Melbourne/Ligation Protocol
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====Method from primary and secondary reagents==== | ====Method from primary and secondary reagents==== | ||
=====Primary & secondary Reagents Required including controls===== | =====Primary & secondary Reagents Required including controls===== | ||
| - | *Ligase buffer | + | *[[Melbourne/primary 10X Ligase buffer|T4 Ligase buffer 10X]] or [[Melbourne/primary 2X Ligase buffer|T4 Ligase buffer 2X]] |
*DNA for ligation | *DNA for ligation | ||
| - | *T4 | + | *[[Melbourne/primary Ligase|T4 Ligase]] |
| - | *milliQ water | + | *[[Melbourne/primary milliq|milliQ water]] |
=====Method including controls===== | =====Method including controls===== | ||
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) | |
| - | + | *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 [[Melbourne/primary Ligase|T4 Ligase]] | |
| - | + | *[[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. | ||
=====Equipement Required===== | =====Equipement Required===== | ||
| - | *1. | + | *[[Melbourne/1.7ml microcentrifuge|microcentrifuge: 1.7ml]] |
*Pipettes | *Pipettes | ||
*Fridge | *Fridge | ||
| + | |||
=====References===== | =====References===== | ||
* | * | ||
Latest revision as of 11:37, 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
- microcentrifuge: 1.7ml
- Pipettes
- Fridge
