Apr 13, 2020

Public workspaceSPRI beads preparation

DOI
dx.doi.org/10.17504/protocols.io.bes2jege
  • 1Edmond and Lily Safra Center for Brain Sciences, Hebrew University of Jerusalem;
  • 2Rachel and Selim Benin School of Computer Science, Hebrew University of Jerusalem
Alon Chappleboim
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DOI: dx.doi.org/10.17504/protocols.io.bes2jege
Protocol Citation: Daniel Kitsberg, Miriam Adam, Naomi Habib 2020. SPRI beads preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bes2jege
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: April 07, 2020
Last Modified: April 13, 2020
Protocol Integer ID: 35386
Abstract
SPRI is a technique for extracting nucleic acids from liquid mixtures.
This is a quick and cheap alternative to commerical SPRI beads for RNA extraction using the Sera-mag beads by GE, based on a public protocol found here.

The cost of this SPRI buffer is ~6$/10ml
Materials
MATERIALS
ReagentNaClSigma AldrichCatalog #53014
ReagentTrisodium citrate dihydrateSigma-aldrichCatalog #S1804
Reagentnuclease free water
ReagentSera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic), 15 mLGe HealthcareCatalog #65152105050250
ReagentTween 20SigmaCatalog #P1379
ReagentHCL
ReagentPEG 8000Sigma AldrichCatalog #81268
Before start
Make sure you have stock solutions:
  • Nuclease-free water
  • 5M NaCl
  • 1N HCl
  • 1M Trisodium citrate
  • 50% PEG
  • 10% Tween 20
SPRI buffer preparation
SPRI buffer preparation
20m
20m
Prepare 50 ml RNA Wash Buffer:
1 mM Trisodium citrate, 0.05% Tween 20, pH 6.4 @ 25 °C
Add Amount30 mL nuclease free water
Add Amount50 µL 1M Trisodium citrate
Add Amount250 µL 10% Tween 20
Add Amount21 µL 1N HCl
Complete volume to Amount50 mL with nuclease free water

5m
Prepare 25 ml 50% PEG 8000 solution
Weigh Amount12.5 g of PEG 8000 in a sterile 50 ml tube
Add no more than Amount14 mL of DDW
Rotate for about an hour until all the PEG is dissolved and the solution is homogeneous
Make up the volume to 25ml and allow the tube to rotate for another 10 minutes or so until a homogeneous solution is attained
Note
The 50% PEG solution is very viscous and takes a while to prepare


Bead Preparation
  1. Vortex the Sera-Mag beads thoroughly
  2. Transfer Amount1 mL to a 1.5 ml microcentrifuge tube
  3. Magnetize and discard the supernatant
  4. Add Amount1 mL of RNA wash buffer
  5. Remove the tube from the magnet and resuspend the beads by vortexing for at least Duration00:00:15
  6. Spin down with a microcentrifuge, magnetize and discard the supernatant

Repeat steps 4 to 6 twice, for a total of 3 washes leaving the supernatant in the tube after the last wash
5m
Prepare Amount50 mL SPRI Buffer:
1mM Trisodium citrate, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, Ph6.4 Temperature25 °C
  1. In a 50 mL conical tube, mix the base buffer:
  • Amount4.672 mL nuclease-free water
  • Amount25 mL 5M NaCl
  • Amount28 µL 1N HCl
  • Amount50 µL 1M Trisodium citrate
  1. Remove the supernatant from the beads, add Amount1 mL of base buffer to the bead tube and resuspend by vortexing for Duration00:00:15 . Briefly spin down the liquid without pelleting the beads.
  2. Add the washed beads to base buffer in 50ml tube. Cap and vortex for Duration00:00:30 .
  3. Add Amount20 mL of 50% PEG stock. Dispense slowly and allow the viscous liquid to slide down the inside walls of the pipette to ensure an accurate volume is added.
  4. Add Amount250 µL 10% Tween 20
  5. Cap the tube and mix by inversion
Note
The SPRI Buffer is ready to be used, and can be stored at Temperature4 °C for at least two weeks (probably months). Verify pH before use.

10m