Sep 08, 2020
Amplicon clean-up using SPRI beads
- 1University of Birmingham
External link: https://doi.org/10.1016/j.remle.2020.05.007
Protocol Citation: Josh Quick 2020. Amplicon clean-up using SPRI beads. protocols.io https://dx.doi.org/10.17504/protocols.io.7nxhmfn
Manuscript citation:
Fernández-Rodríguez A, Casas I, Culebras E, Morilla E, Cohen MC, Alberola J, COVID-19 and post-mortem microbiological studies. Spanish Journal of Legal Medicine doi: 10.1016/j.remle.2020.05.007
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: Working
We use this protocol and it's working
Created: September 25, 2019
Last Modified: September 08, 2020
Protocol Integer ID: 28087
Materials
STEP MATERIALS
Elution Buffer (EB)QiagenCatalog #19086
Agencourt AMPure XPBeckman CoulterCatalog #A63880
QuantiFluor(R) ONE dsDNA System, 100rxnPromegaCatalog #E4871
Protocol materials
Elution Buffer (EB)QiagenCatalog #19086
Agencourt AMPure XPBeckman CoulterCatalog #A63880
QuantiFluor(R) ONE dsDNA System, 100rxnPromegaCatalog #E4871
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Elution Buffer (EB)QiagenCatalog #19086
QuantiFluor(R) ONE dsDNA System, 100rxnPromegaCatalog #E4871
Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add 50 µL SPRI beads to a 50 µL reaction.
Pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate for 00:05:00 at room temperature.
Place on magnetic rack and incubate for 00:02:00 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 200 µL of room-temperature 70 % volume ethanol to the pellet.
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
go 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 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 30 µL Elution Buffer (EB), mix gently by either flicking or pipetting and incubate for 00:02:00 .
Elution Buffer (EB)QiagenCatalog #19086
Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
Quantify 1 µL product using the Quantus Fluorometer using the ONE dsDNA assay.
QuantiFluor(R) ONE dsDNA System, 100rxnPromegaCatalog #E4871
Equipment
Quantus
NAME
Fluorometer
TYPE
Promega
BRAND
E6150
SKU
LINK