May 22, 2020

Public workspaceAmplicon clean-up using SPRI beads for RAPID nanopore kit RBK004 V.3

Version 1 is forked from Amplicon clean-up using SPRI beads
DOI
dx.doi.org/10.17504/protocols.io.bgsrjwd6
  • 1Massey University
  • Coronavirus Method Development Community
Nikki Freed
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DOI: dx.doi.org/10.17504/protocols.io.bgsrjwd6
Protocol Citation: Nikki Freed, Olin Silander 2020. Amplicon clean-up using SPRI beads for RAPID nanopore kit RBK004. protocols.io https://dx.doi.org/10.17504/protocols.io.bgsrjwd6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 22, 2020
Last Modified: July 02, 2020
Protocol Integer ID: 37425
Materials
MATERIALS
ReagentAgencourt AMPure XP beads
STEP MATERIALS
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Freshly prepared 80% ethanol
10 mM Tris-HCl pH 8.0 with 50 mM NaCl

Protocol materials
ReagentAgencourt AMPure XP beads
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.

ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880

Ampure XP bead clean up
Ampure XP bead clean up
Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add Amount50 µL room temperature SPRI beads to a Amount50 µL reaction.

Pulse centrifuge to collect all liquid at the bottom of the tube.

Incubate for Duration00:05:00 at room temperature.

Place on magnetic rack and incubate for Duration00:02:00 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 Amount200 µL of freshly prepared room-temperature Concentration80 % volume ethanol to the pellet.

Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and re-form a pellet. Remove the ethanol using a pipette and discard.

Go togo to step #7 and repeat 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 Duration00:01:00 or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).

Remove the tube from the magnetic rack. Resuspend pellet in Amount10 µL 10 mM Tris-HCl pH 8.0 with 50 mM NaCl, mix gently by flicking and incubate at room temperature for Duration00:02:00 .




Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.