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Team:Todai-Tokyo/Protocols/Infusion
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== Infusion Protocol == | == Infusion Protocol == | ||
| - | # Mix the purified PCR insert(50-200ng) and linearized vector(100-400ng) | + | # Mix the purified PCR insert(50-200ng) and linearized vector(100-400ng). |
| - | # Add 1µl of 5x In-Fusion Reaction Buffer and MilliQ up tp 4.5µl | + | # Add 1µl of 5x In-Fusion Reaction Buffer and MilliQ up tp 4.5µl. |
| - | # Add 0.5µl of In-Fusion Enzyme and mix well | + | # Add 0.5µl of In-Fusion Enzyme and mix well. |
| - | + | # Incubate at 37ºC for 15 min, followed by 15min ant 50ºC, then place on ice. | |
| - | Incubate at | + | # Bring the reaction volume up to 25µl with TE buffer(pH8.0) and mix well. |
| + | # Use 5µl of reaction mixture for transformation. | ||
Revision as of 10:37, 20 October 2009
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Infusion Description
Infusion is a method to combine DNA fragments using a combination of PCR and homologous recombination. It can be used as an alternative to ligation with the advantage that it does not require restriction enzyme digests, although specific primers are required for the reaction.
[http://bioinfo.clontech.com/infusion/ Clonetech page on In-Fusion]
Infusion Protocol
- Mix the purified PCR insert(50-200ng) and linearized vector(100-400ng).
- Add 1µl of 5x In-Fusion Reaction Buffer and MilliQ up tp 4.5µl.
- Add 0.5µl of In-Fusion Enzyme and mix well.
- Incubate at 37ºC for 15 min, followed by 15min ant 50ºC, then place on ice.
- Bring the reaction volume up to 25µl with TE buffer(pH8.0) and mix well.
- Use 5µl of reaction mixture for transformation.
