- Materials
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- cDNA Primer (cPRM)
- Elution Buffer (EB)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- Thermolabile Exonuclease I (NEB, cat # M0568)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 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
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
- Agilent Bioanalyzer (or equivalent)
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Thaw the cDNA Primer (cPRM), Elution Buffer (EB). LongAmp Hot Start Taq 2X Master Mix and Thermolabile Exonuclease I at room temperature, spin down and pipette mix. Store the reagents on ice.
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Spin down the reverse-transcribed RNA sample.
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Prepare four fresh 0.2 ml PCR tubes and add 5 μl of reverse-transcribed sample per tube.
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In each of the 0.2 ml PCR tubes containing the reverse-transcribed sample, prepare the following reaction at room temperature:
Reagent Volume Reverse-transcribed sample (from previous step) 5 μl cDNA Primer (cPRM) 1.5 μl Nuclease-free water 18.5 μl 2x LongAmp Hot Start Taq Master Mix 25 μl Total (including all reagents) 50 μl -
Mix gently by pipetting.
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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 10-18* Annealing 62°C 15 secs 10-18* Extension 65°C 60 secs per kb 10-18* Final extension 65°C 6 mins 1 Hold 4°C ∞ *We recommend 14 cycles as a starting point. However, the number of cycles can be adjusted between the values shown according to experimental needs.
For further information, please read The effect of varying the number of PCR cycles in the PCR-cDNA Sequencing Kit document.
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Add 1 μl Thermolabile Exonuclease I directly to each PCR tube. Mix by flicking the tube and briefly spin down.
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Incubate the reaction at 37°C for 5 minutes, followed by 80°C for 2 minutes in the thermal cycler.
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Pool the four PCR reactions (total 204 μl) 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 140 µl of resuspended AMPure XP beads to the reaction.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 1 ml 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 500 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 12 µl of Elution Buffer (EB).
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Incubate at room temperature for 10 minutes.
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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 into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Remove and retain the eluate which contains the cDNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
- Dispose of the pelleted beads
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For each sample, analyse 1 µl of the amplified cDNA for size, quantity and quality using a Qubit fluorometer and Agilent Bioanalyzer (or equivalent) for a QC check.
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Take forward 50 fmol of amplified cDNA and make the volume up to 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 5 ng 16 fmol 5 fmol 3 fmol 10 ng 32 fmol 11 fmol 5 fmol 15 ng 49 fmol 16 fmol 8 fmol 20 ng 65 fmol 22 fmol 11 fmol 25 ng 81 fmol 27 fmol 13 fmol 50 ng 154 fmol 51 fmol 26 fmol 100 ng 324 fmol 108 fmol 54 fmol If the quantity of amplified cDNA is above 50 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.