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  • ‹ Back to Applications and technical notes
  • In-Fusion Cloning applications collection
  • Efficient multiple-fragment cloning
  • Mutagenesis with In-Fusion Cloning
  • Rapid, high-throughput cloning for antibody development
  • Solve a synthesis challenge with easy multiple-insert cloning
  • Direct cloning into large vectors
  • Simplified insertion of a GFP-encoding cassette into a 100-kb plasmid
  • Efficient cloning for sgRNA/Cas9 plasmids
  • In-Fusion Cloning of sgRNAs
  • De novo insertion of small fusion protein tags
Efficient sgRNA cloning Overview: efficient, seamless sgRNA cloning
Home › Learning centers › Cloning › Applications and technical notes › In-Fusion Cloning of sgRNAs

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Efficient sgRNA cloning Overview: efficient, seamless sgRNA cloning
Tech Note

In-Fusion Cloning of sgRNAs

  • Allows restriction site-independent cloning
  • Enables a one-step cloning reaction compatible with multiplexing sgRNAs
  • Provides ready-to-transfect sgRNA constructs in <3 days
Introduction Results Conclusions Methods References

Introduction  

Typical single guide RNA (sgRNA) cloning into Cas9 vectors entails a lengthy process that requires specialized reagents (e.g., specific type IIS restriction enzymes) and multiple cloning steps such as incorporation of new restriction sites, restriction digests, and ligations. The traditional approach is further confounded by low cloning efficiency, resulting in additional time-consuming screens to identify a colony containing your sgRNA of interest. These bottlenecks also render it difficult to move sgRNAs from one vector to another and make it nearly impossible to multiplex several sgRNAs in one plasmid.

However, in a recent publication, Khan et al. were able to improve and streamline their Cas9/sgRNA plasmid construction using our In-Fusion Cloning HD Plus Kits. Using a single-step In-Fusion Cloning reaction, the authors were able to produce ready-to-transfect single or multiplexed sgRNA constructs in less than 3 days.

Results  

Cloning of a single guide RNA

To facilitate streamlined and rapid cloning of sgRNAs into a plant-specific expression vector (pDE-Cas9), the authors devised a cloning strategy that employed one set of target-specific primers and a second set of universal primers (Figure 1). Each primer set generated two fragments: Fragment A, consisting of a U6-26(P) promoter and a 20-nt protospacer sequence at the 3' end; and Fragment B, consisting of an sgRNA and a 15-bp overlap of the 3' end of the protospacer (required for In-Fusion-directed homologous recombination) that extended to the 5' end of the fragment. Fragments A and B were then assembled and placed into the linearized pDE-Cas9 vector in a single-step, single-tube In-Fusion cloning reaction.

Schematic for In-Fusion cloning of a single sgRNA into pDE-Cas9 vector.

Figure 1. Schematic for cloning a single sgRNA into a linearized vector using In-Fusion Cloning HD Plus. Fragments A and B were generated by PCR, then fused and directionally cloned into a linearized pDE-Cas9 vector in a one-step In-Fusion Cloning reaction.


Following cloning, plasmids were isolated from four colonies for restriction digest screens. While ligase-based methods can require screening large numbers of colonies to find a positive clone, all four In-Fusion clones gave the expected digest patterns. Subsequent sequencing of one vector (pDE-Cas9-gYFP1) demonstrated successful integration of the fragments in the correct orientation. These results indicate the successful generation of multiple, ready-to-transfect vectors in less than three days' time.

Cloning of multiple guide RNAs

The authors then modified this method to allow them to clone two sgRNAs into the pDE-Cas9 vector by using two sets of target-specific primers and two sets of universal primers (Figure 2). A first round of PCR generated four fragments—A, B, C, and D. Fragments A and C each consisted of a U6-26(P) promoter and a 20-nt protospacer, while fragments B and D each represented a single sgRNA sequence. In a second round of PCR, Fragments A and B were fused into fragment AB, while Fragments C and D were fused into fragment CD. Following second-round PCR, fragments AB and CD were gel purified, then assembled and inserted into the linearized pDE-Cas9 vector in a single-step, single-tube In-Fusion Cloning reaction.

