One-pot native barcoding of amplicons v4 (LoCost)
Josh Quick, Lauren Lansdowne
Abstract
This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.
Before start
Attachments
Steps
In a new PCR strip-tube/plate set up the following reaction for each sample:
| A | B |
|---|---|
| Component | Volume |
| PCR dilution from previous step | 3.3 µL |
| Ultra II End Prep Reaction Buffer | 1.2 µL |
| Ultra II End Prep Enzyme Mix | 0.5 µL |
| Nuclease-free water | 5 µL |
| Total | 10 µL |
Incubate at room temperature for 0h 15m 0s
Incubate at 65°C for 0h 15m 0s
Incubate on ice for 0h 1m 0s
In a new PCR strip-tube/plate set up the following reaction for each sample:
| A | B |
|---|---|
| Component | Volume |
| End-preparation reaction mixture | 0.75 µL |
| NBXX barcode | 1.25 µL |
| Blunt/TA Ligase Master Mix | 5 µL |
| Nuclease-free water | 3 µL |
| Total | 10 µL |
Incubate at room temperature for 0h 20m 0s
Incubate at 65°C for 0h 10m 0s
Incubate on ice for 0h 1m 0s
In a new 1.5mLEppendorf tube pool all one-pot barcoding reactions together.
Add 0.4x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add 96µL SPRI beads to 240µL pooled one-pot barcoding reactions.
Mix by vortexing and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 0h 5m 0s at room temperature.
Place on magnetic rack and incubate for 0h 2m 0s 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 250µL SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Remove supernatant and discard.
Repeat steps 11.9 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
Add 200µL of room-temperature 70% volumeethanol to bathe the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
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 0h 1m 0s or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).
Resuspend pellet in 30µL 10millimolar (mM) Tris pH 8.0, mix gently by either flicking or pipetting and incubate for 0h 2m 0s.
Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
