Aug 22, 2015
HiFi DNA Assembly (NEB)
Forked from NEBuilder HiFi DNA Assembly Reaction (E2621)
- Northeastern iGEM 2015
External link: https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
Protocol Citation: Josh Timmons: HiFi DNA Assembly (NEB). protocols.io https://dx.doi.org/10.17504/protocols.io.dn85hv
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
Created: August 22, 2015
Last Modified: March 19, 2018
Protocol Integer ID: 1472
Abstract
This is the protocol for DNA Assembly using the NEBuilder® HiFi DNA Assembly Master Mix (E2621).
Guidelines
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.
Materials
MATERIALS
NEBuilder HiFi DNA Assembly Master Mix - 10 rxnsNew England BiolabsCatalog #E2621S
Set up the following reaction on ice (to 20μl total volume):
| Recommended Amount of Fragments Used for Assembly | ||||
| 2–3 Fragment Assembly* | 4–6 Fragment Assembly** | Positive Control✝ | ||
| Recommended DNA 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 |
Note
NEU iGEM found that a HiFi DNA Assembly with 7 pieces, each at 0.05pmol, was successful. Plasmid fragments, post-coloumn purification, were diluted to 0.1pmol/uL in water. 0.5uL of each fragment was then pipette into the reaction tube on ice.
Protocol
NAME
E2621 DNA Assembly ReactionCREATED BY
New England Biolabs
Vector DNA
Insert fragments DNA
NEBuilder HiFi DNA Assembly Master Mix
NEBuilder HiFi DNA Assembly Master Mix - 10 rxnsNew England BiolabsCatalog #E2621S
Deionized H2O
Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled).
01:00:00
Following incubation, store samples on ice or at –20°C for subsequent transformation.
Transform NEB 5-alpha Competent E. coli cells (provided in the cloning kit or purchased separately from NEB) with 2 μl of the assembled product, following the transformation protocol.