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  • ‹ Back to In-Fusion Cloning and competition
  • In-Fusion Snap Assembly vs. GeneArt Gibson Assembly HiFi
  • In-Fusion Snap Assembly vs. NEBuilder HiFi
  • Sequence accuracy in seamless cloning
  • Choosing a seamless cloning method
  • Improving background over Gibson Assembly
  • A successful alternative to ligation cloning
  • Single- and multiple-insert cloning
  • Easy cloning into lentiviral vectors
  • Outperforming TOPO cloning
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Home › Learning centers › Cloning › In-Fusion Cloning general information › In-Fusion Cloning and competition › Easy cloning into lentiviral vectors

In-Fusion Cloning general information

  • In-Fusion Cloning overview
  • In-Fusion Cloning guide
  • In-Fusion Cloning and competition
    • In-Fusion Snap Assembly vs. GeneArt Gibson Assembly HiFi
    • In-Fusion Snap Assembly vs. NEBuilder HiFi
    • Sequence accuracy in seamless cloning
    • Choosing a seamless cloning method
    • Improving background over Gibson Assembly
    • A successful alternative to ligation cloning
    • Single- and multiple-insert cloning
    • Easy cloning into lentiviral vectors
    • Outperforming TOPO cloning
  • In-Fusion Cloning citations
  • Stellar Competent Cells product overview and performance data
  • EcoDry reagents and sustainability
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TECH NOTE

In-Fusion Cloning: overcoming restriction site limitations in lentiviral vectors

Data kindly provided by: Jun Yang
Instructor, University of Texas Medical Branch

Introduction Results Conclusions Methods

Introduction  

In the following experiment, In-Fusion Cloning was used to clone five different PCR fragments into a lentiviral vector for eukaryotic expression. This vector had only one restriction site suitable for subcloning, which made it difficult and time-consuming to perform PCR cloning using traditional ligation methods. The In-Fusion protocol used below made it possible to eliminate those constraints. Each insert fragment was amplified with the included high-fidelity polymerase and cloned directly into the final lentiviral vector. Colonies of each transformant were screened by restriction digest in order to identify positive clones. Final vectors were complete in just three days, with only two to three hours of hands-on time required per day.

I have not tried ligation-based cloning for any of my current cloning experiments, but I have done a lot before we tested the In-Fusion kit. The major differences between these two cloning methods in our lab are the additional TA cloning, transformation, minipreps, digestion, and ligation needed for ligation-based cloning, which means at least three more working days. Typically we needed to screen at least nine colonies for each cloning experiment, with 70–80% positive confirmed by restriction digest. In my experience, the In-Fusion method is very convenient, reliable, and time-saving."

—Jun Yang, University of Texas Medical Branch

Results  

Five unique inserts, ranging in size from ~400 bp to 1.6 kb, were PCR-amplified to incorporate specific 5' and 3' ends designed for cloning into the destination lentiviral vector (~8 kb). Each PCR reaction generated strong, specific bands under a single set of PCR conditions. The inserts were cloned into the linearized vector with In-Fusion enzymes and transformed into the provided competent cells with very high efficiency. Positive clones were identified by screening three to five colonies each with restriction digest (Figure 1).

Restriction digest of clones. Screening of transformant colonies was performed via XbaI/EcoRI restriction digest. Results show only one negative colony (E1) out of all 18 colonies resulting from five individual cloning reactions.   

Figure 1. Restriction digest of clones. Screening of transformant colonies was performed via XbaI/EcoRI restriction digest. Results show only one negative colony (E1) out of all 18 colonies resulting from five individual cloning reactions.

Conclusions  

In-Fusion Cloning generated five positive lentiviral clones with a success rate of nearly 100%. PCR conditions did not need to be modified for each insert, and direct cloning into a large vector was highly efficient. The cloning locus was based on experimental preference, instead of being limited by available restriction sites. Identification of positive clones was done with minimal screening, indicating high accuracy. Final vectors were complete in just three days, with only two to three hours of hands-on time required per day.

