- Materials
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- <100-200 fmol of each DNA sample to be barcoded in 24 µl
- PCR Barcodes (BC01-96, at 10 µM)
- Consumables
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- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Equipment
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- Centrifuge capable of taking 96-well plates
- Thermal cycler
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Prepare the DNA in nuclease-free water.
- Transfer <100-200 fmol of each DNA sample to be barcoded into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume to 24 μl with nuclease-free water
- Mix thoroughly by flicking the tube to avoid unwanted shearing
- Spin down briefly in a microfuge
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Capping and decapping the 96 well format
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Layout of barcodes in the 96 tube plate
The wells of the 96 tube plate correspond to the barcodes in the following way. All barcodes are supplied at 10 µM concentration and to be used at a final concentration of 0.2 µM.
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In a 0.2 ml 96 well plate, set up a barcoding PCR reaction as follows for each library:
The following is written for LongAmp Taq, but can be adapted for use with other polymerases.
Between each addition, pipette mix 10-20 times.
Reagent Volume PCR Barcode (one of BC1-BC96, at 10 µM) 1 µl <100-200 fmol first-round PCR product 24 µl LongAmp Taq 2x master mix 25 µl Total volume 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.
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Seal the plate with adhesive film or PCR strip caps and briefly spin down in a plate spinner.
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Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95 °C 3 mins 1 Denaturation 95 °C 15 secs 12-15 (b) Annealing 62 °C (a) 15 secs (a) 12-15 (b) Extension 65 °C (c) dependent on length of target fragment (d) 12-15 (b) Final extension 65 °C dependent on length of target fragment (d) 1 Hold 4 °C ∞ a. This is specific to the Oxford Nanopore primer and should be maintained
b. Adjust accordingly if input quantities are altered
c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)
d. Adjust accordingly for different lengths of amplicons and the type of polymerase that is being used. Oxford Nanopore R&D teams standardly use 8 min for DNA fragmented to 8 kb.
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Purify the barcoded DNA using standard methods which are suitable for the fragment size.
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Quantify the barcoded library using standard techniques, and pool all barcoded libraries in the desired ratios in a 1.5 ml DNA LoBind Eppendorf tube.
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Prepare 1 µg of pooled barcoded libraries in 47 µl nuclease-free water.
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Optional actionIf the volume of your pool exceeds the 49 µl required for the end-prep reaction, consider a 2.5x AMPure XP bead purification of the pool to concentrate your sample.