Home DNA Assembly Cloning and Mutagenesis Kits Products NEBuilder^® HiFi DNA Assembly Master Mix

NEBuilder^® HiFi DNA Assembly Master Mix

NEBuilder HiFi DNA Assembly Master Mix has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10238675.

Did you know this product can be purchased in larger volumes? Submit an inquiry to find out more.

NEBuilder^® HiFi DNA Assembly Master Mix allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. 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.

Also available with chemically competent cells at a discounted price.
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.)
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
Easily adapted for multiple DNA manipulations, including site-directed mutagenesis
No licensing fee requirements from NEB for NEBuilder products

Catalog #	Concentration	Size	List Price	Your Price
E2621S	Not Applicable	10 reactions	$173.00	Sign In Sign In
E2621L	Not Applicable	50 reactions	$683.00	Sign In Sign In
E2621X	Not Applicable	250 reactions	$2,680.00	Sign In Sign In

Please enter a quantity for at least one size

Want to try this product? Request a sample

Product Information

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. 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 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 15 kb 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 15 kb. 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:

To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.

For help designing your primers for use with NEBuilder, please view our primer design video.

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.

View additional performance data compared to GeneArt Gibson Assembly and In-Fusion Snap Assembly

This product is related to the following categories:: DNA Assembly, Cloning and Mutagenesis Kits Products

This product can be used in the following applications:: NEBuilder® HiFi DNA Assembly,; High-throughput cloning and automation solutions,; Genome Editing Applications

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

E2621S

-20

	NEBuilder^® Positive Control	N2611AVIAL	-20	1 x 0.05 ml	Not Applicable
	NEBuilder^® HiFi DNA Assembly Master Mix	M5520AVIAL	-20	1 x 0.1 ml	2 X

E2621L

-20

	NEBuilder^® Positive Control	N2611AVIAL	-20	1 x 0.05 ml	Not Applicable
	NEBuilder^® HiFi DNA Assembly Master Mix	M5520AAVIAL	-20	1 x 0.5 ml	2 X

E2621X

-20

	NEBuilder^® Positive Control	N2611AVIAL	-20	5 x 0.05 ml	Not Applicable
	NEBuilder^® HiFi DNA Assembly Master Mix	M5520AAVIAL	-20	5 x 0.5 ml	2 X

Properties & Usage

Materials Required but not Supplied
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.
Advantages and Features
Features

Benefits over GeneArt Gibson Assembly and In-Fusion Snap Assembly:
- 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.
Product Notes
1. 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.
2. 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.
3. Transformation: NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) provided with the NEBuilder HiFi DNA Assembly Cloning Kit are recommended for use for assembled products of less than 15kb. It is also possible to use other NEB competent E. coli strains, with the exception of BL21, BL21(DE3), Lemo21(DE3), Nico(DE3), and SHuffle®. When using competent E. coli from a vendor other than NEB, we have been decreased robustness of transformation with high-fidelity DNA assembled products.
4. Electroporation: Electroporation can increase transformation efficiency by several logs. When using the NEBuilder HiFI DNA Assembly Master Mix, use 1μl of the assembled product for electroporation, and plate multiple dilutions.
  
  Should you require the use of Electrocompetent cells, please use the "Electrocompetent Cells Transformation Protocol"
5. 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.