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Team:Cambridge/Gibson/Protocol
From 2010.igem.org
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==Step 4) Gibson Assembly== | ==Step 4) Gibson Assembly== | ||
| - | *Prepare Master Mix | + | *Prepare [[Team:Cambridge/Gibson/MasterMix | Master Mix]] |
| - | + | *Add DNA to be ligated and Master Mix in volumetric ratio 1:3 | |
| - | + | ||
| - | + | ||
| - | *Add DNA to be ligated and | + | |
*Incubate for 1 hour at 50°C | *Incubate for 1 hour at 50°C | ||
Revision as of 13:18, 30 September 2010
Gibson Assembly: Protocols
The formal paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].
Step 1) Design Primers
- 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.
- The standard way to do this is with PCR with specialised primers
- We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]
Step 2) Order Primers
- This step can take a while, so Gibson Assembly requires some planning ahead
Step 3) PCR
Step 4) Gibson Assembly
- Prepare Master Mix
- Add DNA to be ligated and Master Mix in volumetric ratio 1:3
- Incubate for 1 hour at 50°C
e.g. If you were ligating two fragments (A and B) you could put:
| 2.5µl | fragment A |
| 2.5µl | fragment B |
| 15µl | Gibson Master Mix |
