Oct 23, 2020
Gibson Assembly
- 1Northeast Forest University
- 2020 iGEM NEFU China
Protocol Citation: Zhujun Wei 2020. Gibson Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.bnwtmfen
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: October 23, 2020
Last Modified: October 23, 2020
Protocol Integer ID: 43699
Abstract
This protocol includes two different kits. Choose one to use.
Materials
The old kit: Trelief™ SoSoo Cloning Kit Ver.2
The new kit: ClonExpress®II One Step Cloning Kit
Safety warnings
Please wear gloves for the experiment.
Set up a small box with ice, put DNA and Gibson Mix into it before going into the metal bath or the Bio-rad S1000TM thermocycler cycler .
Add the following reagent to a PCR tube (4 μl).
| Gibson Mix | 2 μl | |
| insert | X μl | |
| vector | Y μl |
X+Y=2 ul , X : Y=3 : 1~5 : 1.
Temperature : Time= 50℃ : 15-30 min
Set up a small box with ice, put DNA and Gibson enzyme and Gibson buffer into it before going into the metal bath or the Bio-rad S1000TM thermocycler cycler .
Add the following reagent to a PCR tube (10 μl).
| Gibson enzyme | 1 μl | |
| Gibson buffer | 2μl | |
| insert | X μl | |
| vector | Y ul |
X=0.02× vector length /vector concentration
Y=0.02× insert length /insert concentration
Temperature : Time= 37℃ : 30-60 min