Methods  

The following inserts were amplified via PCR with CloneAmp HiFi PCR Premix:

•  RSV NS1 and RSV NS2 fragments (~400 bp each)
•  human IRF3, IRF7, and IRF7 mutant cDNAs (~1.4–1.6 kb each)

Takara Bio's Primer Design Tool was used to design amplification primers that included 5'- and 3'-cloning ends to match the ends of the linearized lentiviral vector, such that they facilitated a successful In-Fusion reaction. PCR conditions were programmed with a denaturation step at 98°C, an annealing step of 55°C for 15 seconds, and an extension time of 1 minute at 72°C, for a total of 30 cycles.The insert PCR products were analyzed on an agarose gel and purified using the provided NucleoSpin Gel and PCR Clean-Up kit. Linearization of the destination vector was performed with XbaI and EcoRI according to the manufacturer's instructions.The purified PCR inserts were cloned into the linearized lentiviral vector with the In-Fusion HD Cloning Plus (discontinued and replaced with In-Fusion Snap Assembly) enzyme mix and then transformed into the included Stellar Competent Cells. Both protocols followed the instructions in the user manual. Three to five colonies were screened by restriction digest mapping with XbaI and EcoRI.

Learn more about our In-Fusion Cloning products by visiting the product page »

Related Products

Cat. # Product Size Price License Quantity Details
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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638943: In-Fusion Snap Assembly Master Mix

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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.

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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638944: In-Fusion Snap Assembly Master Mix

638944: In-Fusion Snap Assembly Master Mix

Back

The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

638945 In-Fusion® Snap Assembly Starter Bundle 10 Rxns USD $312.00

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

638945: In-Fusion Snap Assembly Starter Bundle

638945: In-Fusion Snap Assembly Starter Bundle
638946 In-Fusion® Snap Assembly Value Bundle 50 Rxns USD $1157.00

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

638946: In-Fusion Snap Assembly Value Bundle

638946: In-Fusion Snap Assembly Value Bundle

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.

638947 In-Fusion® Snap Assembly Master Mix 10 Rxns USD $188.00

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.

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Back

638947: In-Fusion Snap Assembly Master Mix

638947: In-Fusion Snap Assembly Master Mix

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.

638948 In-Fusion® Snap Assembly Master Mix 50 Rxns USD $750.00

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

638948: In-Fusion Snap Assembly Master Mix

638948: In-Fusion Snap Assembly Master Mix

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.

638949 In-Fusion® Snap Assembly Master Mix 250 Rxns USD $2979.00

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 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
  • Real-time PCR
  • Stem cell research
  • mRNA and cDNA synthesis
  • PCR
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  • Nucleic acid purification
  • Gene function
  • Protein research
  • Antibodies and ELISA
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  • 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
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  • Spatial omics
  • RNA-seq
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  • Single-cell NGS automation
  • Reproductive health
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  • Immune profiling
  • Real-time PCR
  • Great value master mixes
  • Signature enzymes
  • High-throughput real-time PCR solutions
  • Detection assays
  • References, standards, and buffers
  • Stem cell research
  • Media, differentiation kits, and matrices
  • Stem cells and stem cell-derived 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
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  • Nucleic acid purification
  • Automated platforms
  • Plasmid purification kits
  • Genomic DNA purification kits
  • DNA cleanup kits
  • RNA purification kits
  • 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
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  • Cloning
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  • Stem cell research
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  • Gene function
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  • Viral transduction
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  • Antibody immunoprecipitation
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  • APPLICATIONS
  • Molecular diagnostics
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  • mRNA and protein therapeutics
  • Characterizing the viral genome and host response
  • Identifying and cloning protein targets
  • Expressing and purifying protein targets
  • Immunizing mice and optimizing vaccines
  • Pathogen detection
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  • 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
  • Kickstart your cancer research with long-read sequencing
  • Sample prep from FFPE tissue
  • Sample prep from plasma
  • Cancer biomarker quantification
  • Single cancer cell analysis
  • Cancer transcriptome 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
  • Embgenix FAQs
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  • Infectious diseases
  • Develop vaccines for HIV
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