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.