Melbourne/Ligation Protocol
From 2007.igem.org
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Add to a sterile 1.7ml Eppendorf | Add to a sterile 1.7ml Eppendorf | ||
#roughly equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified) | #roughly equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified) | ||
| - | #2ul of 10X buffer | + | #2ul of 10X buffer or 10ul of 2X buffer |
#1ul of T4 DNA ligase | #1ul of T4 DNA ligase | ||
#milliQ water to make up to 20ul | #milliQ water to make up to 20ul | ||
Revision as of 09:12, 28 September 2007
<Return to list of protocols> <Team home page>
- Applications: Ligate DNA fragments together
- Time to complete protocol: Overnight, 2 hours, or 10 minutes
- Lab time: 5 minutes
- Waiting time: Overnight, 2h, 10min
- Approximate cost of materials: $
Contents |
Method from primary and secondary reagents
Primary & secondary Reagents Required including controls
- Ligase buffer (10X or 2X)
- DNA for ligation
- T4 DNA ligase
- milliQ water
Method including controls
Add to a sterile 1.7ml Eppendorf
- roughly equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
- 2ul of 10X buffer or 10ul of 2X buffer
- 1ul of T4 DNA ligase
- milliQ water to make up to 20ul
Incubate Overnight (if using 10X buffer) at 4 degrees, 2h or 5min (if using 2X buffer) at room temprature.
Equipement Required
- 1.5mL Microfuge tubes
- Pipettes
- Fridge
