Team:Cambridge/Protocols/Gibson Assembly

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No bacteria are used during the reaction there is therefore little or no biological hazard. Nevertheless, it is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature, and this may present a risk, depending on the PCR machine employed.
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Revision as of 20:47, 20 September 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

No bacteria are used during the reaction there is therefore little or no biological hazard. Nevertheless, it is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature, and this may present a risk, depending on the PCR machine employed.