Team:Cambridge/Protocols/Gibsonassembly

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Revision as of 10:43, 13 August 2012

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 50°c for 1 hour.


1.33x Master Mix for Gibson Assembly
ReagentVolume (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
Total375


5x isothermal reaction buffer contains:
ReagentAmount
25% PEG-80000.75 g
500 mM Tris-HCl pH 7.51.5 ml
50 mM MgCl275 µl
50 mM DDT150 µl
1 mM dATP30 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.




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