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  • Perform Adaptor Ligation (E7530)

    Protocol

    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.