- 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 Taq 2X Master Mix (e.g. NEB cat # M0287)
- Exonuclease I (NEB, Cat # M0293)
- 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
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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In the same reaction tube, prepare the following reaction at room temperature:
Reagent Volume 2x LongAmp Taq Master Mix 25 μl cDNA Primer (cPRM) 1.5 μl Nuclease-free water 18.5 μl Reverse-transcribed RNA sample 5 μl Total 50 μl -
Mix gently by pipetting.
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Amplify using the following cycling conditions. The recommended starting point for Extension is 14 cycles - adjust this depending on experimental needs.
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 50 secs per kb 10-18 Final extension 65 °C 6 mins 1 Hold 4 °C ∞ -
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 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 160 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 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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Analyse 1 µl of the amplified cDNA for size, quantity and quality.
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Make up 100 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 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.