- Materials
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- PCR Adapter (PCA)
- Primer Mix (PRM)
- Consumables
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- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
- 10 mM Tris-HCl pH 8.5
- 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)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
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Add the reagents as follows in the order below:
Reagent Volume End-prepped DNA 15 µl PCR Adapter (PCA) 10 µl Blunt/TA Ligase Master Mix 25 µl Total 50 µl -
Mix by pipetting and briefly spin 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 20 µl of resuspended AMPure XP beads for a 0.4X clean and mix by pipetting up and down ten times.
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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 80% 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 80% 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 25 µl nuclease-free water. Spin down and 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 the eluate once it is clear and colourless. Transfer the eluted sample to a fresh 0.2 ml PCR tube.
- Dispose of the pelleted beads
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Quantify 1 µl of adapted DNA using a Qubit fluorometer.
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Prepare the PCA adapted DNA in nuclease-free water:
- Transfer 20 ng genomic DNA into a 0.2 ml thin-walled PCR tube.
- Adjust the volume to 48 μl with nuclease-free water.
- Mix thoroughly by flicking the tube.
- Spin down briefly in a microfuge.
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Set up the adapted DNA PCR as follows:
Reagent Volume Nuclease-free water 46 µl Primer Mix (PRM) 2 µl 10 ng/µl adapter ligated template 2 µl HotStart 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 94 °C 3 mins 1 Denaturation 94 °C 15 secs 15-18 (a) Annealing 56 °C 15 secs 15-18 (a) Extension 65 °C 6 mins (b) 15-18 (a) Final extension 65 °C 10 mins 1 Hold 4 °C ∞ a. Adjust accordingly if input quantities are altered.
b. 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). Here 6 min is used for ~8 Kbp templates.
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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 80% 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.
-
Keep the samples on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellets. Remove the ethanol using a pipette and discard.
-
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 pellet in 52 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 52 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.