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  • Protocol (E7550)

    Starting Material: 20ul of first strand cDNA synthesized with the NEBNext First Strand RNA Synthesis Module (#E7525)

    Perform Second Strand cDNA Synthesis.

    1. 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
    2. Mix thoroughly by gentle pipetting.
    3. 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 at 4°C.
    Purify the Double-stranded cDNA Using 1.8X Agencourt® AMPure® XP Beads (Recommended).
    1. Vortex AMPure XP beads to resuspend.
    2. 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.
    3. Incubate for 5 minutes at room temperature.
    4. 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.
    5. 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.
    6. Repeat Step 5 once.
    7. Air dry the beads for 10 minutes while the tube is on the magnetic rack with the lid open.
    8. 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.
    9. Remove 55.5 μl of the supernatant and transfer to a clean nuclease-free PCR tube.
      Alternatively, purify the double-stranded cDNA using QIAquick® PCR Column Purification Kit and elute in 60 μl nuclease-free water. 
    10. Proceed to the NEBNext Ultra End Repair/dA-Tailing Module (NEB #E7442).