ONT dA-tailing for Fungal Barcoding

Put a 1.5mL tube of molecular water on a heat block at 55°C.

Thaw the End-prep Reaction Buffer and End-prep Enzyme Mix at room temperature. (Won't take long.)

Also if continuing on with adapter ligation (next protocol) once this one is done, it would be good to set the chems from that next step on ice so they can begin to thaw as well. This includes AMX H, Quick T4 Ligase, LNB, EB, and SFB.

Optional: Thaw DNA CS at room temperature, spin down for 0h 0m 5s, mix by pipetting, and place on ice.

DNA CS -

(DNA CS consists of a standard DNA sequence that can be used to provide quality control for sequencing and alignment. Generally unnecessary for DNA barcoding efforts with hundreds of repeats of a single amplicon. I skip it)

Mix your amplicon DNA pool throroughly with a pipette. Briefly spin down for 0h 0m 5s.

Important: Vortex the Ultra II End-prep Reaction Buffer for 30 seconds.

(Do not vortex the End-prep Enzyme Mix)

In a 0.2mL thin wall, sterile, nuclease-free tube, combine the following in order. Mix each reagent together after it is added by gently pipetting the entire volume up and down 10-20 times for each addition.

Ideal amplicon DNA concentration is 0.5ng per 50mL for Flongle or 1ug DNA per 50uL for R10.3. At this concentration you can use the volumes described below.

Component Flongle Volume R10.4.1 Flowcell Volume

DNA CS (optional) 0.5uL 1uL

Amplicon DNA 24.5uL (0.5ng) 49uL (1ng)

Ultra II End-prep reaction buffer 3.5uL 7uL

Ultra II end-prep enzyme mix 1.5uL 3uL

Total 30uL 60uL

The NEB protocol this is based on can be found here.

Spin down the tube in a mini centrifuge for 0h 0m 5s.

Incubate in a thermocycler using the following program:

20°C for 5 minutes

65°C for 5 minutes

4°C Hold

Spin down the tube for 5 seconds in a mini centrifuge.

Transfer the entire 30µL / 60µL reaction to a new 1.5mL LoBind eppi tube.

Resuspend magnetic beads in solution by vortexing. Add 30µL - 60µL of beads to the reaction (1X bead clean) and mix gently by pipetting up and down.

Incubate at room temperature for 0h 5m 0s. (Can put the tube in a rotator [hula] mixer if one is available.)

Spin down the tube in a mini centrifuge for 5 -10 seconds.

Place sample tube on the magnetic separator for 0h 2m 0s or until the solution clears. Beads should now be on the side of the tube.

With the tube still on the magnet, remove the liquid from the tube and discard. Be sure not to disturb the beads.

With the tube still on the magnet, add 200µL of 80% ethanol to the tube and let sit for 0h 2m 0s. Try to minimize disturbance of the beads. Fill gently with liquid stream from the pipette tip on opposite side of the beads.

Remove ethanol by pipetting and discard.

Repeat the ethanol wash one time.

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.

Remove the tube from the magnet and add 31µL for Flongle or 61µL of molecular water for MinION. Pipette up and down five times to mix until the pellet is fully suspended.

The DNA will now be released from the beads and suspended in the water.

Incubate for 0h 2m 0s at room temperature.

Place the tube back on the magnet for 0h 2m 0s or until the solution is clear.

Transfer the water containing the DNA to a new 1.5mL LoBind eppi tube.

You should now have your A-tailed DNA template.

If you have access to a Quantus or Qubit fluorometer, now is a good time to quantify the resulting amount of DNA in your purified sample.

If not, the31µL / 61µL of molecular water added above should put you in the ballpark of the right DNA concentration.

I have a concentration of 11-33 ng/uL at this point and used it at this level for the next step.

It is possible to break and store the sample at 4C overnight if needed. It would be ideal to continue on to adapter ligation at this time.