Team:Cambridge/Protocols/Gibson Assembly

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OVERVIEW
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Gibson Assembly

Theory

Gibson Assembly as a scar free method of DNA recombination that is highly efficient and readily copes with combination of multiple DNA fragments at once.

Practice

Master Mix for Gibson Assembly

Reagent Volume (µl)
Taq ligase (40u/µl) 50
5x isothermal buffer 100
T5 exonuclease (1u/µl) 2
Phusion polymerase (2u/µl) 6.25
Nuclease-free water 216.75
Total 375

Master Mix is 1.33x concentrated

DNA and Gibson Master Mix should be combined with a volumetric ration of 1:3 in a PCR tube. The total volume can be 20-50µl.

Set thermocycler containing the PCR tubes to 50 degrees C for 1 hour.

Safety