Gibson Assembly

Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases）

Applications in our project: Assembly of BBa_K2309004, BBa_K2309005, BBa_K2309022 and BBa_K2309028 with pNZ8148 respectively.

Procedure:

Prepare an ice box, put every material on ice.
Add 0.02-0.5 pmols DNA fragment (which has been amplified by PCR using primer 1 and primer 2—see at PCR protocol) into PCR tube. (The mass of DNA can be calculated by NEB-calculator)
Add 1/3 amount of DNA inserts PstI-digested pNZ8148 plasmid in the tube.
Add 2X Gibson Assembly Master Mix (produced by NEB) to the tube.
Fill the tube with dH₂O to a total volume of 20 μl.
Incubate the samples in a thermocycler at 50℃ for 15 minutes. (can be longer) Store samples on ice or at -20℃ for subsequent examination or Transformation.

Collaborators and Supporters

Team:XJTLU-CHINA/Protocols Gibson Assembly