Difference between revisions of "Template:HelpPage/Assembly Methods"
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===3A Assembly=== | ===3A Assembly=== | ||
| + | [[Image:3AAssembly.png|thumb|right|300px|'''Schematic of 3A Assembly''']] | ||
3A Assembly (which stands for three antibiotic assembly) is a method for assembling two parts, specifically BioBrick parts, and selecting for correct assemblies through antibiotics. 3A assembly is currently the preferred assembly method for BioBrick parts at iGEM. | 3A Assembly (which stands for three antibiotic assembly) is a method for assembling two parts, specifically BioBrick parts, and selecting for correct assemblies through antibiotics. 3A assembly is currently the preferred assembly method for BioBrick parts at iGEM. | ||
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The Registry has been providing iGEM teams and Registry labs with linearized plasmid backbones to further improve this assembly method. | The Registry has been providing iGEM teams and Registry labs with linearized plasmid backbones to further improve this assembly method. | ||
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| − | The advantages of 3A assembly include... | + | '''The advantages of 3A assembly include...''' |
*No PCR | *No PCR | ||
*No Gel purification | *No Gel purification | ||
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====Protocol==== | ====Protocol==== | ||
[[Help:Protocol/3A_Assembly|3A assembly protocol]] | [[Help:Protocol/3A_Assembly|3A assembly protocol]] | ||
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===Gibson Assembly=== | ===Gibson Assembly=== | ||
====Protocol==== | ====Protocol==== | ||
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| + | ===Standard Assembly=== | ||
| + | [[Image:StdAss.png|thumb|right|300px|'''Schematic of Standard Assembly''']] | ||
| + | For Standard assembly a part is cut out from its plasmid backbones and inserted into the plasmid backbone of another part. Two restriction digests are done, one for the part that will be moved and one for the parent. The digests are then run on a gel and using gel purification the required fragments are isolated (the part and the cut plasmid backbone). The purified insert and cut plasmid backbone are ligated and the resulting composite part can be transformed into E.coli cells. | ||
====Protocol==== | ====Protocol==== | ||
Revision as of 15:06, 28 February 2012
There are different methods for assembling parts: some are due to different standards requiring different materials and methods (i.e. restriction enzymes), others are due to preferences in protocol (faster assembly, higher efficiency, ease of use, etc).
3A Assembly
3A Assembly (which stands for three antibiotic assembly) is a method for assembling two parts, specifically BioBrick parts, and selecting for correct assemblies through antibiotics. 3A assembly is currently the preferred assembly method for BioBrick parts at iGEM.
It uses effective antibiotic selection to eliminate unwanted background colonies and eliminates the need for gel purification and colony PCR of the resulting colonies. In theory, about 97% of the colonies should be the desired assembly.
The Registry has been providing iGEM teams and Registry labs with linearized plasmid backbones to further improve this assembly method.
The advantages of 3A assembly include...
- No PCR
- No Gel purification
- Success Rate (compared to _____?)
See the 3A assembly page for more information
Protocol
Gibson Assembly
Protocol
Standard Assembly
For Standard assembly a part is cut out from its plasmid backbones and inserted into the plasmid backbone of another part. Two restriction digests are done, one for the part that will be moved and one for the parent. The digests are then run on a gel and using gel purification the required fragments are isolated (the part and the cut plasmid backbone). The purified insert and cut plasmid backbone are ligated and the resulting composite part can be transformed into E.coli cells.