- Materials
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- <100-200 fmol of each DNA sample to be barcoded in 48 µl
- PCR Barcode Primers (BC01-12, at 10 µM)
- Consumables
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- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Thermal cycler
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Prepare the DNA in nuclease-free water.
- Transfer <100-200 fmol DNA into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume to 48 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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Set up a barcoding PCR reaction as follows for each pool:
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 BC01-BC12, at 10 µM) 2 µl <100-200 fmol first-round PCR product 48 µl LongAmp Taq 2x master mix 50 µl Total volume 100 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 gently by flicking the tube, and spin down.
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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 amplicon size.
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Quantify the barcoded library using standard techniques, and pool all barcoded amplicons in the desired ratios.
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Prepare 1 µg of pooled barcoded libraries in 47 µl nuclease-free water.