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Team:Cambridge/Protocols/Gibson Assembly
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Revision as of 15:31, 3 August 2011
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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.
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.
Set thermocycler containing the PCR tubes to 50 degrees C for 1 hour.
Safety
