User:Andrew Perry/Notebook/bbGibson/Gibson assembly protocol

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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.