Schematic for In-Fusion cloning of multiple sgRNA constructs into pDE-Cas9.

Figure 2. Schematic for cloning multiple sgRNAs into a linearized vector with In-Fusion Cloning HD Plus. Fragments A, B, C, and D were amplified (Panel A) and fused into fragments AB and CD (Panel B) by PCR. Fragments AB and CD were then fused and directionally cloned into a linearized pDE-Cas9 vector in a one-step In-Fusion Cloning reaction.

Following cloning, five colonies were analyzed by restriction digestion and all five clones gave the expected digestion pattern. Subsequent sequencing of each clone revealed the successful integration of fragments in the correct orientation 100% of the time. These findings demonstrate the utility of this protocol in efficiently generating ready-to-transfect constructs with both single and multiple sgRNAs in less than three days' time.

Conclusions  

Traditional sgRNA cloning methods are inefficient, time- and labor-intensive, require screening of large numbers of colonies to identify successful inserts, and cannot be easily shuttled between expression vectors. The In-Fusion Cloning methods described here eliminate these difficulties and allow the rapid, highly efficient generation of vectors containing single or multiple sgRNAs in a single-step, single-tube cloning reaction. We also note that the authors' methods may be adapted to your vector(s) of choice by simply modifying the universal primer sequences, providing a way to quickly and efficiently make constructs for your CRISPR/Cas-9 experiments.

To view more resources on sgRNA cloning with In-Fusion technology, visit our overview page.

Methods  

In-Fusion seamless cloning reactions were performed using a wide variety of insert to vector ratios, all of which were successful (though we recommend a 2:1 ratio of insert:vector). Cloning reaction mixes were assembled using fragment AB or fragments AB and CD (15–180 fmoles and 0.5 µl each fragment), 0.5 µl linearized pDE-Cas9 vector (15–90 fmoles), and 0.3 µl 5X In-Fusion HD Cloning Plus mix. These reactions were incubated for 15 minutes at 50°C, inactivated for 10 minutes at 80°C, then 1 µl of the reaction mix was transformed into 20 µl of Stellar Competent Cells. The authors noted that using gel-purified fragments resulted in a 44-fold increase of colonies compared to unpurified fragments: ~200 per reaction gel-purified, compared to ~4.5 per reaction for unpurified fragments. The authors selected four single sgRNA or five multiple sgRNA clones, all of which contained the expected fragment sizes restriction fragment length polymorphism analyses. Sequencing analyses of these constructs revealed 100% of the clones contained the correct sequence and orientation of the targeted inserts.

References  

Khan, A. A. et al. A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants. Plant Methods 13, 86 (2017).

Related Products

Cat. # Product Size License Quantity Details
636763 Stellar™ Competent Cells 10 x 100 uL USD $222.00

Stellar Competent Cells are an E. coli HST08 strain that provides high transformation efficiency. These cells can be used in a wide variety of applications—from preparation of cDNA and genomic libraries, to construction of longer-length genomic libraries, to subcloning, and even methylated DNA cloning. Stellar Competent Cells lack the gene cluster for cutting foreign methylated DNA (mrr-hsdRMS-mcrBC and mcrA), and are therefore useful for cloning methylated DNA. The cells can also be used for blue/white screening (i.e., α-complementation) when transformed with vectors containing the lacZα gene. A pUC19 vector is provided as well as SOC Medium. Stellar Competent Cells are recommended for use with Clontech's In-Fusion PCR Cloning Kits.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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636763: Stellar Competent Cells

636763: Stellar Competent Cells

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Stellar Competent Cells provided transformation efficiencies equivalent to or better than those obtained using DH5α or DH10B competent cells

Stellar Competent Cells provided transformation efficiencies equivalent to or better than those obtained using DH5α or DH10B competent cells

The high efficiency of Stellar Competent Cells was especially noticeable in the transformation of 10 kb and 20 kb plasmids. Using DNA plasmids of 2 kb (100 pg), 10 kb (1 ng), and 20 kb (1 ng), each strain of competent cells was transformed and plated on ampicillin-containing LB agar plates. Transformation efficiencies were determined based on the colony counts obtained.

