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NovoHelix® HiFi DNA Assembly Master Mix

NovoHelix HiFi DNA Assembly Master Mix

NovoHelix 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.


Catalog Nr




 HiFi DNA Assembly Master Mix
 10 reactions
 HiFi DNA Assembly Master Mix
 50 reactions
 HiFi DNA Assembly Master Mix
 250 reactions
 HiFi DNA Assembly Master Mix
 1000 reactions
 HiFi DNA Assembly Master Mix

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Product Information
Product Literature & Protocol
FAQs & Troubleshooting
Supporting Documents
Product Information
Advantages & Features

  • 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 NovoHelix 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
  • No licensing fee requirements for NovoHelix 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.


HiFi DNA Assembly Master Mix contains multiple engineered enzymes including a flap endonuclease, hyperthermophilic family B DNA polymerase and hyperthermophilic ligase improved through protein engineering.  These engineered enzymes were expressed heterologously in an E. coli strain from codon-optimized synthetic genes.  The individual components containing each recombinant protein were tested and normalized to standard activity assays and assembled into the final mixture.

Heat Inactivation:

No, individual component enzymes canNOT be heat inactivated.   

NovoHelix recommends purifying the assembled DNA over a micro-spin column that facilitates the removal of modifying enzymes including DNA polymerases and nucleases if [1] the assembled DNA is less than 30-kb, [2] the assembled DNA will be transformed by electroporation, [3] the assembled DNA will not be used immediately (under ~1 hour at 4°C) and needs to be stored. DNA eluted from these micro-spin columns is typically suitable for electroporation if Type 1 ultrapure water with a resistivity of >18.2 MΩ·cm and conductivity of 0.05501 μS/cm is used for final elution on the silica membrane. If the assembled DNA is greater than 30-kb, the DNA can be desalted on a dialysis membrane with ultrapure water prior to electroporation to reduce arcing. Contaminants in the sample can be decreased to acceptable or negligible levels if the external buffer (ultrapure water) volume is large (typically 30–500 times the sample volume). It is not necessary to desalt or clean up the assembled DNA for electroporation or chemical transformation, but including this step may improve transformation efficiency.

Recommended Reaction Conditions:

  • Primer Design

The overlap sequence needs to have between 20 – 150 bp homology to the adjoining part.  NovoHelix HiFi DNA assembly primers are typically 60 bp in length (20 bp binds directly to the specific DNA element and 40 bp homology to adjoining part).

  • Optimal Quantities

NovoHelix recommends a total of 0.03–0.2 pmol of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–1 pmol of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of picomoles of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula:

pmol = (weight in ng) x 1,000 / (base pairs x 650 daltons)
50 ng of 5000 bp dsDNA is about 0.015 pmols
50 ng of 500 bp dsDNA is about 0.15 pmols

The mass of each fragment can be measured using the NanoDrop spectrophotometer by absorbance at 260 nm or estimated from agarose gel electrophoresis via comparison to a known standard such as band intensity from a DNA ladder.
Product Literature & Protocol

HiFi DNA Assembly Protocol

                                                                                                Recommended Amount of Fragments Used for Assembly

 2–3 Fragment Assembly*4–6 Fragment Assembly**
Positive Control✝
Recommended DNA Molar Ratio
 vector:insert = 1:2 vector:insert = 1:1
Total Amount of Fragments
0.03–0.2 pmols*
X μl
0.2–0.5 pmols**
X μl
10 μl
HiFi DNA Assembly Master Mix
 10 μl10 μl
10 μl
 Deionized H2O10-X μl
 10-X μl0
 Total Volume20 μl✝✝
 20 μl✝✝ 20 μl
Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.

**To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).

Control reagents are provided for 5 experiments.

†† If greater numbers of fragments are assembled, increase the volume of the reaction linearly by using additional NovoHelix HiFi DNA Assembly Master Mix. Alternatively, pool the DNA fragments into an equimolar mix first and then re-purify these pooled equimolar fragments over a micro-column and elute with a minimum volume (~10-µl). The eluate may be reapplied to the same micro-column  membrane to improve elution of large DNA fragments without increasing the final volume..

