Team:Cambridge/Gibson/Mechanism

From 2010.igem.org

(Difference between revisions)
(New page: {{:Team:Cambridge/Templates/headerMinimalprototype}} {{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Gibson Assembly: Introduction}} Gibson Assembly is a technique for assemblin...)
Line 1: Line 1:
{{:Team:Cambridge/Templates/headerMinimalprototype}}
{{:Team:Cambridge/Templates/headerMinimalprototype}}
-
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Gibson Assembly: Introduction}}
+
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Gibson Assembly: Mechanism}}
-
Gibson Assembly is a technique for assembling DNA with short (c. 40 bp) overlapping sequences together.  Since these overlapping regions can be easily added by PCR with primers which have added "flaps", any DNA sequences can be joined by this mechanism.
+
 +
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.
 +
 +
===Creating overlapping DNA sequences===
 +
Overlapping DNA sequences can be created by PCR.  We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.
 +
<pre>
 +
20 bp of sequence to add -> 20 bp of template to anneal to.
 +
</pre>
 +
By using two such primers we can add 20 bp of sequence A to sequence B and 20 bp of sequence B to sequence A.  We are then ready to use Gibson Assembly.
 +
 +
[[Image:Cambridge-Gib1.png]]
 +
 +
== Gibson Assembly==
 +
Gibson Assembly mix contains 3 enzymes:
 +
* T5 exonuclease
 +
* Phusion polymerase
 +
* Taq ligase
 +
[[Image:Cambridge-Gib2.png]]
 +
[[Image:Cambridge-Gib3.png]]
 +
[[Image:Cambridge-Gib4.png]]
 +
[[Image:Cambridge-Gib5.png]]
 +
[[Image:Cambridge-Gib6.png]]
 +
[[Image:Cambridge-Gib7.png]]
 +
[[Image:Cambridge-Gib8.png]]
 +
[[Image:Cambridge-Gib9.png]]
-
== Advantages ==
 
-
* No scar created - useful '''fusion proteins''' and '''adding an RBS''', where scars can be problematic.
 
-
* Can religate linear DNA to a circle - useful for '''site-directed mutagenesis'''
 
-
* Faster than Biobrick assembly, and works with less DNA.
 
{{:Team:Cambridge/Templates/footer}}
{{:Team:Cambridge/Templates/footer}}

Revision as of 15:23, 18 September 2010

Retrieved from "http://2010.igem.org/Team:Cambridge/Gibson/Mechanism"