Ligation
Updated 1/13/2015 09:50 PM
Introduction
Materials
- Ligation Reaction (10µL)
- 25ng Linearized Vector
- 75ng Insert
- 1µL 10X Ligase Buffer
- 0.5-1µL T4 DNA Ligase
- dH2O
- Negative Control (10µL)
- 25ng Linearized Vector
- 5µL 2X Ligase Buffer
- 0.5-1µL T4 DNA Ligase
- dH2O
Procedure
- Mix each reaction described in the materials.
Assuming a 3:1 insert-to-vector ratio, the amount of insert to add can be calculated as 3 x (amount of vector) x (length of insert) / (length of vector).
Scale the reaction if DNA concentrations are low.
Add the ligase last.
Avoid repeated freeze/thaw cycles of the ligase buffer.
- Incubate following the ligase manufacturer's instructions.
When using NEB Quick ligase, 15 mins at room temperature (25°C) will be adequate. More complicated ligation reactions can be helped by incubation at 37°C.
- Denature the ligase by incubating at 65°C for 10 min.
- Immediately proceed with transformation or store at -20°C.
If many colonies result from the negative (vector + ligase) control, the vector was not completely digested and/or phosphatase treatment did not work.
Additional controls can also be used to troubleshoot failed ligations.
DNA ligation joins DNA fragments by forming phosphodiester bonds between nucleotides, catalyzed by a ligase. It is most often used to join DNA fragments produced via restriction digest. The fragments can either have compatible sticky ends or blunt ends.