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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.
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After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min.
