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Latest revision as of 08:10, 31 October 2017
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
