Team:Cambridge/Gibson/Protocol

From 2010.igem.org

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==Step 1) Design Primers==
==Step 1) Design Primers==
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*If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap with respect to each other.
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*If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.
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//Insert diagram of two pieces of DNA with overlap
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*The standard way to do this is with PCR with specialised primers
*The standard way to do this is with PCR with specialised primers
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==Step 4) Gibson Assembly==
==Step 4) Gibson Assembly==
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*Prepare Master Mix
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=== Master mix ===
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== Master mix ==
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{|class="wikitable"
{|class="wikitable"
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|=375
|=375
|}  
|}  
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Master Mix is 1.33x concentrated
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*Add DNA to be ligated and master mix in volumetric ratio 1:3
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<i>e.g. If you were ligating two fragments (A and B) you could put
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{|class="wikitable"
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|-
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|2.5µl
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|fragment A
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|-
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|2.5µl
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|fragment B
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|-
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|15µl
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|Gibson Master Mix
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|}
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</i>
{{:Team:Cambridge/Templates/footer}}
{{:Team:Cambridge/Templates/footer}}

Revision as of 13:13, 30 September 2010