable reaction setup with fewer pipetting steps, Gateway® BP Clonase® Enzyme Mix has been replaced by Gateway® BP Clonase® II Enzyme Mix, which contains enzymes and buffer in a single mix.

    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)

    TA Cloning™ Kit, with pCR™2.1 Vector, without competent cells Invitrogen™

    The TA Cloning® Kit with pCR™2.1 vector provides a quick, one-step cloning strategy for directly inserting a Taq-amplified PCR product into a plasmid vector. The TA Cloning® Kit uses the pCR™2.1 cloning vector and ExpressLink™ T4 DNA Ligase to generate a ligation product in a fifteen-minute, room-temperature ligation step. Reactions typically yield >80% recombinants containing inserts.

    Features of the TA Cloning® Kit with pCR™2.1 vector:
    Fast & convenient—15-minute, room-temperature ligation
    Efficient—blue/white screening and >80% clones with correct insert
    Flexible—choice of kanamycin or ampicillin resistance for flexible antibiotic selection
    Hassle-free—eliminates any enzymatic modifications of the PCR product
    Streamlined—does not require the use of PCR primers that contain restriction sites

    The pCR™2.1 vector provides:
    • 3'-T overhangs for direct ligation of Taq-amplified PCR products
    • T7 promoter for in vitro RNA transcription and sequencing
    • A versatile polylinker with flanking EcoR I sites for easy excision of inserts
    • M13 forward and reverse primer sites for sequencing

    How TA Cloning® Works
    Taq polymerase has a non-template-dependent activity that adds a single deoxyadenosine (A) to the 3' ends of PCR products. The linearized vector supplied in this kit has single 3' deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

    Kit Configurations
    The TA Cloning® Kit is offered in a variety of configurations: without competent cells (K2020-20 and K2020-40), with One Shot® INVF' Chemically Competent E. coli (K2000-01 and K2000-40), with One Shot® TOP10F' Chemically Competent E. coli (K2030-01 and K2030-40), and with One Shot® TOP10 Chemically Competent E. coli (K2040-01 and K2040-40) in 20- and 40-reaction kit sizes.

    CloneJET PCR Cloning Kit Thermo Scientific™

    Thermo Scientific CloneJET PCR Cloning Kit, also available with DH10B cells (K123120), is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Any other blunt or sticky-end DNA fragment can be cloned. It is ideal for phosphorylated or non-phosphorylated DNA fragments. Ligation into the included positive selection vector takes only 5 minutes, yielding more than 99% recombinant clones. Blunt-ended PCR products generated with a proofreading enzyme are ligated directly into the cloning vector.

    PCR products generated either with non-proofreading DNA polymerases or mixtures of DNA polymerases are blunted prior to ligation in 5 minutes with the thermostable DNA Blunting Enzyme provided with the kit. All common laboratory E. coli strains can be directly transformed with the ligation product.

    Features

    The CloneJET PCR Cloning Kit contains a novel, ready-to-use positive selection cloning vector pJET1.2/blunt. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Recircularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme, which kills the host E. coli cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection.

    For convenience in mapping and manipulation of the insert, the pJET1.2/blunt cloning vector multiple cloning site contains two BglII recognition sequences that flank the insertion site. In addition, the vector contains a T7 promoter for in vitro and in vivo transcription as well as sequencing of the insert.

    Highlights

    Fast—PCR cloning in only 5 minutes
    Highest efficiency – > 99% of positive clones
    No cloning background—positive selection vector
    Versatile—ideal for blunt-end or sticky-end cloning
    Economical—no expensive blue/white screening

    Applications

    • Cloning of blunt-end or 3'-dA tailed PCR products up to 10 kb
    • Cloning of DNA fragments generated by restriction enzymes
    • Sequencing of cloned DNA
    in vitro and in vivo transcription of cloned inserts from the T7 promoter

    Includes

    • pJET1.2/blunt Cloning Vector
    T4 DNA Ligase
    • 2X Reaction Buffer
    • DNA Blunting Enzyme
    • pJET1.2 Forward Sequencing Primer (5'-CGACTCACTATAGGGAGAGCGGC-3')
    • pJET1.2 Reverse Sequencing Primer (5'-AAGAACATCGATTTTCCATGGCAG-3')
    • Control PCR Product
    Water, nuclease-free
    • Detailed Protocol

    Prior to electroporation, always column-purify the ligation mixture using e.g. GeneJET PCR Purification Kit #K0701 or chloroform to extract it. Electroporation is inhibited by the presence of proteins and salts in the mixture.

