Perform USER Excision and PCR Library Enrichment (E7420)
ProtocolNote: The Universal PCR primer and Index (X) Primer are contained in the NEBNext SinglePlex (NEB #E7350) or NEBNext Multiplex (NEB #E7335 or NEB #E7500) Oligos for Illumina.
- To the size selected cDNA (20 μl) add the following components:
NEBNext USER Enzyme 3 µl
NEBNext High-Fidelity PCR Master Mix, 2X 25 μl
Universal PCR Primer (25 μM) 1 μl
Index (X) Primer (25 μM)* 1 μl
Total volume 50 μl
* If you are using the NEBNext Multiplex Oligos for Illumina (E7335 or E7500) for each reaction, only one of the 12 PCR primer indices is used during the PCR step.
- PCR Cycling Conditions
CYCLE STEP TEMP TIME CYCLES User Digestion 37°C 15 minutes 1 Initial Denaturation 98°C 30 seconds 1 Denaturation 98°C 10 seconds 12-15*,** Annealing 65°C 30 seconds Extension 72°C 30 seconds Final Extension 72°C 5 minutes 1 Hold 4°C ∞
** It is important to limit the number of PCR cycles to avoid overamplification. If overamplification occurs, larger molecular weight products (> 500 bp) will appear on the bioanalyzer trace.
Note: X refers to the original sample volume from the above step.
- Vortex AMPure XP beads to resuspend.
- Add 50 μl (1.0X) of resuspended AMPure XP beads to the PCR reaction (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least times.
- Incubate for 5 minutes at room temperature.
- Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the 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 5 minutes while the tube is on the magnetic rack with the lid open.
- Elute the DNA target from the beads into 23 μl nuclease free water. Mix well on a vortex mixer or by pipetting up and down, quickly spin the tube and place the magnetic rack until the solution is clear.
- Transfer 20 μl of the supernatant to a clean PCR tube, and store at -20°C.