• Introduction
  • Home
  • The Team
  • Photo Gallery
• Our Project
  • Results Summary
  • Selection System
  • In-Vitro Characterization
  • In-Vivo Characterization
  • Microfluidics
  • Data
  • Attributions
• Tools
  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
  • May
  • June
  • July
  • August
  • September
  • October
• Considerations
  • Human Practices
  • Safety
• Acknowledgements
  • Sponsors
  • Partners

Gibson assembly

We use Gibson assembly to assemble DNA fragments into plasmids. It is a multistep process, requiring first a PCR to create each fragment by copying them out of template plasmids, then a single isothermal assembly to stick the fragments together, then transformation into competent cells and culture for amplification.

Protocol adapted from Nikolaus Obholzer (https://wiki.med.harvard.edu/SysBio/Megason/IsothermalAssembly)

• Take the different DNA parts you want to assemble and measure their concentration with Nanodrop
• We need equal molecuar ratios! Divide the concentration measured above [ng/ul] by the length of each part to have equivalent numbers
• Based on these numbers, calculate the ul you need of each to have the same amount of molecules
• Take 5 ul of the mixed solution and add 15 ul of Gibson mastermix
• Heat at 50°C for 45-60 minutes (there is a programm written in the iGEM folder)
• Purify with Qiagen PCR purification kit

Now the samples are ready to be transformed.

PS: if someone has a less complicated method, please change the protocol :)