Team:EPF-Lausanne/Protocol/Ligation

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1. Download the following spreadsheet : [[File:Team-EPF-Lausanne Ligation.xls]]
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Ligation is a way to combine several DNA fragments into a single plasmid. This is often the
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step following a [[Team:EPF-Lausanne/Protocol/PCR|PCR] 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.
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2. Fill in the pink areas with the vector and fragment concentration, their size and the ratio.
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# Download the following spreadsheet : [[File:Team-EPF-Lausanne Ligation.xls]]
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# Fill in the pink areas with the vector and fragment concentration, their size and the ratio.
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# Add all the suggested ingredients order in a microcentrifuge tube, in the order they appear.
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# Ligate for 2 hours at 14ºC.
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# Immediately transform competent bacteria with the ligation product.
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3. Add all the suggested ingradients in a microcentrifuge tube, in the order they appear.
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Note: This protocol hasn't been optimized for blunt-end ligation (though it might still work).
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4. Ligate for 2 hours at 14ºC.
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5. Inmediatly transform competent bacteria with the ligation product.
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Revision as of 08:19, 25 September 2012

Protocol: Ligation


Ligation is a way to combine several DNA fragments into a single plasmid. This is often the step following a [[Team:EPF-Lausanne/Protocol/PCR|PCR] or a gel extraction. You can also do a "dirty" ligation, where you follow a certain number of digestions directly by a ligation.

  1. Download the following spreadsheet : File:Team-EPF-Lausanne Ligation.xls
  2. Fill in the pink areas with the vector and fragment concentration, their size and the ratio.
  3. Add all the suggested ingredients order in a microcentrifuge tube, in the order they appear.
  4. Ligate for 2 hours at 14ºC.
  5. Immediately transform competent bacteria with the ligation product.

Note: This protocol hasn't been optimized for blunt-end ligation (though it might still work).