Team:Cambridge/Gibson/Protocol

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: