NEBuilder^® HiFi DNA Assembly Cloning Kit

#E5520

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.

Companion Products

• Monarch® Plasmid Miniprep Kit 
• Monarch® DNA Gel Extraction Kit
• Monarch® PCR & DNA Cleanup Kit (5 μg)

1. Upon arrival, store the kit components at -80°C. Before use, thaw and vortex the master mix thoroughly and keep on ice. After first use, store the HiFi DNA Assembly Master Mix and postive controls at -20°C, store the SOC Outgrowth Medium at room temperature. Store the competent cells at -80°C.

2. 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.
   

3. 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.
   
   

4. 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), Nico21(DE3), and SHuffle^®. When using competent E. coli from a vendor other than NEB, we have seen decreased robustness of transformation with high-fidelity DNA assembled products.

5. Electroporation

   : Electroporation can increase transformation efficiency by several logs. When using the NEBuilder HiFi DNA Assembly Master Mix, use 1 μl 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"
   
   

6. 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.