NovoHelix® HiFi DNA Assembly Master Mix

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.

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.

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).

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.