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Team:Cambridge/Protocols/Gibson Assembly
From 2011.igem.org
Contents |
Gibson Assembly
Theory
Gibson Assembly is a scar-free method of DNA recombination that is highly efficient and surpasses standard assembly in utility by easily assembling multiple fragments simultaneously. This protocol is adapted from [http://www.cambridgeigem.org/RFC57.pdf RFC57] by the 2010 Cambridge iGEM team.
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
DNA and Gibson Master Mix should be combined with a volumetric ration of 1:3 in a PCR tube. The total volume can be 20-50µl.
Set thermocycler containing the PCR tubes to 50 degrees C for 1 hour.
Safety
No bacteria are used during the reaction there is therefore little or no biological hazard. Nevertheless, it is important to observe correct laboratory procedure and wear appropriate clothing and gloves; nucleases are present on human skin.
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