- Materials
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- Barcode Primers (BP01-BP12)
- Elution Buffer (EB)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- LongAmp Taq 2X Master Mix (e.g. NEB cat # M0287)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Thermal cycler
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Ice bucket with ice
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The PCR steps outlined below adds barcodes to each reverse transcribed RNA (cDNA) sample. The Barcode Primers provided in the PCR-cDNA Barcoding kit (SQK-PCB109) are used to barcode/multiplex up to 12 individual samples on a single flow cell.
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It is recommended that any remaining reverse transcription reaction is retained to allow for further PCR reactions if greater yield is required.
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For each sample (up to 12), prepare the following reaction at room temperature:
Reagent Volume Reverse-transcribed RNA sample 5 μl Barcode Primers (BP01-BP12) 1.5 μl Nuclease-free water 18.5 μl 2x LongAmp Taq Master Mix 25 μl Total 50 μl -
Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95° C 30 secs 1 Denaturation 95° C 15 secs 11-18* Annealing 62° C 15 secs 11-18* Extension 65° C 50 secs per kb 11-18* Final extension 65° C 6 mins 1 Hold 4° C ∞ *The recommended starting point is 14 cycles - adjust this depending on experimental needs.
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Add 1 μl Exonuclease I directly to each PCR tube. Mix by pipetting.
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Incubate the reaction at 37°C for 15 minutes, followed by 80°C for 15 minutes in the thermal cycler.
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Pool any PCR reactions containing the same barcoded sample in a clean 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the AMPure XP beads by vortexing.
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Add 0.8X equivalents of resuspended AMPure XP beads to the reaction and mix by pipetting.
E.g. You should use 40.8 µl AMPure XP beads per 51 µl reaction mix.
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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.
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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.
If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
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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 70% ethanol. Briefly allow to dry.
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Remove the tube from the magnetic rack and resuspend pellet in 12 µl of Elution Buffer (EB).
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 12 µl of 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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Analyse 1 µl of the amplified cDNA for size, quantity and quality.
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In a 1.5 ml Eppendorf DNA LoBind tube, pool together a total of 100 fmol of the amplified cDNA barcoded samples to a final volume of 11 μl in Elution Buffer (EB).
Mass Molarity if fragment length = 0.5 kb Molarity if fragment length = 1.5 kb Molarity if fragment length = 3 kb 50 ng 154 fmol 51 fmol 26 fmol 100 ng 308 fmol 103 fmol 51 fmol 200 ng 616 fmol 205 fmol 103 fmol 300 ng 925 fmol 308 fmol 154 fmol If the quantity if amplified cDNA is above 100 fmol, the remaining cDNA can be frozen and stored for another sequencing experiment (in this case, library preparation would start from the Adapter Addition step). We recommend avoiding multiple freeze-thaw cycles to prevent DNA degradation.