- Materials
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- 100-200 fmol of each adapter-ligated sample
- OR 100-200 fmol of each amplicon sample
- PCR Barcodes (BC01-96, at 10 µM)
- Consumables
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- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Freshly prepared 80% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Equipment
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- Thermal cycler
- Magnetic rack
- Microfuge
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
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Please note, this protocol is written for a template input of 100–200 fmol with PCR Barcodes (BC01-96) used at a final concentration of 0.2 µM. However, the input mass and the number of PCR cycles may be adjusted as appropriate depending on the requirements of the experiment.
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Thaw the PCR Barcodes (BC01-96) required for your number of samples at room temperature. Individually mix the barcodes by pipetting, spin down, and place on ice.
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Prepare the samples in nuclease-free water:
- Transfer 100-200 fmol of each sample to a clear 0.2 ml PCR tube or plate
- For 1–12 samples: Adjust the volume to 48 μl with nuclease-free water
- For 13–96 samples: Adjust the volume to 24 μl with nuclease-free water
- Mix thoroughly by flicking the tube or plate to avoid unwanted shearing
- Spin down briefly in a microfuge
- Transfer 100-200 fmol of each sample to a clear 0.2 ml PCR tube or plate
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Select a unique barcode for each sample to be run together on the same flow cell.
Note: Only use one barcode per sample.
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Set up a barcoding PCR reaction as follows for each library in fresh 0.2 ml PCR tubes or plate:
Between each addition, pipette mix 10-20 times.
Reagent Volume per sample for using 1–12 barcodes Volume per sample for using 13 barcodes or more PCR Barcode (one of BC1-BC96, at 10 µM) 2 µl 1 µl Adapter ligated DNA or amplicons with tailed primers 48 µl 24 µl HotStart LongAmp Taq 2x master mix 50 µl 25 µl Total volume 100 µl 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 by pipetting and briefly spin down.
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Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 94 °C 3 mins 1 Denaturation 94 °C 15 secs 15-18 (a) Annealing 56 °C 15 secs 15-18 (a) Extension 65 °C 6 mins (b) 15-18 (a) Final extension 65 °C 10 mins 1 Hold 4 °C ∞ a. Adjust accordingly if input quantities are altered.
b. Adjust accordingly for different lengths of amplicons and the type of polymerase that is being used (LongAmp Taq amplifies at a rate of 50 seconds per kb). Here 6 min is used for ~8 Kbp templates.
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Resuspend the AMPure XP beads by vortexing.
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Add 0.4X volume of resuspended AMPure XP Beads to each reaction and mix by flicking the tube.
Reagent Volume for 100 µl samples Volume for 50 µl samples AMPure XP Beads 40 µl 20 µl -
Incubate at room temperature for 5 minutes.
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Prepare sufficient fresh 80% ethanol in nuclease-free water for all of your samples. Allow enough for 400 µl per sample, with some excess.
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Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Keep the samples on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellets. 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 samples back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellets to the point of cracking.
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Remove the samples from the magnetic rack and resuspend each pellet in 25 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnetic rack until the eluate is clear and colourless.
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Remove and retain 25 µl of each eluate into clean 0.2 ml PCR tubes or plate.
Dispose of the pelleted beads
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Quantify the barcoded library using a Qubit fluorometer and pool all barcoded libraries in the desired ratios in a 1.5 ml DNA LoBind Eppendorf tube.
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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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Prepare 1 µg of pooled barcoded libraries in 50 µl nuclease-free water.
If the volume of your pool exceeds the 50 µl required for the end-prep reaction, consider a 2.5X AMPure XP Bead purification of the pool to concentrate your sample.