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Thaw the native barcodes at room temperature. Use one barcode per sample. Individually mix the barcodes by pipetting, spin down, and place them on ice.
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Select a unique barcode for every sample to be run together on the same flow cell, from the provided 24 barcodes. Up to 24 samples can be barcoded and combined in one experiment.
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Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:
Reagent |
Volume |
End-prepped DNA |
22.5 µl |
Native Barcode |
2.5 µl |
Blunt/TA Ligase Master Mix |
25 µl |
Total |
50 µl |
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Mix well by pipetting and spin down.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing.
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Add 20 µl of resuspended AMPure XP beads to the reaction and mix by gently flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare sufficient 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 when clear and colourless.
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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.
-
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 21 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Quantify 1 µl of eluted sample using a Qubit fluorometer.
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Analyse 1 µl of sample using the Agilent Bioanalyzer. Determine the average amplicon size from this data, and use this to calculate the input sample volume for the next step.
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Pool equimolar amounts of each barcoded sample into a 1.5 ml Eppendord DNA LoBind tube, ensuring that sufficient sample is combined to produce a pooled sample of 0.2 pmol total.
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Quantify 1 µl of pooled and barcoded DNA using a Qubit fluorometer.
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Dilute 100–200 fmol pooled sample to 65 µl in nuclease-free water.