Protocol for NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module (E7550)

Note: This protocol has changed to be compatible with the NEBNext Ultra II Directional RNA Workflow. If you need access to the previous version of the manual, please contact [email protected].

Starting Material: 20ul of first strand cDNA synthesized with the NEBNext Ultra II RNA First Strand RNA Synthesis Module (#E7771, Chapter 1).

1. Second Strand cDNA Synthesis

1.1. Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis reaction product.

SECOND  STRAND  SYNTHESIS REACTION

VOLUME

First-Strand Synthesis Product 20 µl
 (orange) NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix 8 µl
 (orange) NEBNext Second Strand Synthesis Enzyme Mix 4 µl
Nuclease-free Water 48 µl
Total Volume 80 µl

1.2. Keeping the tube on ice, mix thoroughly by pipetting the reaction up and down at least 10 times.

1.3. Incubate in a thermal cycler for 1 hour at 16°C with the heated lid set at ≤ 40°C (or off).


2. Purification of Double-stranded cDNA using SPRIselect Beads or NEBNext Sample Purification Beads

2.1. Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.

2.2. Add 144 μl (1.8X) of resuspended beads to the second strand synthesis reaction (~80 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

2.3. Incubate for 5 minutes at room temperature.

2.4. Briefly spin the tube in a microcentrifuge to collect any sample from the sides of the tube. Place the tube on a magnetic rack to separate beads from the supernatant. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads, which contain DNA.

Caution: Do not discard the beads!

2.5. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

2.6. Repeat Step 2.5 once for a total of 2 washing steps.

2.7. Air dry the beads for 5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not overdry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visble liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

2.8. Remove the tube from the magnetic rack. Elute the DNA from the beads by adding 53 μl 0.1X TE Buffer (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down at least 10 times. Quickly spin the tube and incubate for 2 minutes at room temperature. Place the tube on the magnetic rack until the solution is clear. 

2.9 Remove 50 μl of the supernatant and transfer to a clean nuclease-free PCR tube.

2.10 Proceed to the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546).