Cell‑free enzymatic DNA production: Fast, clean, and fermentation‑free for streamlined manufacturing.
Backbone free dbDNA™ (doggybone™ DNA): Lacks bacterial sequence and antibiotic markers to simplify regulation and boost safety.
High‑purity DNA: Ideal for sensitive applications, including mRNA therapeutic workflows.
Closed-ended structure: Provides enhanced stability and reduced NHEJ (in gene editing applications).
Scalable from R&D to GMP applications: Move from benchtop testing to commercial‑scale production with ease.
Low DNA input and high yields: Reduces material cost.
Flexible cloning solutions: Includes a versatile workflow-ready destination vector with best‑practice guidance supported by our robust cloning portfolio products.
Product Information
In collaboration with Touchlight, NEB has developed the EnClose Cell-free dbDNA Synthesis Kit. It includes everything needed to enzymatically generate closed-ended linear double-stranded (doggybone) dbDNA (Figure 1) containing a sequence of interest (SOI). The robust combination of phi29-XT DNA Polymerase for high-yield rolling circle amplification (RCA) and TelN Protelomerase for deconcatenation and covalent closure of linear dsDNA ends enables a streamlined one-day workflow (Figure 2) that produces cell-free dbDNA for downstream applications such as in vitro transcription (IVT), LV vector and AAV vector production and more.
Figure 1: dbDNA (doggybone DNA) is a linear, double-stranded DNA with covalently-closed ends
Figure 2: Overview of the EnClose Cell-free dbDNA Synthesis Kit workflow
The dbDNA Vector (Figure 3) provided with this kit is designed to serve as both an input source for positive control reactions, and as an optimized, flexible destination vector for your own sequence of interest (SOI) upstream of dbDNA production. The dbDNA Vector utilizes a high-copy pUC origin (ori) for higher yields and contains the Kanamycin resistance gene (KanR) for reliable transformant selection following cloning.
Figure 3: EnClose Cell-free dbDNA Synthesis Kit: Scalable from screening to high-yield production
dbDNA synthesis reactions scale linearly across a wide volume range, supporting workflows from 10 µl screening assays to 2,000 µl high-yield production needs. Figure 4: dbDNA yield scales linearly with RCA reaction volume between 10 µl and 100 µl
Quadruplicate dbDNA reactions were initiated using RCA reaction volumes of 10, 20, 50 or 100 µl with the dbDNA™ Vector as a control input. The volumes of reaction components added for each subsequent step were scaled proportionately to the protocol in the E9301 kit manual. The dbDNA products were purified using the Monarch® Spin High-Capacity DNA Cleanup Kit (NEB #T1135) and eluted in 100 µl of 0.1X Tris-EDTA.
A. Products were run on a 1.2% agarose gel to verify dbDNA size and purity. B. Product yields were quantified using an Unchained Labs® Lunatic® spectrophotometer. Figure 5: dbDNA reactions generate high yields of product in a short period of time; NEBuilder HiFi DNA Assembly reactions allow for completely cell-free process
Duplicate dbDNA reactions were carried out using the indicated amount of dbDNA™ Vector as a control input or diluted NEBuilder® HiFi reaction DNA. NEBuilder HiFi reactions were diluted 1:10 with 0.1X Tris-EDTA, and 0.4 µl of diluted NEBuilder HiFi reaction was used per 20 µl RCA reaction. 20 µl RCA reactions were carried out for the indicated times.
A. Products were run on a 1.2% agarose gel to verify dbDNA size and purity. B. Product yields were quantified using an Unchained Labs Lunatic spectrophotometer.
Cloning your SOI into the dbDNA Vector is uniquely flexible. It can be achieved through a variety of cloning modalities supported by New England Biolabs, including traditional restriction enzyme (RE) cloning, NEBuilder® HiFi DNA Assembly, and NEBridge® Golden Gate Assembly (for insertion of high-complexity or repetitive elements). Visit Cloning into the dbDNA™ Vector for more information.
Figure 6: dbDNA Vector plasmid map & sequence
A. dbDNA Vector plasmid map. B. Upstream (top) and downstream (bottom) sequence of dbDNA Vector for traditional restriction enzyme cloning and NEBuilder® HiFi DNA Assembly primer design.
This product is related to the following categories:
Workflow can be made entirely cell-free by going straight from NEBuilder HiFi DNA Assembly Reaction directly into dbDNA workflow and increasing RCA step from 3 to 6 hours
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Licenses
This product is licensed for commercial or non-commercial research and development activities only from Touchlight IP Limited under Patent No. EP2391731 and other patents pending and granted worldwide, provided that no rights are granted for use of the product in research and development activities for the treatment and control of the health and/or behavior of and the diagnosis, prevention, treatment and control of diseases and/or conditions affecting animals other than human beings. For information on dbDNA™ and its ability to fast track your project with cell-free DNA please contact Touchlight at info@touchlight.com or visit Touchlight’s website at https://touchlight.com.
Based on your Freezer Program type, you are trying to add a product to your cart that is either not allowed or not allowed with the existing contents of your cart. Please review and update your order accordingly. If you have any questions, please contact Customer Support at freezers@neb.com or 1-800-632-5227 x 8.
Choose your country
North America
Europe
Asia-Pacific
Session Expired
Your session has expired
You have been idle for more than 20 minutes, for your security you have been logged out. Please sign back in to continue your session.
Please sign in to continue shopping.
Institution Changed
Your profile has been mapped to an Institution, please sign back for your profile updates to be completed.
Sign in to your NEB account
To save your cart and view previous orders, sign in to your NEB account. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site.
Powered by Bioz