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Stellar Competent Cells provided the highest transformation efficiencies for all the ligation reaction mixtures tested.

Stellar Competent Cells provided the highest transformation efficiencies for all the ligation reaction mixtures tested.

The difference in transformation efficiencies of the competent cells was particularly prominent when cloning 20 kb fragments. Ligation reactions were set up with either 100 ng of a 2 kb, Hind III-cut DNA fragment + 50 ng of Hind III/BAP-cut pUC118 vector, or 75 ng of a 20 kb, Hind III-cut DNA fragment + 25 ng of Hind III/BAP-cut pUC118 vector. All reactions were performed at 16℃ for 6 hours using the Takara DNA Ligation Kit (Cat# 6023 or 6024). Each strain of competent cells was transformed using portions of these reaction mixtures and plated on ampicillin-containing LB agar (+ X-gal). Transformation efficiencies were determined based on the white colony counts obtained.

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Stellar Competent Cell colonies showed a faster growth rate than DH10B colonies

Stellar Competent Cell colonies showed a faster growth rate than DH10B colonies

Stellar colonies transformed with larger plasmid clearly showed a faster growth rate. DNA plasmids of 2 kb (Panel A) and 10 kb (Panel B) were each used to transform Stellar and DH10B competent cells with similar genetic characteristics, including methylation requiring restriction. This experiment used the same method as in Figure 1. Photographs of colonies on agar were taken after 15 hours of incubation.

636766 Stellar™ Competent Cells 50 x 100 uL USD $770.00

Stellar Competent Cells are an E. coli HST08 strain that provides high transformation efficiency. These cells can be used in a wide variety of applications-from preparation of cDNA and genomic libraries, to construction of longer-length genomic libraries, to subcloning, and even methylated DNA cloning. Stellar Competent Cells lack the gene cluster for cutting foreign methylated DNA (mrr-hsdRMS-mcrBC and mcrA), and are therefore useful for cloning methylated DNA. The cells can also be used for blue/white screening (i.e., α-complementation) when transformed with vectors containing the lacZα gene. This package includes 50 tubes of competent cells (100 μl/tube), SOC Medium and a pUC19 vector.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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636766: Stellar Competent Cells

636766: Stellar Competent Cells

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Stellar Competent Cells provided transformation efficiencies equivalent to or better than those obtained using DH5α or DH10B competent cells

Stellar Competent Cells provided transformation efficiencies equivalent to or better than those obtained using DH5α or DH10B competent cells

The high efficiency of Stellar Competent Cells was especially noticeable in the transformation of 10 kb and 20 kb plasmids. Using DNA plasmids of 2 kb (100 pg), 10 kb (1 ng), and 20 kb (1 ng), each strain of competent cells was transformed and plated on ampicillin-containing LB agar plates. Transformation efficiencies were determined based on the colony counts obtained.

Back

Stellar Competent Cells provided the highest transformation efficiencies for all the ligation reaction mixtures tested.

Stellar Competent Cells provided the highest transformation efficiencies for all the ligation reaction mixtures tested.

The difference in transformation efficiencies of the competent cells was particularly prominent when cloning 20 kb fragments. Ligation reactions were set up with either 100 ng of a 2 kb, Hind III-cut DNA fragment + 50 ng of Hind III/BAP-cut pUC118 vector, or 75 ng of a 20 kb, Hind III-cut DNA fragment + 25 ng of Hind III/BAP-cut pUC118 vector. All reactions were performed at 16℃ for 6 hours using the Takara DNA Ligation Kit (Cat# 6023 or 6024). Each strain of competent cells was transformed using portions of these reaction mixtures and plated on ampicillin-containing LB agar (+ X-gal). Transformation efficiencies were determined based on the white colony counts obtained.

