Protocol for NEBNext® Ultra™ II Non-Directional RNA Second Strand Synthesis Module (E6111)
|This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.|
|This caution sign signifies a step in the protocol that has two paths leading to the same end point.|
|Colored bullets indicate the cap color of the reagent to be added.|
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 firstname.lastname@example.org.Starting Material: 20 µl of first strand cDNA synthesized with the NEBNext Ultra II RNA First Strand Synthesis Module (NEB #E7771; Chapter 2).
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||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 thermocycler 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 at least 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 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 up to 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 visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.
2.8 Remove the tube from the magnet. Elute the DNA target from the beads by adding 53 μl 0.1X TE Buffer to the beads. Mix well on a vortex mixer or by pipetting up and down ten times. Briefly 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.
Note: If you need to stop at this point in the protocol, samples can be stored at –20°C
2.10 Proceed to the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546).