Protocol for DNA (E7500)
End Repair and dA-Tailing Steps
- Perform end repair and dA-Tailing according to the appropriate protocol.
Adaptor Ligation of dA-Tailed DNA
- Mix the following components in a sterile microfuge tube:
Reagent Set Protocol (NEB #E6000 )
dA-Tailed DNA 10 μl
Quick Ligation Reaction Buffer (2X) 25 μl
Adaptor (15 μM) 10 μl
Quick T4 DNA Ligase 5 μl
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Total volume should be 50 μl
Master Mix Protocol (NEB #E6040 )
dA-Tailed DNA 25 μl
Quick Ligation Reaction Buffer (5X) 10 μl
Adaptor (15 μM) 10 μl
Quick T4 DNA Ligase 5 μl
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Total volume should be 50 μl
- Incubate in a thermal cycler for 15 minutes at 20°C.
- Add 3 μl of USER enzyme mix by pipetting up and down, and incubate at 37°C
for 15 minutes.
- Vortex AMPure XP beads to resuspend.
- Add 90 μl of resuspended AMPure XP beads to the ligation reaction (~ 53 μl).
Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times.
- Incubate for 5 minutes at room temperature.
- Put the tube/PCR plate on an appropriate magnetic stand 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 80% freshly prepared ethanol to the tube/PCR plate while in
the magnetic stand. Incubate at room temperature for 30 seconds, and then
carefully remove and discard the supernatant.
- Repeat Step 8 once.
- Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic
stand with the lid open.
- Elute DNA target by adding 102 μl water to the beads for bead-based size
selection as detailed in the next section, or at desired volume for size
selection using E-Gel size select gels or standard 2% agarose gels. Mix well on
a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the
magnetic stand until the solution is clear.
- Transfer 100 μl of supernatant (or desired volume) to a new tube/well, and proceed to bead-based size selection.
Size Select Adaptor Ligated DNA Using AMPure XP Beads.
Caution: The following size selection protocol is for libraries with 200 bp inserts only. For libraries with larger fragment inserts, please optimize bead:DNA ratio accordingly.
Note: X refers to original sample volume (100 μl).
- Add 80 μl (0.8X) resuspended AMPure XP beads to 100 μl DNA solution. Mix
well on a vortex mixer or by pipetting up and down at least 10 times.
- Incubate for 5 minutes at room temperature.
- Place the tube/PCR plate on an appropriate magnetic stand to separate beads
from supernatant. After the solution is clear (about 5 minutes), carefully
transfer the supernatant to a new tube/well (Caution: do not discard the
supernatant). Discard beads that contain the large fragments.
- Add 20 μl (0.2X) resuspended AMPure XP beads to the supernatant, mix well
and incubate for 5 minutes at room temperature.
- Put the tube/PCR plate on an appropriate magnetic stand 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 (Caution: do not discard beads).
- Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in
the magnetic stand. Incubate at room temperature for 30 seconds, and then
carefully remove and discard the supernatant.
- Repeat Step 6 once.
- Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic
stand with the lid open.
- Elute DNA target from beads into 22 μl water or 0.1X TE buffer. Mix well on
a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the
magnetic stand until the solution is clear.
- Transfer 20 μl of the supernatant to a clean PCR tube and proceed to enrichment.
PCR Enrichment of Adaptor Ligated DNA
- Mix the following components in a sterile microfuge tube:
DNA 20 μl
Universal PCR Primer (25 μM) 2.5 μl
Index Primer (X)* (25 μM) 2.5 μl
NEBNext High-Fidelity 2X PCR Master Mix** 25 μl
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Total volume 50 μl
* Note: The NEBNext Multiplex Oligos for Illumina (Index Primers Set 2) contains 12 PCR Primers, each with a different index. For each reaction, only one of the 12 PCR primer indices is used during the PCR step.
** NEBNext High-Fidelity 2X PCR Master Mix will be replacing Phusion® High-Fidelity PCR Master Mix. Both vials will be supplied for a limited time only.
- PCR cycling conditions:
CYCLE STEP TEMP TIME CYCLES Initial Denaturation 98°C 30 seconds 1 Denaturation
Annealing
Extension98°C
65°C
72°C10 seconds
30 seconds
30 seconds4–8* Final Extension 72°C 5 minutes 1 Hold 4°C ∞
*If library construction was performed with 5 μg of starting material, use 4 cycles of amplification. If starting material was 1 μg, use 6–8 cycles of amplification. However, optimization of PCR cycle number may be required to avoid over-amplification.
- Vortex AMPure XP beads to resuspend.
- Add 50 μl (1X) of resuspended AMPure XP beads to the PCR reactions (~ 50
μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
- Incubate for 5 minutes at room temperature.
- Put the tube/PCR plate on an appropriate magnetic stand 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 80% ethanol to the tube/PCR plate while in the magnetic stand.
Incubate at room temperature for 30 seconds, and then carefully remove and
discard the supernatant.
- Repeat Step 7 once
- Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic
stand with the lid open.
- Elute DNA target from beads into 27 μl of 0.1X TE. Mix well on a vortex
mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic
stand until the solution is clear.
- Transfer 25 μl of the supernatant to a clean LoBind tube, and store at
-20°C.
Alternately, adaptor ligated DNA can be purified on one purification column. Elute in 30 µl of 0.1X TE Buffer.
- Dilute the library 20 fold with nuclease free water, and assess the library quality on a Bioanalyzer (Agilent high sensitivity chip). Check that the electropherogram shows a narrow distribution with a peak size approximately 300–320 bp.