Team:Macquarie Australia/Protocols/GibsonAssembly

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 5000 bp dsDNA is approx 0.015 pmols.

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. '''