Home Resources Video Library NEBNext® Tech Tips - Adaptor Ligation Tips

NEBNext® Tech Tips: Adaptor Ligation

This video provides best practices for NGS library prep adaptor ligation and adaptor dilution.

Script

When setting up a ligation reaction, it’s important to note the adaptor dilution recommendation, which can be found at the beginning of the ligation portion of the manual for each section. NEB recommends diluting the non-indexed, loop-shaped NEBNext® Adaptor in 10 mM Tris-HCl or adaptor dilution buffer supplied with some of the NEBNext library prep kits. NEBNext Adaptor Dilution Buffer is also available separately. If using the NEBNext Unique Dual indexed UMI adaptors, please only use the NEBNext UMI Adaptor Dilution Buffer that is supplied with them, the NEBNext UMI Adaptor Dilution Buffer differs in formulation from the standard NEBNext Adaptor Dilution Buffer mentioned previously. An example adaptor-dilution calculation might be, to make a 10-fold adaptor dilution add 1 µl of adaptor and to a separate tube containing 9 µl of dilution buffer.  To successfully set up a ligation reaction do not premix the adaptor and ligation master mix. Pre-mixing the adaptor or diluted adaptor with the ligation master mix can cause adaptor dimers to form, even when the mixture is on ice. Instead, you may add the adaptor or diluted adaptor to the sample following the end prep step. Ligation Master Mix and ligation enhancer may be premixed prior to reaction set up. Note that not all NEBNext protocols require ligation enhancer, please check the protocol for the purchased kit for details. NEB recommends that the sample containing the adaptor or diluted adaptor and inserts is mixed by pipetting up and down or by vortexing briefly. Once the sample is thoroughly mixed, add the ligation master mix and ligation enhancer, and mix again. Place the well-mixed sample into a prewarmed thermocycler and incubate per your protocol with heated lid off. Thanks for watching. If you have any questions, please reach out to us at info@neb.com.

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