Starting Material: 20ul of first strand cDNA synthesized with the NEBNext
First Strand RNA Synthesis Module (#E7525 )
Perform Second Strand cDNA Synthesis.
Purify the Double-stranded cDNA Using 1.8X Agencourt® AMPure® XP
- Add the following reagents to the First Strand Synthesis reaction (20 μl):
Nuclease-free Water 48 μl
Second Strand Synthesis Reaction
Buffer with dUTP Mix (10X) 8 μl
Second Strand Synthesis Enzyme Mix 4 μl
Total volume 80 μl
- Mix thoroughly by gentle pipetting.
- Incubate in a thermocycler for 1 hour at 16°C.
Note: If you need to stop at this point in the protocol, after the 1 hour
incubation at 16°C, samples can be left in the thermocycler overnight
- Vortex AMPure XP beads to resuspend.
- Add 144 μl (1.8X) of resuspended Agencourt AMPure XP 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.
- Incubate for 5 minutes at room temperature.
- Quickly spin the tube in a microcentrifuge to collect any sample on the
sides of the tube. Place the tube on an appropriate magnetic rack to
separate beads from supernatant. After the solution is clear (about 5
minutes), carefully remove and discard the supernatant. Be careful not to
disturb the beads that contain DNA targets.
- Add 200 μl of freshly prepared 80% ethanol to the tube while in the
magnetic rack. Incubate at room temperature for 30 seconds, and then
carefully remove and discard the supernatant.
- Repeat Step 5 once.
- Air dry the beads for 10 minutes while the tube is on the magnetic rack
with the lid open.
- Elute the DNA target from the beads into 60 μl nuclease-free water. Mix
well on a vortex mixer or by pipetting up and down. Quickly spin the tube
and then place it in the magnetic rack until the solution is clear.
- Remove 55.5 μl of the supernatant and transfer to a clean nuclease-free
Alternatively, purify the double-stranded cDNA using QIAquick® PCR
Column Purification Kit and elute in 60 μl nuclease-free water.
- Proceed to the NEBNext Ultra End Repair/dA-Tailing Module