Melbourne/Ligation Protocol
From 2007.igem.org
(Difference between revisions)
(→Primary & secondary Reagents Required including controls) |
(→Method including controls) |
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Add to a sterile 1.7ml Eppendorf | Add to a sterile 1.7ml Eppendorf | ||
*Roughly an equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified) | *Roughly an 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 | + | *2ul of [[Melbourne/primary 10X Ligase buffer|T4 Ligase buffer 10X]] or 10ul of [[Melbourne/primary 2X Ligase buffer|T4 Ligase buffer 2X]] |
| - | *1ul of T4 | + | *1ul of [[Melbourne/primary Ligase|T4 Ligase]] |
| - | *milliQ water to make up to 20ul | + | *[[Melbourne/primary milliq|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. | Incubate Overnight (if using 10X buffer) at 4 degrees, 2h or 5min (if using 2X buffer) at room temprature. | ||
Revision as of 11:35, 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
- T4 Ligase buffer 10X or T4 Ligase buffer 2X
- DNA for ligation
- T4 Ligase
- milliQ water
Method including controls
Add to a sterile 1.7ml Eppendorf
- Roughly an equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
- 2ul of T4 Ligase buffer 10X or 10ul of T4 Ligase buffer 2X
- 1ul of T4 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
