ONT dA-tailing for Fungal Barcoding
Stephen Douglas Russell
Abstract
This protocol is for dA-tailing, which is an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. In other words, this puts A-chains on the end of our PCR product, creating a site for the ligation adapter to attach to. Simple process - create a reaction with three chemicals, cleanup the product with beads.
Time required: ~45 minutes
The NEB protocol this is based on can be found here.
Steps
End repair/A-tailing
Put a 1.5mL tube of molecular water on a heat block at 55°C. This will be used after the cleanup step towards the end of this protocol.
Thaw the NEB End-prep Reaction Buffer and NEB End-prep Enzyme Mix at room temperature. (Won't take long.)
Also if you are continuing on with adapter ligation (next protocol) once this step is done, it would be good to set the chems for next step on ice so they can begin to thaw as well.
This includes LA, Quick T4 Ligase, LNB, EB, and SFB. (if using V14 chemistry)
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 usually skip it)
Mix your amplicon DNA pool thoroughly with a pipette (pipette up and down 5 times). 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 R10.4.1. At this concentration you can use the volumes described below.
Component R10.4.1 Flongle Volume R10.4.1 MinION 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 (Flongle) - 60µL (MinION) of beads to the reaction (1X bead cleanup) 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. I typically do not use one; just let it sit in a tube rack.)
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 for 0h 0m 5s 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.
DNA Quantification
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, the 31µL / 61µL of molecular water added above should put you in the ballpark of the right DNA concentration.
I typically have a concentration of 11-33 ng/uL at this point and used it at this level for the next step. No dilutions or adjustments.
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.
