"
Team:Todai-Tokyo/Protocols/Infusion
From 2009.igem.org
(Difference between revisions)
(→Infusion Protocol) |
M.Salvador (Talk | contribs) |
||
| (2 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
{{:Team:Todai-Tokyo/Template}} | {{:Team:Todai-Tokyo/Template}} | ||
| - | + | {{Team:Todai-Tokyo/protocol_temp}} | |
== Infusion Description == | == Infusion Description == | ||
| Line 10: | Line 10: | ||
== Infusion Protocol == | == Infusion Protocol == | ||
| - | # Mix the purified PCR insert(50-200ng) and linearized vector(100-400ng) | + | # PCR amplify the DNA sequence to be recombined into the vector using primers with sequences of the vector on 5' ends (refer to the Clonetech In-Fusion page above). |
| - | # Add 1µl of 5x In-Fusion Reaction Buffer and MilliQ up tp 4.5µl | + | # Linearize the vector using appropriate restriction enzymes. |
| - | # Add 0.5µl of In-Fusion Enzyme and mix well | + | # 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. | |
| - | Incubate at | + | # 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. | ||
Latest revision as of 16:30, 20 October 2009
| Home | The Team | The Project | Parts Submitted to the Registry | Modeling | Notebook | Protocols | Ethics |
|---|
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
- PCR amplify the DNA sequence to be recombined into the vector using primers with sequences of the vector on 5' ends (refer to the Clonetech In-Fusion page above).
- Linearize the vector using appropriate restriction enzymes.
- 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.
