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Team:Cambridge/Protocols/Gibson Assembly
From 2011.igem.org
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Gibson Assembly
Theory
Gibson Assembly as a scar free method of DNA recombination that is highly efficient and readily copes with combination of multiple DNA fragments at once.
The reaction relies on a temperature sensitive exonuclease known as T5, sets of primers with tails and a highly processive polymerase called phusion polymerase.
The reaction starts at 0 degrees C,
Practice
Master Mix for Gibson Assembly
| Reagent | Volume/µl | ||
|---|---|---|---|
| Taq ligase (40u/µl) | 50 | ||
| 5x isothermal buffer | 100 | ||
| T5 exonuclease (1u/µl) | 2 | ||
| Phusion polymerase (2u/µl) | 6.25 | ||
| Nuclease-free water | 216.75 | Total | 375 |
Master Mix is 1.33x concentrated
In order to join 3 fragments together add 1µl of each fragment (previously amplified by PCR) to a PCR tube along with 9µl of the master mix above.
Safety
