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.
- 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
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Total volume 83.5 μl
*The adaptor is provided in NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina.
- Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.
- Incubate 15 minutes at 20°C in a thermal cycler.
- Add 3 µl of USER Enzyme to the ligation mixture from Step 3.
- Mix well and incubate at 37°C for 15 minutes.