User:Andrew Perry/Notebook/bbGibson/Gibson assembly protocol
Reproduced based on RFC57 and Gibson et al, 2009.
5x isothermal reaction buffer:
- 25% PEG-8000
- 500 mM Tris-HCl pH 7.5
- 50 mM MgCl2,
- 50mM DTT
- 5mM NAD
- 1mM each of the four dNTPs
1.33x Gibson Master Mix:
- Taq ligase (40u/ul): 50 ul
- 5x isothermal buffer: 100 ul
- T5 exonuclease (1u/ul): 2 ul
- Phusion polymerase (2u/ul): 6.25 ul
- Nuclease-free water: 216.75 ul
Total volume: 375 ul
Gibson reports that this master mix can be stored at -20 °C for up to a year without significant loss of activity.
DNA assembly reaction (20 ul total volume):
- 15 ul 1.33x Gibson master mix
- DNA fragments (kit purified PCR products): approximately 10–100 ng of each for 6 kb DNA segments, equimolar amounts. For larger DNA segments, proportional amounts of DNA should be used (for example, 250 ng of each 150 kb DNA segment).
(this suggests for a 2.5 kb pSB1x3 series vector and ~ 1 kb parts, 10 - 20 ng each should be enough, given that these are all smaller than 6 kb ?)
- Sterile nuclease-free water up to a volume of 20 ul
Incubate at 50 °C for 15 to 60 min, (60 min incubations are reported to be optimal).
Transform the entire reaction into competent cells.