    Related Products
    CloneJET PCR Cloning Kit
    Fast DNA End Repair Kit

    MicroAmp™ 96-Well Tray/Retainer Set Applied Biosystems™

    Applied Biosystems® MicroAmp® Tray/Retainer Sets, available in a 96- or 24-well format, hold MicroAmp® Reaction Tubes without Caps for processing on the Veriti™ 96-Well Thermal Cycler, 96-Well GeneAmp® PCR System 9700, or the Applied Biosystems 2720 Thermal Cycler.

    • Available in 96- or 24-well format, offering flexibility
    • Designed for compatibility with MicroAmp® Reaction Tubes and MicroAmp® 8-Strip Reaction Tubes

    Convenience and Performance
    The MicroAmp® Tray/Retainer Sets allow you to organize your tubes while adding samples and mixing. This handy tool reduces spillage and tube mix-ups by securing and ordering your tubes on your lab bench. When the samples are ready, simply put the tray of tubes directly on the block of the GeneAmp Thermal Cycler. Not only does the tray eliminate the hassle of transferring the tubes but it also provides the thermal contact necessary for high-performance thermal cycling.


    Note: See user's manual or package insert for limited label license, and trademark information. For Research Use Only. Not for use in diagnostics procedures.

    CloneJET™ PCR Cloning Kit with DH10B Competent Cells Thermo Scientific™

    This package includes the CloneJET PCR Cloning Kit (20 or 40 reactions) and DH10B Competent Cells that are optimized for use with the kit and can be directly transformed with the ligation product.

    CloneJET PCR Cloning Kit
    The Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive-selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Any blunt or sticky-end DNA fragment can be cloned. The kit works well with both phosphorylated and non-phosphorylated DNA fragments. Ligation into the included positive-selection vector takes only five minutes, yielding more than 99% recombinant clones. Blunt-ended PCR products generated with a proofreading enzyme are ligated directly into the cloning vector.

    PCR products generated either with non-proofreading DNA polymerases or mixtures of DNA polymerases need to be blunted prior to ligation in a five minute reaction with the thermostable DNA Blunting Enzyme provided with the kit. DH10B Competent Cells are optimized for use with the CloneJET PCR Cloning Kit and can be directly transformed with the ligation product.

    The CloneJET PCR Cloning Kit contains the novel, ready-to-use positive selection cloning vector pJET1.2/blunt. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Re-circularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme, which kills the host E. coli cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection.

    Fast—PCR cloning in only five minutes
    Highest efficiency—>99% of positive clones
    No cloning background—positive selection vector
    Versatile—ideal for blunt-end or sticky-end cloning
    Economical—no expensive blue/white screening

    Applications include:
    • Cloning of blunt-end or 3' dA-tailed PCR products up to 10 kb
    • Cloning of DNA fragments generated by restriction enzymes
    • Sequencing of cloned DNA
    • in vitro and in vivo transcription of cloned inserts from the T7 promoter

    Learn more about the CloneJET PCR Cloning Kit ›

    DH10B Competent Cells
    Thermo Scientific DH10B Competent Cells are high efficiency, chemically competent E.coli cells. The DH10B strain is suitable for cloning DNA containing methylcytosine and methyladenine, allowing both prokaryotic and eukaryotic genomic DNA to be cloned efficiently. These cells are ideal for the construction of cDNA libraries using plasmid-derived vectors. Genotype: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG.

