Perform Adaptor Ligation (E7530)


Dilute the NEBNext Adaptor for Illumina (15 μM) to 1.5 μM with a 10- fold dilution (1:9) with sterile water for immediate use.

  1. To the dA-Tailed cDNA (65 μl), add the following components:

    Blunt/TA Ligase Master Mix   15 μl
    Diluted NEBNext Adaptor*  1 μl
    Nuclease-free Water   2.5 μl
    Total volume   83.5 μl

    *The adaptor is provided in NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina.

  2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.
  3. Incubate 15 minutes at 20°C in a thermal cycler.
  4. Add 3 µl of USER Enzyme to the ligation mixture from Step 3.
  5. Mix well and incubate at 37°C for 15 minutes.