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Team:Macquarie Australia/Protocols/GibsonAssembly
From 2013.igem.org
Gibson Assembly Protocol
Before beginning Gibson assembly, all agar plate requirements, concentration calculations and equipment requirements were prepared. Once the gBlocks had arrived Gibson assembly was performed on all fragments.
Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer. The table below shows each tube with vector & volume, volume of Gibson Master Mix, water volume & volume of each gBlock fragment. After incubation, these were transformed into Top10 cells using the Transformation Protocol found here -
Transformation Protocol.
5. After addition of all components (shown in table) incubation was preformed at 50°C for 60 min followed.
| Element | ChlM | ChlI1 | CTH1 | POR | ChlG | DVR1 | ChlP | ChlI2 | Gene | Gene |
|---|---|---|---|---|---|---|---|---|---|---|
| Fragment 1 | 3.3uL Gblock1 | 5.0uL Gblock1 | 2.3uL Gblock1 | 4.8uL Gblock1 | ||||||
| Fragment 2 | 3.3uL Gblock2 | 3.3uL Gblock2 | 3.2uL Gblock2 | 3.0uL Gblock2 | ||||||
| Fragment 3 | 3.1uL Gblock3 | |||||||||
| 10X T4 DNA ligase buffer | 2 µL | |||||||||
| Vector (0.05pmol) | 2.7uL | 2.7uL | 2.7uL | 2.7uL | 2.7uL | 2.7uL | 2.7uL | 2.7uL | 2.7uL | 2.7uL |
| H2O | 0.7uL | |||||||||
| Gibson Master Mix | 10 uL | 11 uL | 11.3 uL | 10.5 uL |