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Stellar Competent Cell colonies showed a faster growth rate than DH10B colonies

Stellar Competent Cell colonies showed a faster growth rate than DH10B colonies

Stellar colonies transformed with larger plasmid clearly showed a faster growth rate. DNA plasmids of 2 kb (Panel A) and 10 kb (Panel B) were each used to transform Stellar and DH10B competent cells with similar genetic characteristics, including methylation requiring restriction. This experiment used the same method as in Figure 1. Photographs of colonies on agar were taken after 15 hours of incubation.

638943 In-Fusion® Snap Assembly Master Mix 500 Rxns Inquire for Quotation *

In-Fusion Snap Assembly Master Mix is designed for fast, directional cloning of one or more fragments of DNA into any vector. This proprietary master mix fuses DNA fragments (e.g., PCR-generated sequences and linearized vectors) efficiently and precisely by recognizing a 15-bp overlap at their ends. This 15-bp overlap can be engineered into the primers designed for PCR amplification of the desired sequences. In Fusion Snap Assembly Master Mix offers high efficiency, even for applications that can be challenging, including the cloning of long fragments, short oligonucleotides, and multiple fragments.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

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The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

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638943: In-Fusion Snap Assembly Master Mix

638943: In-Fusion Snap Assembly Master Mix
638944 In-Fusion® Snap Assembly Master Mix 1,000 Rxns Inquire for Quotation *

In-Fusion Snap Assembly Master Mix is designed for fast, directional cloning of one or more fragments of DNA into any vector. This proprietary master mix fuses DNA fragments (e.g., PCR-generated sequences and linearized vectors) efficiently and precisely by recognizing a 15-bp overlap at their ends. This 15-bp overlap can be engineered into the primers designed for PCR amplification of the desired sequences. In Fusion Snap Assembly Master Mix offers high efficiency, even for applications that can be challenging, including the cloning of long fragments, short oligonucleotides, and multiple fragments.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

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The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

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638944: In-Fusion Snap Assembly Master Mix

638944: In-Fusion Snap Assembly Master Mix
638945 In-Fusion® Snap Assembly Starter Bundle 10 Rxns USD $286.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

In-Fusion Snap Assembly Master Mix enables high-efficiency, high-fidelity, directional cloning of one or more PCR fragments into any vector. In addition to the cloning kit, this package includes:

- A NucleoSpin Gel and PCR Clean-Up kit: This kit is suitable for gel extraction as well as PCR purification. Kits are provided with individual purification columns.

- Stellar Competent Cells: High-efficiency competent cells are essential to the success of In-Fusion Cloning. An E. coli HST08 strain is included that provides high transformation efficiency (greater than 5 x 10^8 cfu/µg) and complements the efficiency of all In-Fusion Snap Assembly kits. Cells are provided in 100-μl aliquots in individual tubes.

- PrimeSTAR Max DNA Polymerase: This convenient 2X liquid master mix offers exceptionally accurate, efficient, and fast DNA amplification. The premix contains dNTPs and an optimized buffer, allows rapid setup of PCR reactions, and facilitates successful cloning. The polymerase master mix is provided in 625-μl aliquots.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

Back

The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

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638945: In-Fusion Snap Assembly Starter Bundle

638945: In-Fusion Snap Assembly Starter Bundle
638946 In-Fusion® Snap Assembly Value Bundle 50 Rxns USD $1063.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

In-Fusion Snap Assembly Master Mix enables high-efficiency, high-fidelity, directional cloning of one or more PCR fragments into any vector. In addition to the cloning kit, this package includes:

- A NucleoSpin Gel and PCR Clean-Up kit: This kit is suitable for gel extraction as well as PCR purification. Kits are provided with individual purification columns.

