Team:Cambridge/Protocols/Gibson Assembly

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Master Mix is 1.33x concentrated
Master Mix is 1.33x concentrated
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In order to join 3 fragments together add 1µl of each fragment (previously amplified by PCR) to a PCR tube along with 9µl of the master mix above.
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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.
Set thermocycler containing the PCR tubes to 50 degrees C for 1 hour.

Revision as of 15:30, 25 August 2011

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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


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