    • High transformation efficiency: >1x109 cfu/µg pUC19 DNA
    • Suitable for routine and high-throughput cloning applications
    • Convenient two reactions per vial packaging
    • S.O.C. Outgrowth medium included

    Related products
    CloneJET PCR Cloning Kit without competent cells
    aLICator LIC Cloning and Expression System

    FirstChoice™ RLM-RACE Kit Invitrogen™

    The FirstChoice® RLM-RACE Kit is designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band after PCR. This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol. The RLM-RACE procedure selects only full-length mRNA—no rRNA, tRNA or degraded RNA—and facilitates the cloning of sequences from the 5' ends of messages.

    Features of the FirstChoice® RLM-RACE Kit:

    • From RNA to PCR product in less than a day
    • Yields single, specific product from rare transcripts
    • Selects 5' and/or 3' ends of true messages
    • Efficient—all enzymatic reactions are optimized to ensure detection of even the rarest mRNA

    RACE
    Rapid amplification of cDNA ends (5'-RACE) is a polymerase chain reaction-based technique developed to facilitate the cloning of sequence from the 5'-ends of messages. The FirstChoice® RLM-RACE Kit is a major improvement to the basic RACE protocol.

    Selects full-length mRNA—no rRNA, tRNA or degraded mRNA
    In the FirstChoice® RLM-RACE procedure, full-length mRNAs are selected by treating total or poly(A) RNA with calf intestinal phosphatase (CIP) to remove the 5'-phosphate from all molecules which contain free 5'-phosphates (ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA). Full-length mRNAs are unaffected. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from the full-length mRNA leaving a 5'-monophosphate. A synthetic RNA adapter is ligated to the RNA population—only molecules containing a 5'-phosphate, the uncapped, full-length mRNAs, will accept the adapter. Random-primed, reverse transcription reactions and nested PCR are then performed to amplify the 5'-end of your specific transcript.

    From RNA to PCR in less than a day
    Each step of the FirstChoice® RLM-RACE procedure has been optimized, so you can complete all the enzymatic reactions and even start PCR in no more than 5 hours. The FirstChoice® RLM-RACE Kit contains reagents and enzymes to produce 5 RLM-RACE-ready cDNA preparations, in addition to primers and nested RACE adapter primers for 100 PCR reactions. Reverse transcription reagents are also included. Each kit contains control RNA and primers to test the kit's performance along with a detailed Instruction Manual. SuperTaq™ Thermostable Taq DNA Polymerase is available separately. For optimal amplification of fragments ≥1 kb, use SuperTaq-Plus. Also included are a 3' RACE Adapter and Primers for 3' RACE.

    TA Cloning™ Kit, with pCR™2.1 Vector and One Shot™ TOP10F' Chemically Competent E. coli Invitrogen™

    The TA Cloning® Kit with pCR™2.1 vector provides a quick, one-step cloning strategy for directly inserting a Taq-amplified PCR product into a plasmid vector. The TA Cloning® Kit uses the pCR™2.1 cloning vector and ExpressLink™ T4 DNA Ligase to generate a ligation product in a fifteen-minute, room-temperature ligation step. Reactions typically yield >80% recombinants containing inserts.

    Features of the TA Cloning® Kit with pCR™2.1 vector:
    Fast & convenient—15-minute, room-temperature ligation
    Efficient—blue/white screening and >80% clones with correct insert
    Flexible—choice of kanamycin or ampicillin resistance for flexible antibiotic selection
    Hassle-free—eliminates any enzymatic modifications of the PCR product
    Streamlined—does not require the use of PCR primers that contain restriction sites

    The pCR™2.1 vector provides:
    • 3'-T overhangs for direct ligation of Taq-amplified PCR products
    • T7 promoter for in vitro RNA transcription and sequencing
    • A versatile polylinker with flanking EcoR I sites for easy excision of inserts
    • M13 forward and reverse primer sites for sequencing

    How TA Cloning® Works
    Taq polymerase has a non-template-dependent activity that adds a single deoxyadenosine (A) to the 3' ends of PCR products. The linearized vector supplied in this kit has single 3' deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

    Kit Configurations
    The TA Cloning® Kit is offered in a variety of configurations: without competent cells (K2020-20 and K2020-40), with One Shot® INVF' Chemically Competent E. coli (K2000-01 and K2000-40), with One Shot® TOP10F' Chemically Competent E. coli (K2030-01 and K2030-40), and with One Shot® TOP10 Chemically Competent E. coli (K2040-01 and K2040-40) in 20- and 40-reaction kit sizes.

