EnClose® Cell-free dbDNA™ Synthesis Kit
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
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.
Visit Practical Tips for Making and Working with dbDNA for expert guidance and best practices.
Figure 3: EnClose Cell-free dbDNA Synthesis Kit provides high dbDNA yield across constructs
The dbDNA workflow supports a range of input plasmid and product sizes, delivering > 90% yield of the desired dbDNA; yield variation depends on sequence of interest (SOI) size and features. Product yields were quantified using an Unchained Labs® Lunatic® spectrophotometer. 50 ng of DNA sample was loaded into each lane of a BioAnalyzer® 12000 to determine purity of correct dbDNA product.
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.
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.
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.
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 into the dbDNA™ Vector
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 7: 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.
Closed-ended DNA & Protelomerases
Kit Components
The following reagents are supplied with this product:
| NEB # | Component Name | Component # | Stored at (°C) | Amount | Concentration |
|---|---|---|---|---|---|
| E9301S | phi29-XT DNA Polymerase | M0572MVIAL | -20 | 1 x 0.2 ml | 10,000 units/ml |
| E9301S | EnClose® TelN Protelomerase | M0768MVIAL | -20 | 1 x 0.16 ml | 25,000 units/ml |
| E9301S | dbDNA™ Reaction Buffer | B9301SVIAL | -20 | 1 x 1.25 ml | 10 X |
| E9301S | dbDNA™ Vector | N9301SVIAL | -20 | 1 x 0.05 ml | 500 µg/ml |
| E9301S | dbDNA™ Primer Mix | S9301SVIAL | -20 | 1 x 0.2 ml | 500 µM |
| E9301S | Deoxynucleotide (dNTP) Solution Mix | N0447AAVIAL | -20 | 1 x 0.4 ml | 10 mM each dNTP |
| E9301S | Pyrophosphatase, Inorganic (E. coli) | M0361SVIAL | -20 | 1 x 0.1 ml | 100 units/ml |
| E9301S | XbaI | R0145TVIAL | -20 | 1 x 0.03 ml | 100,000 units/ml |
| E9301S | T5 Exonuclease | M0663SVIAL | -20 | 1 x 0.1 ml | 10,000 units/ml |
Advantages and Features
Features
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
Protocols, Manuals & Usage
Protocols for this product
- Cloning into dbDNA™ Vector using Restriction Enzymes (NEB #E9301)
- Cloning into dbDNA™ Vector (NEB #E9301) using NEBuilder® HiFi DNA Assembly (NEB #E2621)
Manuals
The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.
Usage Guidelines
Tools & Resources
Web Tools
FAQs & Troubleshooting
FAQs
- When digesting my dbDNA™ vector with PspOMI (NEB #R0653), the cleavage is incomplete. How can I improve the reaction?
- Can the RCA step in the EnClose® Cell-free dbDNA™ Synthesis Kit (NEB #E9301) protocol be performed at a different temperature?
- Why does my RCA reaction appear viscous when performing the EnClose® Cell-free dbDNA™ Synthesis protocol?
- Why does my RCA reaction appear cloudy when performing the EnClose® Cell-free dbDNA™ Synthesis protocol (NEB #E9301)?
- Will I get a higher yield if I allow my RCA step in the EnClose® Cell-free dbDNA™ Synthesis (NEB #E9301) protocol go for more than 3 hours (e.g., overnight)?
- How can I determine if my RCA step in the EnClose® Cell-free dbDNA™ Synthesis Kit (NEB #E9301) protocol was successful?
- Can I use my own specific primers for my RCA step in the EnClose® Cell-free dbDNA™ Synthesis protocol (NEB #E9301) instead of the provided primers?
- Can I dilute the RCA reaction before the EnClose® TelN Protelomerase reaction step in the EnClose Cell-free dbDNA™ Synthesis protocol (NEB #E9301)?
- Does the EnClose® Cell-free dbDNA™ Synthesis Kit (NEB #E9301) work with other buffers?
- Can I use the phi29-XT DNA Polymerase RCA Kit (NEB #E1603) to generate dbDNA™?
- What is the structure at the cleavage site of the dsDNA after incubation with EnClose® TelN Protelomerase?
- My sequence of interest (SOI) contains an XbaI recognition site. How can I remove the plasmid backbone without cutting my dbDNA™ product with XbaI during the enzymatic cleanup step?
- What could be causing my dbDNA™ yield to be low (NEB #E9301)?
- What yields can I expect from the EnClose® Cell-free dbDNA™ Synthesis Kit (NEB #E9301)
- What is the largest DNA construct that the EnClose® Cell-free dbDNA™ Synthesis Kit can accommodate?
Quality, Safety & Legal
Quality Control Assays
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:
Certificate of Analysis
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:
Safety Data Sheets
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
- phi29-XT DNA Polymerase
- Deoxynucleotide (dNTP) Solution Mix
- Pyrophosphatase, Inorganic (E. coli)
- XbaI
- T5 Exonuclease
Legal and Disclaimers
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.
