TITLE: ONT dA-tailing for Fungal Barcoding
AUTHORS: Stephen        Douglas Russell, Stephen    Douglas Russell

[DESCRIPTION]
Tailing is an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. 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, PCR bead cleanup.

Time required: ~45 minutes

[STEPS]
SECTION: End repair/A-tailing
1. 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.

SECTION: End repair/A-tailing
2. Optional: Thaw DNA CS at room temperature, spin down for 5 s, 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)
SECTION: End repair/A-tailing
3. Mix your amplicon DNA pool throroughly with a pipette. Briefly spin down for 5 s.

SECTION: End repair/A-tailing
4. Important: Vortex the Ultra II End-prep Reaction Buffer for 30 seconds.

(Do not vortex the End-prep Enzyme Mix)
SECTION: End repair/A-tailing
5. 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.
SECTION: End repair/A-tailing
6. Spin down the tube in a mini centrifuge for 5 s. 

SECTION: End repair/A-tailing
7. Incubate in a thermocycler using the following program:

20 °C for 5 minutes
65 °C for 5 minutes
4 °C Hold
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8. Spin down the tube for 5 seconds in a mini centrifuge.

SECTION: End repair/A-tailing
9. Transfer the entire 30 µL / 60 µL reaction to a new 1.5mL LoBind eppi tube.

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

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

SECTION: End repair/A-tailing
12. Spin down the tube in a mini centrifuge for 5 -10 seconds.


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13. Place sample tube on the magnetic separator for 2 min or until the solution clears. Beads should now be on the side of the tube.
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14. With the tube still on the magnet, remove the liquid from the tube and discard. Be sure not to disturb the beads.
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15. With the tube still on the magnet, add 200 µL of 80% ethanol to the tube and let sit for 2 min. Try to minimize disturbance of the beads. Fill gently with liquid stream from the pipette tip on opposite side of the beads.

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16. Remove ethanol by pipetting and discard.
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17.  Repeat the ethanol wash one time.   

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18. 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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19. Remove the tube from the magnet and add 31 µL for Flongle or 61 µL 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.

SECTION: End repair/A-tailing
20. Incubate for 2 min at room temperature.
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21. Place the tube back on the magnet for 2 min or until the solution is clear.

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

You should now have your A-tailed DNA template.
SECTION: DNA Quantification
23. 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, the31 µL / 61 µL 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.

SECTION: DNA Quantification
24. 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.