TITLE: One-pot native barcoding of amplicons v2
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.</div></div>

[STEPS]
?. Set up the following reaction for each sample:Component                                          VolumePCR dilution from previous step       Nuclease-free water                           Ultra II End Prep Reaction Buffer     Ultra II End Prep Enzyme Mix           Total
5 µl
7.5 µl
1.75 µl
0.75 µl
15 µl
?. Incubate at room temperature for  Incubate at  for Incubate on ice for
65 °C
?. In a new 1.5mL Eppendorf tube set up the following reaction:Component                               VolumePrevious reaction mixture       Nuclease-free water                NBXX barcode                          Ultra II Ligation Master Mix    Ligation Enhancer                    Total
1.5 µl
5.7 µl
2.5 µl
10 µl
0.3 µl
20 µl
Use one native barcode from the EXP-NBD104 (1-12) or EXP-NBD114 (13-24) per sample. Use from 6 to 24 barcodes in a library, any fewer and there will be insufficient total material to achieve good yields.
?. Incubate at room temperature for Incubate at  for Incubate on ice for
65 °C
The 65°C incubation is to inactivate the DNA ligase to prevent barcode cross-ligation when reactions are pooled in the next step.
?. In a new 1.5 ml Eppendorf tube pool all  one-pot barcoding reactions together.
20 µl
?. Add 0.4x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add  SPRI beads to  12-plex pooled one-pot native barcoding reactions.
96 µl
240 µl
0.4x volume of SPRI will only bind 400 bp amplicons in the presence of ligation buffer as in a one-pot reaction, do not use 1x as this will result in excessive native barcode carryover.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Incubate for  at room temperature.
?. Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. Add 700 µl SFB and resuspend beads completely by pipette mixing.
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Remove supernatant and discard.
?. Repeat steps 11-13 to perform a second SFB wash.
?. Pulse centrifuge and remove any residual SFB.
You do not need to allow to air dry with SFB washes.
?. Add 200 µl of room-temperature 70 % volume ethanol to bathe the pellet.
?. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Only perform 1x 70% ethanol wash
?. Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
?. With the tube lid open incubate for 00:01:00 or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).
?. Resuspend pellet in Elution Buffer (EB), mix gently by either flicking or pipetting and incubate for .
30 µl
?. Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.