Note: This protocol has been changed to be compatible with the NEBNext Ultra II RNA Workflow if you need access to the previous version of the manual, please contact NEBNext@neb.com.
20 µl of first strand cDNA synthesized with the NEBNext Ultra II RNA First Strand Synthesis Module (NEB #E7771
; Chapter 2).
. 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
First-Strand Synthesis Product
(orange) NEBNext Second Strand Synthesis Reaction Buffer
(orange) NEBNext Second Strand Synthesis Enzyme Mix
1.2. Keeping the tube on ice
, mix thoroughly by pipetting the reaction up and down several times.
1.3. Incubate in a thermal cycler for 1 hour at 16°C
with the heated lid set at
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.
2.5. Add 200 μ
l 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.
2.8. Proceed to the NEBNext Ultra II End/Repair/dA-Tailing Module (NEB #E7546