Protocol (E6240) for Use With NEBNext Singleplex (#E7350) or Multiplex (#E7335, #E7500) Oligos for Illumina
Protocol
Starting Material: 10 ng of chromatin-immunoprecipitated (ChIP) qPCR verified or control DNA, in < 40 μl of water or elution bufferEnd Repair of ChIP DNA
- In a sterile microfuge tube mix the following components:
ChIP DNA 1–40 μl
NEBNext End Repair Reaction Buffer 5 μl
NEBNext End Repair Enzyme Mix 1 μl
Sterile H2O variable
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Total volume 50 μl - Incubate in a thermal cycler for 30 minutes at 20°C.
- Vortex AMPure XP beads to resuspend.
- Add 90 μl (1.8X) of resuspended AMPure XP beads to the ligation reaction (~50 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 the 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 5 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 50 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 44 μl of the supernatant to a clean LoBind® tube (Eppendorf AG), and store at -20°C.
dA-Tailing of End Repaired DNA
- Mix the following components in a sterile microfuge tube:
End Repaired DNA 44 μl
NEBNext dA-Tailing Reaction Buffer (10X) 5 μl
Klenow Fragment (3´→ 5´ exo–) 1 μl
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Total volume 50 μl - Incubate at 37°C for 30 minutes.
- Vortex AMPure XP beads to resuspend.
- Add 90 μl (1.8X) of resuspended AMPure XP beads to the dA-tailing reaction (~ 50 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 the 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 5 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 25 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 19 μl of the supernatant to a clean LoBind tube, and store at -20°C.
Adaptor Ligation of dA-Tailed DNA
- Mix the following components in a sterile microfuge tube:
End Repaired, dA-Tailed DNA 19 μl
Quick Ligation Reaction Buffer (5X) 6 μl
Diluted NEBNext Adaptor (1.5 μM) 1 μl
Quick T4 DNA Ligase 4 μl
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Total volume 30 μl
- Incubate at 20°C for 15 minutes.
- 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 54 μl of resuspended AMPure XP beads to the ligation reaction (~30 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 the 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 5 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 110 μl of dH2O. 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 to a clean tube/PCR plate.
Size Selection of Adaptor Ligated DNA Using AMPure XP Beads
Note: (X) refers to original sample volume of 100 μl.
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Add 90 μl (0.9x) 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.
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Incubate for 5 minutes at room temperature.
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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.
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Add 20 μl (0.2x) resuspended AMPure xP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
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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).
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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.
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Repeat Step 6 once.
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Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with lid open.
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Elute DNA target from beads into 30 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.
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Transfer 23 μl of the supernatant to a clean PCR tube and proceed to enrichment.
Note: Be sure not to transfer any beads. Trace amounts of bead carry-over may affect the optimal performance of the polymerase used in the NEBNext High-Fidelity 2X PCR Master Mix in the subsequent PCR step.
PCR Enrichment of Adaptor Ligated DNA
- Mix the following components in a sterile microfuge tube:
Adaptor ligated DNA 23 μl
NEBNext High-Fidelity 2X PCR Master Mix** 25 μl
Universal PCR Primer (25 μM) 1 μl
Index 1 Primer* (25 μM) 1 μL
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Total volume 50 μl
* If you are using the NEBNext Multiplex Oligos for Illumina (#E7335 , #E7500 ), 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 sec
1
Denaturation
Annealing
Extension
98°C
65°C
72°C
10 sec
30 sec
30 sec
15 Final Extension
72°C
4°C
5 min
hold
1
Clean Up Using AMPure XP Beads
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Vortex beads to resuspend.
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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 5 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 20 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 15 μl of the supernatant to a clean LoBind tube, and store at –20°C.
Dilute the library 20 fold with nuclease free water, and assess the library quality on a Bioanalyzer® (Agilent high sensitivity chip) (Agilent technologies, Inc.). Check that the electropherogram shows a narrow distribution with a peak size around 275 bp is expected (an example is shown below).