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Team:Macquarie Australia/Protocols/GibsonAssembly
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Gibson Assembly Protocol
Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on NEB guidlines.
NOTE: For efficiency with reference to the NEB Gibson protocol, we considered:
0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.
A calculation provided was used furthermore to determine the pmols required based on the length of fragments:
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
The cells were appropriately transformed and concentrations were determined prior. (transformation protocol can be found with the following link)
-Transformation Protocol.
50ng of 500 bp is approx 0.15 pmol
50-100ng of vector recommended with excess insert of 2-3 fold
Use 5 x more insert if <200bps.
** Control reagents
*** Additional master mix may be required for larger bp fragments.
After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min. '''
| 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 |
