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Ligation Protocol
ligation protocol
- Thaw all reagents on ice.
- Assemble reaction mix into 10 µL volume in a microfuge tube. Reaction may be scaled up to 20 µL if DNA concentrations are low.
- Add reagents in following order: water, buffer, insert, vector, T4 ligase.
- Gently mix by stirring gently with pipette tip.
- Typical Incubation time and temperature is 15°C for at least 4 hours. Incubation at 4°C for overnight is commonly used.
- Prepare a control that excludes the insert. This allows for detection of re-annealing due to incomplete vector digestion (If vector is not completely digested the colonies generated by the vector reaction will greatly outnumber that of the vector + insert reaction).
- Ligation reaction can be inactivate d by incubation at 65°C for 20 minutes. This step is optional.
- Proceed directly to transformation reaction.
Component | Final concentration/amount |
---|---|
water |
to 10 µL |
Ligase buffer (with ATP) |
1X |
Vector |
25 ng |
Insert |
75 ng |
T4 DNA ligase |
0.1 to 1 Weiss unit |