- Stellar Competent Cells: High-efficiency competent cells are essential to the success of In-Fusion Cloning. An E. coli HST08 strain is included that provides high transformation efficiency (greater than 5 x 10^8 cfu/µg) and complements the efficiency of all In-Fusion Snap Assembly kits. Cells are provided in 100-μl aliquots in individual tubes.

- PrimeSTAR Max DNA Polymerase: This convenient 2X liquid master mix offers exceptionally accurate, efficient, and fast DNA amplification. The premix contains dNTPs and an optimized buffer, allows rapid setup of PCR reactions, and facilitates successful cloning. The polymerase master mix is provided in 625-μl aliquots.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

Back

The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

Back

638946: In-Fusion Snap Assembly Value Bundle

638946: In-Fusion Snap Assembly Value Bundle
638947 In-Fusion® Snap Assembly Master Mix 10 Rxns USD $177.00

In-Fusion Snap Assembly Master Mix is designed for fast, directional cloning of one or more fragments of DNA into any vector. This proprietary master mix fuses DNA fragments (e.g., PCR-generated sequences and linearized vectors) efficiently and precisely by recognizing a 15-bp overlap at their ends. This 15-bp overlap can be engineered into the primers designed for PCR amplification of the desired sequences. In Fusion Snap Assembly Master Mix offers high efficiency, even for applications that can be challenging, including the cloning of long fragments, short oligonucleotides, and multiple fragments.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data

Back

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

Back

638947: In-Fusion Snap Assembly Master Mix

638947: In-Fusion Snap Assembly Master Mix

Back

The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

638948 In-Fusion® Snap Assembly Master Mix 50 Rxns USD $712.00

In-Fusion Snap Assembly Master Mix is designed for fast, directional cloning of one or more fragments of DNA into any vector. This proprietary master mix fuses DNA fragments (e.g., PCR-generated sequences and linearized vectors) efficiently and precisely by recognizing a 15-bp overlap at their ends. This 15-bp overlap can be engineered into the primers designed for PCR amplification of the desired sequences. In Fusion Snap Assembly Master Mix offers high efficiency, even for applications that can be challenging, including the cloning of long fragments, short oligonucleotides, and multiple fragments.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data

Back

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

Back

638948: In-Fusion Snap Assembly Master Mix

638948: In-Fusion Snap Assembly Master Mix

Back

The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

638949 In-Fusion® Snap Assembly Master Mix 250 Rxns USD $2836.00

In-Fusion Snap Assembly Master Mix is designed for fast, directional cloning of one or more fragments of DNA into any vector. This proprietary master mix fuses DNA fragments (e.g., PCR-generated sequences and linearized vectors) efficiently and precisely by recognizing a 15-bp overlap at their ends. This 15-bp overlap can be engineered into the primers designed for PCR amplification of the desired sequences. In Fusion Snap Assembly Master Mix offers high efficiency, even for applications that can be challenging, including the cloning of long fragments, short oligonucleotides, and multiple fragments.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data

Back

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

Back

638949: In-Fusion Snap Assembly Master Mix

638949: In-Fusion Snap Assembly Master Mix

Back

The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

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Takara Bio is proud to offer GMP-grade manufacturing capabilities at our award-winning facility in Kusatsu, Shiga, Japan.

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That's GOOD Science!

