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Team:Cambridge/Protocols/Gibsonassembly
From 2012.igem.org
Gibson Assembly:
Safety:
There are no cells involved in this procedure so biohazard risks are very small. Coats should still be worn however and gloves are particularly important to prevent contamination from the nucleases found on human skin.
Protocol
Add DNA and Master Mix to a PCR tube in a 1:3 (Total DNA: Master Mix) volumetric ratio. E.g. for a total 20 µl sample, add 5 µl of DNA (containing 10-100ng DNA fragments, including the backbone, made up to volume) and 15 µl Master Mix. Incubate at 50oc for 1 hour.
| 1.33x Master Mix for Gibson Assembly | |
| Reagent | Volume (µl) |
| 5x isothermal reaction buffer (see below) | 100 |
| T5 exonuclease (1.0u/µl) | 2 |
| Phusion DNA polymerase (2.0u/µl) | 6.25 |
| Taq DNA ligase (40u/µl) | 50 |
| H2O (nuclease free) | 216.75 |
| Total | 375 |
| 5x isothermal reaction buffer contains: | |
| Reagent | Amount |
| 25% PEG-8000 | 0.75 g |
| 500 mM Tris-HCl pH 7.5 | 1.5 ml |
| 50 mM MgCl2 | 75 µl |
| 50 mM DDT | 150 µl |
| 1 mM dATP | 30 µl |
Notes: Master Mix can be made up in advance and should be stored at -20°c until it is used. At this stage (pre-freezing), it is worth splitting it up into aliquots of the volume you will use in your experiments to prevent the whole sample from repeatedly going through the freeze-thaw cycle.
