Introduction
The Quick T4 DNA Ligation Kit enables ligation of cohesive end or blunt end DNA fragments in 15 minutes at room temperature.
Applications
- Routine subcloning.
- Recircularization of linear DNA.
- Library construction.
- Linker ligation.
Quick Ligation Protocol
- 1. Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 µL with dH2O.
- 2. Add 10 µL of 2× Quick Ligation Buffer and mix.
- 3. Add 1 µL of Quick T4 DNA Ligase and mix thoroughly.
- 4. Centrifuge briefly and incubate at room temperature (25°C) for 15 minutes.
- 5. Chill on ice, then transform or store at -20°C.
- 6. Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.
Transformation Protocol
- 1. Thaw competent cells on ice.
- 2. Chill approximately 5 ng (2 µL) of the ligation mixture in a 1.5 mL microcentrifuge tube.
- 3. Add 50 µL of competent cells to the DNA and mix gently by pipetting up and down.
- 4. Incubate on ice for 30 minutes.
- 5. Heat shock for 2 minutes at 37°C, chill on ice for 5 minutes.
- 6. Add 950 µL of recovery media (e.g. SOC) to the tube and incubate at 37°C for 1 hour.
- 7. Spread 100 µL onto the appropriate solid medium.
- 8. Incubate overnight at 37°C.
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