"
Team:Cambridge/Gibson/Introduction
From 2010.igem.org
(Difference between revisions)
(→Advantages) |
|||
| Line 7: | Line 7: | ||
* No scar created - useful for '''fusion proteins''' and '''adding an RBS''', where scars can be problematic. | * No scar created - useful for '''fusion proteins''' and '''adding an RBS''', where scars can be problematic. | ||
* Can re-ligate linear DNA into a circle - useful for '''site-directed mutagenesis''' | * Can re-ligate linear DNA into a circle - useful for '''site-directed mutagenesis''' | ||
| - | * Although it is roughly the same speed as [http://partsregistry.org/Help:BioBrick_Assembly standard BioBrick assembly] for ligating two fragments, Gibson is perfect for doing multi part systems, since it takes the same amount of time to ligate n pieces of DNA together as it does for | + | * Although it is roughly the same speed as [http://partsregistry.org/Help:BioBrick_Assembly standard BioBrick assembly] for ligating two fragments, Gibson is perfect for doing multi part systems, since it takes the same amount of time to ligate n pieces of DNA together as it does for two pieces. |
<html> | <html> | ||
Revision as of 14:53, 23 October 2010
Gibson Assembly: Introduction
Gibson Assembly [1] 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.
Advantages
- No scar created - useful for fusion proteins and adding an RBS, where scars can be problematic.
- Can re-ligate linear DNA into a circle - useful for site-directed mutagenesis
- Although it is roughly the same speed as standard BioBrick assembly for ligating two fragments, Gibson is perfect for doing multi part systems, since it takes the same amount of time to ligate n pieces of DNA together as it does for two pieces.
| Assembly Method | Number of steps to ligate N pieces of DNA |
| Standard Assembly | 
|
| Parallel Assembly |  (rounded up)
|
| Gibson Assembly | 
|
Disadvantages
- The need for planning, primers must be ordered in advance
- More expensive than BioBrick assembly
- Important: the flexibility that this assembly method offers is a great thing. However, this does not mean it replaces the need for standardised prefixes and suffixes. The BioBrick prefix and suffix and the use of standard well-characterised vectors with standard primer sites, etc. are crucial for standardised iGEM parts. If you use Gibson Assembly it is vital that you still add the prefix and suffix to your DNA; in fact sometimes Gibson Assembly is a useful way to do this (using as template an existing standard BioBrick).