What does it take to generate good science? Careful planning, dedicated researchers, and the right tools. At Takara Bio, we thoughtfully develop best-in-class products to tackle your most challenging research problems, and have an expert team of technical support professionals to help you along the way, all at superior value.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

Clontech, TaKaRa, cellartis

  • Products
  • COVID-19 research
  • Next-generation sequencing
  • Diagnostic solutions
  • Real-time PCR
  • Stem cell research
  • mRNA and cDNA synthesis
  • PCR
  • Cloning
  • Nucleic acid purification
  • Gene function
  • Protein research
  • Antibodies and ELISA
  • New products
  • Special offers
  • COVID-19 research
  • Viral detection with qPCR
  • SARS-CoV-2 pseudovirus
  • Human ACE2 stable cell line
  • Viral RNA isolation
  • Viral and host sequencing
  • Vaccine development
  • CRISPR screening
  • Drug discovery
  • Immune profiling
  • Publications
  • Next-generation sequencing
  • RNA-seq
  • DNA-seq
  • Single-cell NGS automation
  • Reproductive health
  • Bioinformatics tools
  • Whole genome amplification
  • Immune profiling
  • Diagnostic solutions
  • Reproductive health
  • Real-time PCR
  • Real-time PCR kits
  • Reverse transcription prior to qPCR
  • High-throughput qPCR solutions
  • RNA extraction and analysis for real-time qPCR
  • Stem cell research
  • Media and supplements
  • Stem cells and stem cell-derived cells
  • Single-cell cloning of edited hiPS cells
  • mRNA and cDNA synthesis
  • In vitro transcription
  • cDNA synthesis kits
  • Reverse transcriptases
  • RACE kits
  • Purified cDNA & genomic DNA
  • Purified total RNA and mRNA
  • PCR
  • Most popular polymerases
  • High-yield PCR
  • High-fidelity PCR
  • GC rich PCR
  • PCR master mixes
  • Cloning
  • In-Fusion seamless cloning
  • Competent cells
  • Ligation kits
  • Restriction enzymes
  • Nucleic acid purification
  • Plasmid purification kits
  • Genomic DNA purification kits
  • DNA cleanup kits
  • RNA purification kits
  • Cell-free DNA purification kits
  • Microbiome
  • Gene function
  • Gene editing
  • Viral transduction
  • Fluorescent proteins
  • T-cell transduction and culture
  • Tet-inducible expression systems
  • Transfection reagents
  • Cell biology assays
  • Protein research
  • Purification products
  • Two-hybrid and one-hybrid systems
  • Mass spectrometry reagents
  • Antibodies and ELISA
  • Primary antibodies and ELISAs by research area
  • Fluorescent protein antibodies
  • Special offers
  • Free samples
  • TB Green qPCR sale
  • PrimeSTAR enzyme promo
  • Try BcaBEST DNA Polymerase ver.2.0
  • RNA purification sale
  • Capturem IP and Co-IP sale
  • Baculovirus titration kits early access program
  • NGS bundle and save
  • Free sample: PrimePath Direct Saliva SARS-CoV-2 Detection Kit
  • TALON his-tag purification resin special offer
  • GoStix Plus special offers
  • PCR samples
  • Services & Support
  • Instrument services
  • Stem cell services
  • Gene and cell therapy manufacturing
  • Customer service
  • Sales
  • Shipping & delivery
  • Technical support
  • Feedback
  • Online tools
  • Partnering & Licensing
  • Vector information
  • Instrument services
  • Apollo services
  • ICELL8 services
  • SmartChip services
  • Stem cell services
  • Clinical-grade stem cell services
  • Research-grade stem cell services
  • Outsourcing stem cell-based disease model development
  • Gene and cell therapy manufacturing
  • Services
  • Facilities
  • Our process
  • Resources
  • Sales
  • Make an appointment with your sales rep
  • Online tools
  • GoStix Plus FAQs
  • Vector information
  • Vector document overview
  • Vector document finder
  • Learning centers
  • Automation systems
  • Next-generation sequencing
  • cDNA synthesis
  • Real-time PCR
  • Nucleic acid purification
  • PCR
  • Cloning
  • Stem cell research
  • Gene function
  • Protein research
