Description:

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 routineassemblies 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:

• Site-directed mutagenesis
• Construction of an sgRNA-Cas9 expression vector | Animation
• Assembly of linear yeast expression cassettes

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 MethodFigure 2: NEBuilder HiFi DNA Assembly offers improved efficiency and accuracy over NEB Gibson AssemblyReactions 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 AssemblyFigure 3: NEBuilder HiFi delivers higher colony yield than In-Fusion HD Two-fragment reactions were set up using the positive control from theIn-Fusion HD Cloning Kit (Clontech Takara Bio USA, Inc), according torecommended protocols. 2 μl of assembly reaction was transformed intosupplied competent cells. 1/50 of outgrowth was spread on an Ap^R plate.View additional performance data compared to In-Fusion HD 

Comparison of DNA Assembly Reaction Types

 

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

Notes:

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.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.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\"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.

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