Team:Cambridge/Protocols/Gibson Assembly

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==Gibson Assembly==
==Gibson Assembly==
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===Theory===
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Gibson Assembly is a scar-free method of DNA recombination that is highly efficient and surpasses standard assembly in utility by easily assembling multiple fragments simultaneously.  This protocol is adapted from [http://www.cambridgeigem.org/RFC57.pdf RFC57] by the 2010 Cambridge iGEM team.
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===Practice===
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Master Mix for Gibson Assembly
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{| border="1px"
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! scope="col" width="220" style="text-align:center;"|Reagent
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! scope="col" width="150" style="text-align:center;" |Volume (µl)
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|-
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|Taq ligase (40u/µl)
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|50
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|-
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|5x isothermal buffer
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|100
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|-
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|T5 exonuclease (1u/µl)
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|2
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|-
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|Phusion polymerase (2u/µl)
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|6.25
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|-
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|Nuclease-free water
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|216.75
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|-
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|'''Total'''
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|'''375'''
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|-
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|}
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Master Mix is 1.33x concentrated
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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.
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Set thermocycler containing the PCR tubes to 50 degrees C for 1 hour.
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===Safety===
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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; nucleases are present on human skin.
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Latest revision as of 03:22, 22 September 2011

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OVERVIEW
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Contents

Gibson Assembly

Theory

Gibson Assembly is a scar-free method of DNA recombination that is highly efficient and surpasses standard assembly in utility by easily assembling multiple fragments simultaneously. This protocol is adapted from [http://www.cambridgeigem.org/RFC57.pdf RFC57] by the 2010 Cambridge iGEM team.

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; nucleases are present on human skin.

Back to Protocols

Retrieved from "http://2011.igem.org/Team:Cambridge/Protocols/Gibson_Assembly"