Recommended Storage Condition:   
This assembly mixture can be stored at -20 °C for at least one year.  The enzymes remain active following at least 10 freeze-thaw cycles.
FAQs & Troubleshooting
1: Ellis T, Adie T, Baldwin GS. DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol (Camb). 2011 Feb;3(2):109-18. doi: 10.1039/c0ib00070a. Epub 2011 Jan 19. Review. PubMed PMID: 21246151.

2: AlMalki FA, Flemming CS, Zhang J, Feng M, Sedelnikova SE, Ceska T, Rafferty JB, Sayers JR, Artymiuk PJ. Direct observation of DNA threading in flap endonuclease complexes. Nat Struct Mol Biol. 2016 Jul;23(7):640-6. doi: 10.1038/nsmb.3241. Epub 2016 Jun 6. PubMed PMID: 27273516; PubMed Central PMCID:  PMC4939078.

3: Kalva S, Boeke JD, Mita P. Gibson Deletion: a novel application of isothermal in vitro recombination. Biol Proced Online. 2018 Jan 19;20:2. doi:10.1186/s12575-018-0068-7. eCollection 2018. PubMed PMID: 29375275; PubMed Central PMCID: PMC5774033.

4: Schlaak C, Hoffmann P, May K, Weimann A. Desalting minimal amounts of DNA for electroporation in E. coli: a comparison of different physical methods.  Biotechnol Lett. 2005 Jul;27(14):1003-5. PubMed PMID: 16132844.

5: Miller EM, Nickoloff JA. Escherichia coli electrotransformation. Methods Mol Biol. 1995;47:105-13. PubMed PMID: 7550724.

6: Tu Q, Yin J, Fu J, Herrmann J, Li Y, Yin Y, Stewart AF, Müller R, Zhang Y. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency. Sci Rep. 2016 Apr 20;6:24648. doi: 10.1038/srep24648.  PubMed PMID: 27095488; PubMed Central PMCID: PMC4837392.

7: Saraswat M, Grand RS, Patrick WM. Desalting DNA by drop dialysis increases library size upon transformation. Biosci Biotechnol Biochem. 2013;77(2):402-4. Epub 2013 Feb 7. PubMed PMID: 23391930.

8: Guo YY, Shi ZY, Fu XZ, Chen JC, Wu Q, Chen GQ. A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation. Microb Cell Fact. 2015 Feb 12;14:18. doi: 10.1186/s12934-015-0204-x. PubMed PMID: 25896825; PubMed Central PMCID: PMC4455692.

1: Chan WT, Verma CS, Lane DP, Gan SK. A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Biosci Rep. 2013 Dec 12;33(6). pii: e00086. doi: 10.1042/BSR20130098. PubMed PMID: 24229075; PubMed Central PMCID: PMC3860579.

1: Goldsmith M, Kiss C, Bradbury AR, Tawfik DS. Avoiding and controlling double transformation artifacts. Protein Eng Des Sel. 2007 Jul;20(7):315-8. Epub 2007 Jun 15. PubMed PMID: 17575283.

1: Packer MS, Liu DR. Methods for the directed evolution of proteins. Nat Rev Genet. 2015 Jul;16(7):379-94. doi: 10.1038/nrg3927. Epub 2015 Jun 9. Review. PubMed PMID: 26055155.

1: Chen K, Arnold FH. Tuning the activity of an enzyme for unusual environments:  sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5618-22. PubMed PMID: 8516309PubMed Central PMCID: PMC46772.

1: Baek CH, Chesnut J, Katzen F. Positive selection improves the efficiency of DNA assembly. Anal Biochem. 2015 May 1;476:1-4. doi: 10.1016/j.ab.2015.01.021. Epub 2015 Feb 4. PubMed PMID: 25660533.
Supporting Documents

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