- Materials
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- Barcode Adapter (BCA)
- Barcode Primers (BP 01-12, at 10 µM)
- Consumables
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- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl
- (optional) Exonuclease I (NEB, M0293)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Thermal cycler
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Thaw the Blunt/TA Ligase Master Mix, spin down and mix by pipetting the entire volume within the tube up and down 10 times. Check for any precipitate (if any is visible, continue to mix) and place on ice.
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Thaw the Barcode Adapter (BCA), spin down and mix by pipetting the entire volume within the tube up and down 10 times. Place on ice.
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Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:
Reagent Volume End-prepped DNA 15 µl Barcode Adapters (BCA) 10 µl Blunt/TA Ligase Master Mix 25 µl Total 50 µl -
Mix well by gently pipetting the entire volume within the tube up and down 10 times.
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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 to the reaction and mix by pipetting.
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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 pellet in 21 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 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Remove and retain the eluate which contains the DNA library in 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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Calculate how much DNA to take forward into the PCR step for a final DNA concentration of 0.2 ng/µl in a 50 µl reaction.
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Thaw the LongAmp® Hot Start Taq 2X Master Mix at room temperature, spin down and mix by pipetting the entire volume within the tube up and down 10 times. Place on ice.
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Thaw the required Barcode Primers (BP01-12) at room temperature, spin down and mix by pipetting the entire volume within the tube up and down 10 times. Place on ice.
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Set up the adapted DNA PCR as follows:
Reagent Volume Final concentration in 50 µl Adapter ligated DNA, diluted x µl 0.2 ng/µl Nuclease-free water 24-x µl Barcode Primers (BP 01-12, at 10 µM) 1 µl LongAmp® Hot Start Taq 2x Master Mix 25 µl Total 50 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 well by gently pipetting the entire volume within the tube up and down 10 times.
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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 14 (b) Annealing 56 °C (a) 15 secs (a) 14 (b) Extension 65 °C (c) 50 secs/kb 14 (b) Final extension 65 °C 6 mins 1 Hold 4 °C ∞ a. This is specific to the Oxford Nanopore Whole Genome 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)
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Optional action(optional) Add 1 µl of ExoI, mix gently by flicking the tube, spin down briefly and incubate at 37° C for 10 min.
Carrying out this step may give a ~20% improvement in sequencing throughput.
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Resuspend the AMPure XP beads by vortexing.
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Add 30 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
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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.
-
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.
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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 10 µl of 10 mM Tris.HCl pH 8.0 with 50 mM NaCl. 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 10 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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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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Pool all barcoded libraries in the desired ratios to a total of 50-100 fmoles in 10 μl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl.