- Materials
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- PCR Adapter (PCA)
- Primer Mix (PRM)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Microfuge
- Thermal cycler
- Qubit fluorometer (or equivalent for QC check)
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Add the reagents as follows in the order below:
Between each addition, pipette mix 10-20 times.
Reagent Volume End-prepped DNA 30 µl PCR Adapter (PCA) 20 µl Blunt/TA Ligase Master Mix 50 µl Total 100 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing.
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Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the tube from the magnetic rack and resuspend the pellet in 26 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 26 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads.
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Quantify 1 µl of adapted DNA using a Qubit fluorometer.
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Dilute the library to a concentration of 10 ng/µl with nuclease-free water or 10 mM Tris-HCl pH 8.5.
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Set up the adapted DNA PCR as follows:
Between each addition, pipette mix 10-20 times.
Reagent Volume Nuclease-free water 46 µl Primer Mix (PRM) 2 µl 10 ng/µl adapter ligated template 2 µl LongAmp Taq 2x master mix 50 µl Total volume 100 µl If the amount of input material is altered, the number of PCR cycles may need to be adjusted to produce the same yield.
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Mix gently by flicking the tube, and spin down.
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Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95 °C 3 mins 1 Denaturation 95 °C 15 secs 15-18 (b) Annealing 62 °C (a) 15 secs (a) 15-18 (b) Extension 65 °C (c) dependent on length of target fragment (d) 15-18 (b) Final extension 65 °C dependent on length of target fragment (d) 1 Hold 4 °C ∞ a. This is specific to the Oxford Nanopore primer and should be maintained
b. Adjust accordingly if input quantities are altered
c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)
d. Adjust accordingly for different lengths of amplicons and the type of polymerase that is being used (LongAmp Taq amplifies at a rate of 50 seconds per kb)
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Resuspend the AMPure XP beads by vortexing.
-
Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
-
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
-
Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
-
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
-
Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
-
Repeat the previous step.
-
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
-
Remove the tube from the magnetic rack and resuspend pellet in 15 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
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Quantify 1 µl of adapted DNA using a Qubit fluorometer.
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Gel analysis of amplified and ligated DNA
Sometimes a high-molecular weight product is visible in the wells of the gel when the PCR products are run, instead of the expected smear. These libraries are typically associated with poor sequencing performance. We have found that repeating the PCR with fewer cycles can remedy this.
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Dilute 1 µg of PCR product to 47 µl.