For library adapter ligation, is T4 ligase or rapid ligase less prone to forming dimers?

From the experimental protocol perspective, using 50 ng RNA for small RNA library preparation leads to substantial loss during gel excision of adapter dimers. The system can be optimized rather than focusing solely on the enzyme. It is recommended to first perform a small-volume pilot experiment: use the same adapter and RNA with T4 DNA Ligase at 16°C overnight and rapid ligase for 10–15 minutes, respectively, then run PAGE directly after ligation to assess the dimer-to-product ratio. Typically, if the rapid ligase is used according to the manufacturer's recommended time, the probability of adapter self-ligation is slightly higher; T4 ligase overnight ligation gives stable efficiency, but care should be taken to supplement ATP or use fresh buffer. In addition, using pre-adenylated adapters can reduce ATP requirements and self-ligation. If the product yield remains low, reduce adapter input and use PEG8000 to improve molecular crowding. Do not rely solely on gel excision recovery; optimizing the ligation ratio is the key.

First, the core point: a high level of adapter dimers is usually not caused by the ligase itself, but by inappropriate adapter concentration and end design. In the highly efficient 10-minute system of quick ligase, an excessively high adapter ratio can indeed promote adapter self-ligation; T4 DNA Ligase overnight at 16°C has a low reaction temperature and a relatively mild system, with stable ligation efficiency and some benefit in reducing dimers, but the long duration requires prevention of evaporation and degradation. It is recommended to fix the input RNA amount, adapter molar ratio, and ATP concentration, then perform comparisons; reducing the adapter concentration (for example, from 10:1 to 5:1 or 1:1) is more effective than changing the enzyme. It is also necessary to confirm whether your adapter is DNA or RNA. If it is an RNA adapter for small RNA, T4 RNA ligase may be the one to use rather than DNA ligase.

Regarding pricing and trials, NEB T4 DNA Ligase usually comes in small pack sizes, and quick ligase is more expensive but has promotions/trial packs; domestic brands such as Nearshore and TransGen also offer ligase trials. You can contact your local distributor and explain that you are doing small RNA library preparation, and they can often provide a free trial pack or small pack. When requesting a quote, it is advisable to explicitly ask about the number of units in the small pack (e.g., 100-200 units) and cold-chain shipping costs, and not compare by volume. Our lab would first obtain a trial pack to run one ligation system comparison, evaluating by qPCR of the ligation product or library yield, rather than only looking at dimer bands. If you are concerned about slow reagent delivery, you can keep your existing T4 system while applying for a trial, to avoid interruptions in experiments.