- Materials
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- UVP oligos
- Exonuclease-treated UMI tagging reaction
- Consumables
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- 0.2 ml thin-walled PCR tubes
- Platinum™ SuperFi™ II Green PCR Master Mix (ThermoFisher, cat # 12369010)
- Magnesium chloride (M1028)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Equipment
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- Microfuge
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack suitable for 0.2 ml PCR tubes
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Vortex mixer
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In a clean PCR area, prepare the universal amplification mastermix in a 0.2 ml PCR tube:
Reagent Volume 2X Platinum™ SuperFi™ II Green PCR Master Mix 55 µl 50 mM MgCl2 (1 mM final) 2.2 µl 10 µM UVP fwd (100 nM final) 1.1 µl 10 µM UVP rev (100 nM final) 1.1 µl Nuclease-free water 11 µl Total 70.4 µl Note: At 1X concentration the Platinum™ SuperFi™ II Green PCR Master Mix contains 1.5 mM MgCl2, this is further supplemented above to achieve an overall final concentration of 2.5 mM MgCl2.
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Mix by pipetting and store on ice for further use.
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Prepare the following reaction in a 0.2 ml PCR tube:
Reagent Volume Exonuclease treated UMI tagging reaction 18 µl Universal amplification mastermix 32 µl Total 50 µl -
Mix gently by flicking the tube and spin down.
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Incubate using the following program:
Step Temperature Ramp rate Time Cycles Initial denaturation 98°C max 3 min 1 Denaturation
Annealing
Extension98°C
Touchdown from 70°C to 63°C
72°Cmax
0.2°C/sec
max20 sec
45 sec
90 sec5 Denaturation
Extension98°C
72°Cmax
max20 sec
2 min5 Final extension 72°C max 5 min 1 Hold 4°C - - - Note: An AMPure XP bead clean-up is carried out after 10 cycles to reduce off-target amplification, promoting target amplicons.
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Resuspend the AMPure XP beads by vortexing.
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Add 45 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 10 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 the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear, and pipette off the supernatant.
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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 18 µl of nuclease-free water. Incubate for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear.
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Remove and retain 18 µl of eluate containing DNA library into a clean 0.2 ml thin-walled PCR tube.
Dispose of the pelleted beads.