Jan 18, 2017
Gibson Assembly (NEBuilder)
- Aalto-Helsinki 2016
Protocol Citation: Snehadri Sinha: Gibson Assembly (NEBuilder). protocols.io https://dx.doi.org/10.17504/protocols.io.gzebx3e
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: January 18, 2017
Last Modified: March 07, 2018
Protocol Integer ID: 4870
Abstract
Gibson assembly using NEBuilder HiFi DNA assembly kit. Specification of the original NEB protocol.
Set up assembly reaction on ice:
| 2–3 Fragment Assembly | ||
| Recommended DNA Ratio | vector:insert = 1:2 | |
| DNA | X μl (50 ng vector, insert(s) based on that) | |
| NEBuilderHiFi DNA Assembly Master Mix | 10 μl | |
| H2O | 10-X μl | |
| Total Volume | 20 μl |
Incubate samples in a thermocycler at 50 C for 60 minutes
Store samples on ice or -20 C for subsequent transformation.
Transform 2 uL of assembled product into E. coli competent cells. For 10-beta E. coli cells provided with the kit, use NEBuilder transformation protocol.