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NEBuilder HiFi DNA Assembly Master Mix

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货号 E2621S
品牌 NEB
CAS号
规格/包装 10 rxns
售卖单位 1EA
储存条件
现货状态一周

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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 (15–80 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 competent cells (Cloning Kit, NEB #E5520), as a bundle with NEB 10-beta competent cells (Bundle for Large Fragments, NEB #E2623) and without competent cells (Master Mix, NEB #E2621) . NEB 5-alpha competent cells 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:

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.

Figure 1: Overview of the NEBuilder HiFi DNA Assembly Method

Reactions were set up in a 2- and 6- fragment assembly reaction according to recommended reaction conditions. NEBuilder HiFi DNA Assembly results in larger numbers of colonies over NEB Gibson Assembly, for both 2- and 6-fragment assemblies.
View additional performance data compared to NEB Gibson Assembly

Two-fragment reactions were set up using the positive control from the In-Fusion HD Cloning Kit (Clontech Takara Bio USA, Inc), according to recommended protocols. 2 μl of assembly reaction was transformed into supplied competent cells. 1/50 of outgrowth was spread on an Ap^R plate.
View additional performance data compared to In-Fusion HD

Kit Components

The following reagents are supplied with this product:

	Store at (°C)	Concentration
NEBuilder® High-Fidelity Master Mix	-20
NEBuilder® Positive Control	-20	2X

Advantages and FeaturesBack to Top

Features

Benefits over NEB Gibson Assembly Master Mix and/or In-Fusion HD:

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 6 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.