  • Antibodies and ELISA
  • Automation systems
  • SmartChip Real-Time PCR System introduction
  • ICELL8 introduction
  • Next-generation sequencing
  • Technical notes
  • Featured kits
  • Technology and application overviews
  • FAQs and tips
  • DNA-seq protocols
  • Bioinformatics resources
  • Webinars
  • Real-time PCR
  • Overview
  • Reaction size guidelines
  • Guest webinar: extraction-free SARS-CoV-2 detection
  • Guest webinar: developing and validating molecular diagnostic tests
  • Technical notes
  • Nucleic acid purification
  • Nucleic acid extraction webinars
  • Product demonstration videos
  • Product finder
  • Plasmid kit selection guide
  • RNA purification kit finder
  • PCR
  • Citations
  • Selection guides
  • Technical notes
  • FAQ
  • Cloning
  • In-Fusion Cloning general information
  • Primer design and other tools
  • In‑Fusion Cloning tips and FAQs
  • Applications and technical notes
  • Stem cell research
  • Overview
  • Protocols
  • Technical notes
  • Gene function
  • Gene editing
  • Viral transduction
  • T-cell transduction and culture
  • Inducible systems
  • Cell biology assays
  • Protein research
  • Capturem technology
  • Antibody immunoprecipitation
  • His-tag purification
  • Other tag purification
  • Expression systems
  • APPLICATIONS
  • Molecular diagnostics
  • Vaccine development
  • Pathogen detection
  • Immunotherapy research
  • Cancer research
  • Alzheimer's disease research
  • Reproductive health technologies
  • Molecular diagnostics
  • Interview: adapting to change with Takara Bio
  • Applications
  • Solutions
  • Partnering
  • Webinar: Speeding up diagnostic development
  • Contact us
  • Vaccine development
  • Characterizing the viral genome and host response
  • Identifying and cloning vaccine targets
  • Expressing and purifying vaccine targets
  • Immunizing mice and optimizing vaccine targets
  • Pathogen detection
  • Sample prep
  • Detection methods
  • Identification and characterization
  • SARS-CoV-2
  • Antibiotic-resistant bacteria
  • Food crop pathogens
  • Waterborne disease outbreaks
  • Viral-induced cancer
  • Immunotherapy research
  • T-cell therapy
  • Antibody therapeutics
  • T-cell receptor profiling
  • TBI initiatives in cancer therapy
  • Cancer research
  • Sample prep from FFPE tissue
  • Sample prep from plasma
  • Cancer biomarker discovery
  • Cancer biomarker quantification
  • Single cancer cell analysis
  • Cancer genomics and epigenomics
  • HLA typing in cancer
  • Gene editing for cancer therapy/drug discovery
  • Alzheimer's disease research
  • Antibody engineering
  • Sample prep from FFPE tissue
  • Single-cell sequencing
  • Reproductive health technologies
  • Preimplantation genetic testing
  • ESM Collection Kit forms
  • About
  • BioView blog
  • That's Good Science!
  • Our brands
  • Our history
  • Announcements
  • Events
  • Careers
  • Trademarks
  • License statements
  • Quality and compliance
  • Takara Bio affiliates & distributors
  • Need help?
  • Website FAQs
  • BioView blog
  • Automation
  • Cancer research
  • Career spotlights
  • Current events
  • Customer stories
  • Gene editing
  • Research news
  • Single-cell analysis
  • Stem cell research
  • Tips and troubleshooting
  • Women in STEM
  • That's Good Support!
  • About our blog
  • That's Good Science!
  • SMART-Seq Pro Biomarker Discovery Contest
  • DNA extraction educational activity
  • That's Good Science Podcast
  • Season one
  • Season two
  • Season three
  • Our brands
  • Takara
  • Clontech
  • Cellartis
  • Events
  • Biomarker discovery events
  • Calendar
  • Conferences
  • Speak with us
  • Careers
  • Company benefits
  • Takara Bio affiliates & distributors
  • United States and Canada
  • China
  • Japan
  • Korea
  • Europe
  • India
  • Affiliates & distributors, by country
  • Need help?
  • Privacy request
  • Products
  • Services & Support
  • Learning centers
  • APPLICATIONS
  • About
  • Contact Us