Team:Cambridge/Gibson/Protocol

The formal paper in nature describing Gibson Assembly can be found 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: Gibthon

Step 2: Order Primers

• This step can take a while, so Gibson Assembly requires some planning ahead

Step 3: PCR

PCR is a dark art, but we have found that these general principles have served us well over the summer.

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:

Step 5: Transformation