|
|
| (3 intermediate revisions not shown) |
| Line 10: |
Line 10: |
| | <!-- Don't forget to add this protocol to the protocol list page: --> | | <!-- Don't forget to add this protocol to the protocol list page: --> |
| | <!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol --> | | <!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol --> |
| | + | |
| | + | Ligation is a method of combining several DNA fragments into a single plasmid. This is often the |
| | + | step following a [[Team:EPF-Lausanne/Protocol/PCR|PCR]] (and a [[Team:EPF-Lausanne/Protocol/PCRCleanup|PCR cleanup]]) or a [[Team:EPF-Lausanne/Protocol/GelExtraction|gel extraction]]. You can also do a "dirty" ligation, where you follow a certain number of [[Team:EPF-Lausanne/Protocol/RestrictionSiteDigestion|digestions]] directly by a ligation. |
| | | | |
| | # Download the following spreadsheet : [[File:Team-EPF-Lausanne Ligation.xls]] | | # Download the following spreadsheet : [[File:Team-EPF-Lausanne Ligation.xls]] |
| - |
| |
| | # Fill in the pink areas with the vector and fragment concentration, their size and the ratio. | | # Fill in the pink areas with the vector and fragment concentration, their size and the ratio. |
| - | | + | # Add all the suggested ingredients order in a microcentrifuge tube, in the order they appear. |
| - | # Add all the suggested ingradients in a microcentrifuge tube, in the order they appear. | + | |
| - | | + | |
| | # Ligate for 2 hours at 14ºC. | | # Ligate for 2 hours at 14ºC. |
| | + | # Immediately transform competent bacteria with the ligation product. |
| | | | |
| - | # Inmediatly transform competent bacteria with the ligation product.
| + | Note: This protocol hasn't been optimized for blunt-end ligation (though it might still work). |
| | | | |
| | {{:Team:EPF-Lausanne/Template/ProtocolFooter}} | | {{:Team:EPF-Lausanne/Template/ProtocolFooter}} |
| | <noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude> | | <noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude> |
"
