- Materials
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- 10 ng high molecular weight genomic DNA
- 16S Barcodes in 96-well plate, at 1 μM each
- EDTA (EDTA)
- AMPure XP Beads (AXP)
- Elution Buffer (EB)
- Rapid Adapter (RA)
- Adapter Buffer (ADB)
- Consumables
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- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Thermal cycler
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Multichannel pipette and tips
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Check your flow cell.
We recommend performing a flow cell check before starting your library prep to ensure you have a flow cell with enough pores for a good sequencing run.
See the flow cell check instructions in the MinKNOW protocol for more information.
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Take one 96-well plate containing 16S barcodes. Break one set of barcodes (1-24, or as desired) away from the plate and return the rest to storage.
16S barcode plate layout:
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Thaw the desired barcodes at room temperature.
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Briefly centrifuge barcodes in a microfuge to make sure the liquid is at the bottom of the tubes and place on ice.
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Thaw the LongAmp Hot Start Taq 2X Master Mix, spin down briefly, mix well by pipetting and place on ice.
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Prepare the DNA in nuclease-free water.
- Transfer 10 ng of each genomic DNA sample into a 0.2 ml thin-walled PCR tube
- Adjust the volume to 15 μl with nuclease-free water
- Mix thoroughly by flicking avoiding unwanted shearing
- Spin down briefly in a microfuge
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In each 0.2 ml thin-walled PCR tube containing a sample to be tested, prepare the following mixture:
Reagent Volume 10 ng input DNA (from previous step) 15 μl LongAmp Hot Start Taq 2X Master Mix 25 μl Total 40 μl Note: 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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Ensure the components are thoroughly mixed by pipetting and spin down briefly.
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Using clean pipette tips, carefully pierce the foil surface of the required barcodes. Use a new tip for each barcode to avoid cross-contamination. Make a note of which barcode numbers will be run for each sample.
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Using a multichannel pipette, mix the 16S barcodes by pipetting up and down 10 times. Transfer 10 μl of each 16S Barcode into respective sample-containing tubes.
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Ensure the components are thoroughly mixed by pipetting the contents of the tubes 10 times and spin down.
Note: Mix gently to minimise introducing air bubbles to the reactions.
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Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95 °C 1 min 1 Denaturation 95 °C 20 secs 25 Annealing 55 °C 30 secs 25 Extension 65 °C 2 mins 25 Final extension 65 °C 5 mins 1 Hold 4 °C ∞ -
Thaw reagents at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:
Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting or vortexing Rapid Adapter (RA) Not frozen ✓ Pipette Adapter Buffer (ADB) ✓ ✓ Vortex or Pipette AMPure XP Beads (AXP) ✓ ✓ Mix by vortexing immediately before use Elution Buffer (EB) ✓ ✓ Vortex or Pipette EDTA (EDTA) ✓ ✓ Vortex or Pipette Note: Once thawed, keep all reagents on ice.
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Add 4 µl of EDTA to each barcoded sample, mix thorougly by pipetting and spin down briefly.
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Incubate for 5 minutes at room temperature.
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Quantify 1 µl of each barcoded sample using a Qubit fluorometer (or equivalent) for QC check.
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Pool all barcoded samples in equimolar ratios in a 1.5 ml Eppendorf DNA LoBind tube.
Note: Please ensure you have quantified your samples prior to this step and take forward an equimolar concentration of each of the samples for optimal barcode balancing.
Samples may vary in concentration following the barcoded PCR, therefore the volume of each barcoded sample added to the pool will be different. -
Resuspend the AMPure XP Beads (AXP) by vortexing.
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To the pool of barcoded samples, add a 0.6X volume ratio of resuspended AMPure XP Beads (AXP) and mix by pipetting:
Volume of barcoded sample pool 37.5 μl 75 μl 150 μl 300 μl 600 μl Volume of AMPure XP Beads (AXP) 22.5 μl 45 μl 90 μl 180 μl 360 μl Note: Table contains example volumes for reference. Please adjust the volume of AMPure XP Beads (AXP) added for the volume of your barcoded sample pool to ensure a 0.6X volume ratio.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 2 ml of fresh 80% ethanol in nuclease-free water.
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Briefly spin down the sample and pellet on a magnetic rack until supernatant is clear and colourless. Keep the tube on the magnetic rack, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 1 ml of freshly-prepared 80% 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 the pellet by pipetting in 15 µl Elution Buffer (EB). Spin down and incubate for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
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Remove and retain 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Remove and retain the eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
- Dispose of the pelleted beads
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Quantify 1 µl of eluted sample using a Qubit fluorometer.
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Transfer 50 fmol of your eluted sample into a clean 1.5 ml Eppendorf DNA LoBind tube. Make up the volume to 11 µl with Elution Buffer (EB).
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In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:
Reagent Volume Rapid Adapter (RA) 1.5 μl Adapter Buffer (ADB) 3.5 μl Total 5 μl -
Add 1 µl of the diluted Rapid Adapter (RA) to the barcoded DNA.
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Mix gently by flicking the tube, and spin down.
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Incubate the reaction for 5 minutes at room temperature.