Team:Cambridge/Gibson/Protocol

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Revision as of 20:39, 23 October 2010

Gibson Assembly: Protocols

The formal paper in nature describing Gibson Assembly can be found here.

Step 1: Design Primers

Designing Oligos Old-School

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

New and improved Gibthon oligo design

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

Phusion Polymerase

Step 3: PCR

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

Step	Temp	Time
1:Initial Melting	98°C	30s
2:Melting	98°C	10s
3:Annealing	T_m°C	15s
4:Elongation	72	45s per kb DNA
5:GoTo step 2		30 times
6:Final Elongation	72°C	7m30
7:Final Hold	4°C	∞

The Annealing T_m that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.

The Polymerase mixture we used was2x Phusion MasterMix.

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

@@ Line 57: / Line 57: @@
 The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.
+The Polymerase mixture we used was[http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].
 ==Step 4: Gibson Assembly==