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From 2012.igem.org

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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	100
T5 exonuclease (1.0u/µl)	2
Physion DNA polymerase (2.0u/µl)	6.25
Taq DNA ligase (40u/µl)	50
H2O (nuclease free)	216.75
Total	375

5x isothermal reaction buffer
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 -20oc 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.

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