TurboLigase
All-in-one premix in a single tube - fast, easy, and reproducible DNA ligation / Ultra-pure recombinant T4 DNA Ligase
TurboLigase is a proprietary All-in-one premix in a sinle tube of TurboLigase, an ultra-pure recombinant T4 DNA Ligase, with all the components needed for DNA ligation. It enables quick ligation of cohesive-end or blunt-end DNA fragments at room temperature. There is no additional ligation buffer needed. A 5-minute TurboLigation reaction offers the same efficiency as a traditional 16ºC overnight ligation reaction. The reaction product is ready for E. coli chemical transformation without any further treatment. TurboLigase keeps its activity at up to 100 mM NaCl for both cohesive-end and blunt-end ligations.
Features:
Ready-to-use 1-tube premix – All components included for fast and easy ligation
Super-fast ligation – 5 min for cohesive ends, 30 min for blunt ends
High concentration, flexible use – Works efficiently with both high and low DNA concentrations
Components
- 0.25 ml TurboLigase for 100 to 250 TurboLigation reactions. TurboLigase contains ATP.
Applications
- Adding linkers or adapters to DNA
- Blunt or cohesive end ligation of duplex DNA
- DNA cloning
Protocol
- Using an insert to vector molar ratio of 3:1 and total of 50-100 ng DNA.
- Keep TurboLigase at -20ºC Gently mix before use.
- For best result, have the total ligation reaction volume 4-times (4X) of the volume of TurboLigase. More (5X) or less (3X) reaction volume lowers the ligation and transformation efficiency.
- For transformation, thaw competent cells (not included) on ice. Use within 15 min after thaw.
10 μl sample for low concentration insert and vector using 2.5 μl TurboLigaseI:
| Vector | ___ µl | total vector and insert volume up to 7.5 µl |
| Insert | ___ µl | use ∼1:3 molar ratio of vector to insert |
| adjust volume with pure water to | 7.5 µl | |
| TurboLigase | 2.5 µl | add TurboLigase last to increase ligation efficiency |
| Total reaction volume | 10 µl |
4 µl TurboLigation reaction for high concentration insert and vector using 1 µl TurboLigase:
| Vector | ___ µl | total vector and insert volume up to 3 µl |
| Insert | ___ µl | use ∼1:3 molar ratio of vector to insert |
| adjust volume with pure water to | 3 µl | |
| TurboLigase | 1 µl | add TurboLigase last to increase ligation efficiency |
| Total reaction volume | 4 µl |
- Mix gently and incubate the sample at room temperature for 5-10 minutes for cohesive end ligation. Incubation over 15 minutes often decreases the efficiency of cohesive end ligation.
- For blunt end ligation, incubate the at room temperature for 20-60 minutes. Longer time increases the efficiency of blunt end ligation.
- TurboLigase also works in a traditional 16ºC overnight ligation reaction.
- Chill the reaction on ice for 1 minute. Use 0.5-1 ml of the sample for chemical transformation.
Unit Definition
0.015 Weiss unit (1 cohesive end ligation unit) is defined as the amount of TurboLigase required to give 50% ligation of HindIII fragments of l DNA (5´ DNA termini concentration of 0.15 µM) in a total reaction volume of 20 µl in 30 minutes at 16°C in a buffer of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM ATP, 10 mM dithiothreitol.
TurboLigase Specific Activity and Purity:
TurboLigase has a specific activity >12,000 unit/mg.
TurboLigase is purified to homogeneity.
Storage
- Store TurboLigase at -20ºC. It is stable at -20ºC for 12 months from the date of receipt.
The solution may freeze from the top after several months of storage at -20ºC. It does not change the activity. Mix to thaw the solution before use.