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NEBuilder® HiFi DNA Assembly Bundle for Large Fragments
NEB recommends NEB 10-beta Competent E. coli, High Efficiency (NEB #C3019) for assemblies larger than 15 kb. NEBuilder® HiFi DNA Assembly Bundle for Large Fragments offers a discount for purchasing these competent cells with the NEBuilder HiFi DNA Assembly Master Mix.
NEBuilder HiFi DNA Assembly Master Mix was developed to improve the efficiency and accuracy of DNA assembly. This method allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. This method has been used to assemble either single-stranded oligonucleotides or different sizes of DNA fragments with varied overlaps (15–30 bp). It has utility for the synthetic biology community, as well as those interested in one-step cloning of multiple fragments due to its ease of use, flexibility and simple master-mix format. Visit NEBuilderHifi.com for more information.
Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly
Flexible sequence design (scar-less cloning)
No PCR clean-up step required
Join DNA fragments together more efficiently, even with larger fragments or low DNA inputs
Use NEBuilder HiFi in successive rounds of assembly, because it removes 5´ and 3´ end mismatches. (Save time by avoiding time-consuming PCR amplification steps.)
(USA Customers: Please note you will receive two separate boxes when you order this product; one box will contain the competent cells on dry ice; the other box will contain NEBuilder HiFi DNA Assembly Master Mix on wet ice.)
NEB recommends NEB 10-beta Competent E. coli (High
Efficiency, NEB #C3019)
for assemblies larger than 15 kb. NEBuilder HiFi DNA Assembly Bundle for Large Fragments offers a discount
for purchasing these competent cells with NEBuilder HiFi DNA Assembly
Master Mix.
NEBuilder HiFi DNA Assembly was developed to improve the efficiency and accuracy of DNA assembly. This method allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. This method has been used to assemble either single-stranded oligonucleotides or different sizes of DNA fragments with varied overlaps (15-30 bp). It has utility for the synthetic biology community, as well as those interested in one-step cloning of multiple fragments due to its ease of use, flexibility and simple master-mix format. The reaction includes different enzymes that work
together in the same buffer (see Figure 1):
The exonuclease creates single-stranded 3´ overhangs that facilitate the annealing of fragments that share complementarity at one end (the overlap region)
The polymerase fills in gaps within each annealed fragment
The DNA ligase seals nicks in the assembled DNA
The end result is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation of E coli. NEBuilder HiFi DNA Assembly Cloning Kit is supplied with NEB 5-alpha High Efficiency Competent E.coli.
NEBuilder HiFi DNA Assembly kits are available in various formats: with NEB 5-alpha chemically competent cells (Cloning Kit, NEB #E5520), as a bundle with NEB 10-beta chemically competent cells (Bundle for Large Fragments, NEB #E2623) and without competent cells (Master Mix, NEB #E2621)
. NEB 5-alpha chemically competent cells are excellent for routine
assemblies of 15kb or less. NEB recommends NEB 10-beta Competent E. coli
(High Efficiency, NEB #C3019 ) or NEB 10-beta Electrocompetent E. coli (NEB #C3020) for assemblies larger than 15kb. If the assembled genes contain repetitive sequences, NEB Stable Competent E. coli (NEB #C3040) should be used.
NEBuilder has been used in various applications, including:
Figure 2: Overview of the NEBuilder HiFi DNA Assembly method
Figure 3: NEBuilder HiFi DNA Assembly offers improved efficiency in 4-fragment assembly reactions
Four fragments (~20 fmol) with 20 bp overlap were assembled using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621), GeneArt® Gibson Assembly® Mix (Thermo Fisher #A46627) and In-Fusion® Snap Assembly Master Mix (Takara Bio USA #638947) to create a pUC19 vector. Assembly reactions were performed at 50°C for 60 min or 15 min. 2 μl of each assembled mix was transformed into NEB 5-alpha Competent E.coli (NEB #C2987) and spread on LB/Amp plates with IPTG and X-Gal. Blue colonies ( >95% of total colonies) that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Mix yields more colonies than both competitors.
DNA Polymerase (for generating PCR products): For generating PCR Products, we recommend Q5® High-Fidelity DNA Polymerase (NEB #M0491 ) or related products, such as Q5 Hot Start High-Fidelity DNA Polymerase (NEB #M0493 ) or Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494 ).
LB (Luria-Bertani) plates with appropriate antibiotic. For selection of transformed competent cells, we recommend LB plates with appropriate antibiotic.
Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly.
Join DNA fragments together more efficiently, even with larger fragments or low DNA inputs
Use NEBuilder HiFi in successive rounds of
assembly, because it removes 5´ and 3´ end mismatches. (Save time by
avoiding time-consuming PCR amplification steps.)
Bridge two ds-fragments with a synthetic ss-DNA
oligo for simple and fast construction (e.g. linker insertion or gRNA
library)
Use with lower DNA input requirements
Switch from other systems easily, because
NEBuilder HiFi is compatible with Gibson Assembly®- and In-Fusion-
designed fragments
No licensing fee requirements from NEB for the NEBuilder products
Benefits over Traditional Cloning:
Save time with Simple and Fast Seamless Cloning
Use one system for both "standard-size" cloning and larger gene assembly products, up to 11 fragments.
Move on with your workflow, because DNA can be used immediately for transformation or as template for PCR or RCA.
Adapts easily for multiple DNA manipulations, including site-directed mutagenesis.
You will receive 3 separate products when you order this bundle: 2X (NEB #E2621S) and 1X (NEB #C3019H) (competent cells will arrive in a separate box containing dry ice).
Store the NEBuilder HiFi DNA Assembly Master Mix and positive controls at -20°C. Store the NEB 10-beta/ Stable Outgrowth Medium at 4°C. Store the competent cells at -80°C.
To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following:
DNA:PCR product purification is not necessary if the total volume of all PCR products is 20% or less of the assembly reaction volume. Higher volumes of PCR products may reduce the efficiency of high-fidelity DNA assembly and transformation due to the elevated carryover amounts of PCR reaction buffer and unused primers present in the PCR product. Column purification of PCR products may increase the efficiency of both high-fidelity DNA assembly and transformation by 2-10 fold and is highly recommended when performing assemblies of three or more PCR fragments or assembling longer than 5 kb fragments. Purified DNA for assembly can be dissolved in ddH2O (Milli-Q® water or equivalent is preferable), TE or other dilution buffers.
Insert: When directly assembling fragments into a cloning vector,the concentration of assembly fragments should be at least 2 times higher than the concentration of vector. For assembly of 4 or more fragments into a vector, we recommend using an equimolar ratio of fragments.
Biology: Some DNA structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.
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.
NEBuilder HiFi DNA Assembly Mix has been reformulated to be more stable starting with Lot #10111279 and all subsequent (higher number) lots.
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]
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
Limited Warranty: The NEBuilder® HiFi Assembly Master Mix is warranted to perform according to specifications stated on the certificate of analysis. No other warranty is made, whether express or implied, including any warranty of merchant ability or fitness for a particular purpose. This warranty limits NEB’s and its licensors’ liability to only the price of the product. Neither NEB nor its licensors shall have any responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever.
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