Feb 23, 2022
Version 2
NEBuilder HiFi DNA Assembly Reaction (E2621) V.2
- New England Biolabs1
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: [email protected]
External link: https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
Protocol Citation: New England Biolabs 2022. NEBuilder HiFi DNA Assembly Reaction (E2621). protocols.io https://dx.doi.org/10.17504/protocols.io.bfhrjj56Version created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 24, 2020
Last Modified: February 23, 2022
Protocol Integer ID: 36113
Keywords: NEBuilder HiFi DNA Assembly Master Mix, DNA assembly, InFusion, cloning, E2621, nebuilder hifi dna assembly reaction, nebuilder hifi dna assembly master mix, nebuilder hifi dna assembly kit, seamless assembly of multiple dna fragment, dna assembly, assembled dna, accuracy of dna assembly, multiple dna fragment, stranded oligonucleotide, assembled gene, different sizes of dna fragment, fragment the dna ligase seals nick, dna fragment, step cloning of multiple fragment, dna molecule, sealed dna molecule, dna ligase seals nick, including direct transformation ofe coli, direct transformation ofe coli, seamless assembly, dna, step cloning, polymerase, different enzyme, variety of other molecular biology application, cloning kit, enzyme, other molecular biology application, multiple fragment, gene, cloning, template for pcr, utility for the synthetic biology community, synthetic biology community
Abstract
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 in Guidelines):
- 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 ofE coli.
NEBuilder HiFi DNA Assembly kits are available in various formats: with NEB 5-alpha chemically competent cells (Cloning Kit, NEB #E5520), as a bundle with NEB 10-beta chemically competent cells (Bundle for Large Fragments, NEB #E2623) and without competent cells (Master Mix, NEB #E2621) . NEB 5-alpha chemically competent cells are excellent for routine assemblies of 15 kb 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 15 kb. If the assembled genes contain repetitive sequences, NEB Stable Competent E. coli (NEB #C3040) should be used.
Guidelines
Figure 1: Overview of the NEBuilder HiFi DNA Assembly Method
Figure 2: NEBuilder HiFi DNA Assembly offers improved efficiency and accuracy over
NEB Gibson Assembly
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.
Figure 3: NEBuilder HiFi delivers higher colony yield than In-Fusion HD
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 ApRplate.
Materials
MATERIALS
NEBuilder HiFi DNA Assembly Master Mix - 50 rxnsNew England BiolabsCatalog #E2621L
NEBuilder HiFi DNA Assembly Master Mix - 10 rxnsNew England BiolabsCatalog #E2621S
NEBuilder HiFi DNA Assembly Master Mix - 250 rxnsNew England BiolabsCatalog #E2621X
Troubleshooting
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Optimal Quantities
NEB recommends a total of 0.03–0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–0.5 pmols 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 pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula, or using the tool, NEBiocalculator.
pmols = (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 instrument, absorbance at 260 nm or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
Set up the following reaction On ice (to 20 µL total volume):
| A | B | C | D | |
| 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 | |
| NEBuilder HiFi DNA Assembly Master Mix | 10 μl | 10 μl | 10 μl | |
| Deionized H2O | 10-X μl | 10-X μl | 0 | |
| Total Volume | 20 μl ✝✝ | 20 μl ✝✝ | 20 μl | |
* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment.
** To achieve optimal assembly efficiency, design 20-30 bp overlap regions between each fragment with equimolarity of all fragments (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, and use additional NEBuilder HiFi DNA Assembly Master Mix.
Determine whether your reactions will involve assembly of 2-3 fragments or 4-6 fragments and move forward with the following steps:
Assembly of 2 to 3 fragments3 steps
Incubate samples in a thermocycler at 50 °C for 00:15:00 .
Following incubation, store samples On ice or at -20 °C for subsequent transformation.
Note
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.
Transform NEB 5-alpha Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 µL assembled product , following the transformation protocol.