HiFi Taq DNA Ligase (M0647) Protocol
Reaction set-up:
DNA |
X (up to 1 µg of DNA) |
|
10 X HiFi Taq DNA Ligase Buffer |
5 µl |
|
HiFi Taq DNA Ligase |
1 µl |
|
H2O |
X (up to a total of 50 µl reaction) |
Typical nick-ligations can be performed at 60°C for 15 minutes.
LDR and LCR reactions with HiFi Taq DNA Ligase should be performed between 37–75°C. The optimal ligation incubation temperature for a given set of probes is typically within 5°C of the Tm of the probes annealing region and must be determined empirically for the best balance of activity and fidelity in your application. Use of the Thermostable Ligase Reaction Temp Calculator is highly recommended for selection of an incubation temperature. For a typical LDR-type assay, reaction time should be in the range of 10–60 minutes, with 15 minutes being ideal for many applications. For assays requiring denaturation/annealing/ligation cycling, we suggest denaturation @ 95°C for 30 s – 1 min, followed by annealing/ligation for 1 – 5 min at the optimized ligation temperature for your probe set.
Videos
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NEBioCalculator® - Using the Ligation module
This tutorial describes the use of the NEBioCalculator web tool to optimize the molar ratio between vector and insert DNA for use in a ligation reaction.