Gibson Assembly

Goal

Mix preparation

Gibson Assembly reaction

1. Set up the thermocycler at 50°C for one hour.


2. In a 50 µL PCR tube, introduce :
   • 15 µL of Master Mix (1.33 X)
   • 5 µL of DNA fragments

   Optimized cloning efficiency is 50-100 ng of vectors with 2-3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps.
   
   

   SAFETY AND USEFUL RECOMMANDATION

   Those products are not dangerous at first sight, but in case of spreading or contacts with any part of the body, it is recommended to wash this part and to dry it after. Use a different pipette tip for each sample. It is also important to close tubes after putting the different reagents. DNA is more sensitive to its environment while it is not circulared in a plasmid and introduced into a cell. Therefore, a spreading has very few chances to lead to any bad consequences.

   
   
3. Put the 50 µL PCR tube into the thermocycler.


4. Turn on the device.



SAFETY AND USEFUL RECOMMANDATION

It was noticed that it exists a certain number of different thermocycler models. That is why biologists do not know, all the time, how the device they will use, has to be manipulated. This can present risks for thermocycler users. Indeed a thermocycler is an electric device which can become very hot, and in case of misused, people can be injured. Consequently it is very important for users to know how to use the device. It is recommended to read the operating instructions before using it. Besides, the device has protective measures to prevent accidents.

References

• Gibson D. G. (2009). One-step enzymatic assembly of DNA molecules up to several hundred kilobases in size. Nature Protocols.


• Gibson D. G., Young L., Chuang R-Y, Venter J. C., Hutchison C. A., Smith H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. Vol. 6 No. 5.