    MicroAmp™ 96-Well Reaction Tube/Tray/Retainer Set, 0.2 mL Applied Biosystems™

    The Applied Biosystems® MicroAmp® 96-Well Reaction Tube/Tray/Retainer Set is a completely pre-assembled unit containing MicroAmp® Reaction Tubes without caps already loaded in a MicroAmp® 96-Well Tray/Retainer Set. MicroAmp® 96-Well Reaction Tube/Tray/Retainer Sets are optimally designed for precise PCR on Applied Biosystems® 0.2 mL thermal cyclers.

    • Completely pre-assembled for convenience
    • Available in economical 100-unit bulk packs
    • Non-sterile and autoclavable

    Greater Sample Uniformity
    Use with all instruments that have heated sample covers to eliminate condensation and the need for a mineral oil overlay. Proprietary MicroAmp® Reaction Tubes are essential for high-performance PCR and ensure the greatest temperature and amplification uniformity for your samples. The MicroAmp® 96-Well Reaction Tube/Tray/Retainer Set requires caps or full plate covers to seal the tubes.

    Note: See user's manual or package insert for limited label license and trademark information. For Research Use Only. Not for use in diagnostics procedures.

    Rapid DNA Ligation Kit Thermo Scientific™

    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 transformation

    Applications

    • Routine cloning experiments
    • Blunt-end cloning
    • Library construction
    • TA cloning

    Includes

    T4 DNA Ligase
    • 5X Rapid Ligation Buffer
    Water, nuclease-free
    • Detailed Protocol

    Note

    Prior 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 Products
    Rapid DNA Ligation Kit

    GeneRacer™ Kit with SuperScript™ III RT and Zero Blunt™ TOPO™ PCR Cloning Kit for Sequencing Invitrogen™

    GeneRacer® is an advanced RACE (rapid amplification of cDNA ends) technique that improves the efficiency of amplifying full-length 5´ and 3´ cDNA ends. With the GeneRacer® Kit you can:

    • Generate cDNA from transcripts at least 10 kb in length
    • Obtain the full-length 5´ end of rare transcripts at fewer than 30 copies per cell
    • Clone the full-length 5´ and 3´ ends to construct complete cDNA sequence

    The GeneRacer® Kit is available with SuperScript® III Reverse Transcriptase (RT) for improved amplification of the full-length 5´ end from long and complex mRNA. The RNase H portion of SuperScript® III RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScript® III RT is more thermostable than wild-type RTs. This enables reverse transcription at higher temperatures, relaxing secondary structure of complex templates, and allowing cDNA synthesis to go to completion.

    How GeneRacer® Works

    The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (1,2). Figure 1 outlines how the GeneRacer® Kit works. The advanced protocol starts at the RNA level by specifically targeting only 5´ capped mRNA. In subsequent steps the cap is removed and replaced with the GeneRacer® RNA Oligo. During reverse transcription, the GeneRacer® RNA Oligo sequence is incorporated into the cDNA. Only cDNA that is completely reverse transcribed will contain this known sequence. 5´ RACE PCR is then performed using the GeneRacer® 5´ Primer specific to the GeneRacer® RNA Oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5´ cDNA sequence.

    Sensitivity and Length

    To demonstrate the ability of the GeneRacer® Kit to capture the full-length 5fi cDNA end, the 5fi ends of genes with known transcriptional start sites were amplified. Starting with total RNA and following the GeneRacer® protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell were amplified (Figure 2).

    MicroAmp™ Snap-On Optical Film Compression Pad Applied Biosystems™

    The Applied Biosystems® MicroAmp® Snap-On Optical Film Compression Pad is a Optical Film Compression Pad attached to a metal frame. It is designed for use on the Applied Biosystems 7900HT Fast Real-Time System when using the available automation accessory with 96-Well plates.

