Sep 23, 2019
HiFi Gibson Assembly (Protocol for the NEBuilder® HiFi DNA Assembly Master Mix)
- Alba Balletbó1
- 1Wageningen University
- iGEM Wageningen 2019
Protocol Citation: Alba Balletbó 2019. HiFi Gibson Assembly (Protocol for the NEBuilder® HiFi DNA Assembly Master Mix). protocols.io https://dx.doi.org/10.17504/protocols.io.7kchksw
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: September 23, 2019
Last Modified: September 23, 2019
Protocol Integer ID: 28004
Keywords: hifi gibson assembly, protocol for dna assembly, gibson assembly, dna assembly, assembly, dna
Abstract
This is the protocol for DNA Assembly using the NEBuilder® HiFi DNA Assembly Master Mix.
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
Troubleshooting
Set up the following reaction on ice (to 20μl total volume):
| 2-3 Fragment Assembly | 4-6 Fragment Assembly | ||
| DNA Ratio | Vector:Insert = 1:2 | Vector:Insert = 1:1 | |
| Total amount of Fragments | 0.03-0.2 pmols | 0.2-0.5 pmols | |
| NEBuilder HiFi DNA Assembly Master Mix | 10 μL | 10 μL | |
| Deionized H2O | 10 - X μL | 10 - X μL | |
| Total Volume | 20 μL | 20 μL |
Incubate samples in a thermocycler at 50ºC for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled).
Following incubation, store samples on ice or at –20ºC for subsequent transformation.
Transform into chemically competent cells (1-5µL) or in electrocompetent cells (1 µL, diluted 10x or 5 µL, after purification to remove salts).