    MethylMiner™ Methylated DNA Enrichment Kit Applied Biosystems™

    The MethylMiner™ Methylated DNA Enrichment Kit is for highly sensitive enrichment of methylated DNA for downstream analysis including PCR/qPCR based assays, bisuflite conversion followed by amplification, cloning, and sequencing, direct sequencing, library preparation for high-throughput sequencing, and sample-prep for DNA microarray analysis. Patterns of DNA methylation seem to be important for making determinations about development and diseases such as cancer. This kit enables superior enrichment and differential fractionation of double-stranded DNA based on CpG methylation density, with increased sensitivity over antibody-based methods. Fractionation permits important comparisons between samples and enables researchers to focus analysis on only the methylation densities of interest.
    • Partitioning with high affinity binding—at least 4-fold greater sensitivity than antibody-based methods
    • Fractionation based on CpG methylation density—ds DNA capture is achieved with MBD2 protein and facilitates ligation of double-stranded adaptors for next-generation sequencing
    • Rapid and easy elution with salt eliminates the need for proteinase K treatment and phenol:chloroform extraction
    • Precise answers—fractionated DNA permits distinctions to be made regarding methylation status and density
    • Fast protocol—completed in less than 4 hours
    • Easy—simple handling with Dynabeads® the gold standard magnetic beads

    MicroAmp™ Optical Film Compression Pad Applied Biosystems™

    Applied Biosystems® MicroAmp® Optical Film Compression Pads are designed to support a proper thermal seal between the thermal cycler and reaction plates when using adhesive films.


    Note: See user's manual or package insert for limited label license, and trademark information. For Research Use Only. Not for use in diagnostics procedures.

    GeneArt™ Seamless PLUS Cloning and Assembly Kit Invitrogen™

    Like our first generation Seamless Cloning and Assembly Kit, GeneArt® Seamless PLUS Cloning and Assembly Kit is the complete kit for simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute or less, room temperature reaction. However, Seamless PLUS offers several advantages over previous kits:

    Increased Efficiency: pre-cloning option for large fragments for increased cloning efficiency
    Larger Constructs: create constructs up to 40 kb
    Versatility: high capacity, broad-range conjugative vector that replicates in most Gram negative bacteria

    The improvements above are combined with these key benefits shared by all GeneArt® Seamless Cloning and Assembly kits:

    Speed and Ease—clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector; 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
    Free Tools—design your final construct and DNA oligos in silico using our free web-based tool that takes you step-by-step through your project
    • Vector Flexibility—use our linear vector or a vector of your choice
    Diverse Applications—streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

    For cloning of more than 4 DNA fragments or for final molecules larger than 110 Kb, please consider the GeneArt® High-Order Genetic Assembly System.

    Simple and Fast Clone Creation
    GeneArt® Seamless PLUS Cloning is a simple, two-step process, consisting of in vitro assembly followed by transformation into One Shot® DH10B™ T1R SA competent E. coli. The kit employs a proprietary enzyme/buffer mix to assemble DNA fragments with shared terminal end homology without extra sequences or scars in the final construct ('seamless'). Terminal end homology is easily incorporated by PCR amplification with custom DNA oligos engineered using our free web tool.

    Cloning Efficiency, Flexibility, and Precision
    With the GeneArt® Seamless PLUS Cloning and Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA fragments (100 bp to 10 Kb), the total size of the final molecule (≤ 40 Kb), and the quality and specificity of each fragment.

    Typical cloning efficiencies for different numbers of fragments:

    • >95% for 4 fragments, 5 Kb each
    • >90% for 4 fragments, 10 Kb each

    Cloning success 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 PCR. The circularized clones obtained from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

    in silico Design Support
    A key step in GeneArt® Seamless PLUS Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. We provide a free online tool, the GeneArt® Design Tool for Seamless or High-Order Assembly and Mutagenesis, 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 PLUS Cloning and Assembly Kit is designed to empower cloning and DNA assembly 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 construct fusion proteins; delete, replace, or add DNA elements such as restriction sites in an existing vector; and carry out many other techniques that require manipulation of genetic sequences.
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