TITLE: SPRI beads preparation
AUTHORS: Daniel Kitsberg, Miriam Adam, Naomi Habib

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SPRI is a technique for extracting nucleic acids from liquid mixtures.</div><div class = "text-block"><span>This is a quick and cheap alternative to commerical SPRI beads for RNA extraction using the Sera-mag beads by GE, based on a </span><a href="https://openwetware.org/wiki/SPRI_bead_mix" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">public protocol found here</span></a><span style = ":;">.</span></div><div class = "text-block">The cost of this SPRI buffer is ~6$/10ml</div></div>

[STEPS]
?. [SPRI buffer preparation]
Prepare 50 ml RNA Wash Buffer:1 mM Trisodium citrate, 0.05% Tween 20, pH 6.4 @ 25 °CAdd  nuclease free waterAdd  1M Trisodium citrateAdd  10% Tween 20Add  1N HClComplete volume to  with nuclease free water
30 ml
50 µl
250 µl
21 µl
50 ml
?. [SPRI buffer preparation]
Bead PreparationVortex the Sera-Mag beads thoroughly Transfer   to a 1.5 ml microcentrifuge tubeMagnetize and discard the supernatantAdd   of RNA wash bufferRemove the tube from the magnet and resuspend the beads by vortexing for at least  Spin down with a microcentrifuge, magnetize and discard the supernatantTransfer  to a 1.5 ml microcentrifuge tubeMagnetize and discard the supernatantAdd   of RNA wash bufferRemove the tube from the magnet and resuspend the beads by vortexing for at least  Spin down with a microcentrifuge, magnetize and discard the supernatantAdd  of RNA wash bufferRemove the tube from the magnet and resuspend the beads by vortexing for at least Repeat steps 4 to 6 twice, for a total of 3 washes leaving the supernatant in the tube after the last wash
1 ml
1 ml
?. [SPRI buffer preparation]
Prepare  SPRI Buffer:1mM Trisodium citrate, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, In a 50 mL conical tube, mix the base buffer:  nuclease-free water  5M NaCl  1N HCl  1M Trisodium citrateRemove the supernatant from the beads, add   of base buffer to the bead tube and resuspend by vortexing for  . Briefly spin down the liquid without pelleting the beads.Add the washed beads to base buffer in 50ml tube. Cap and vortex for  .Add   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.Add   10% Tween 20Cap the tube and mix by inversion nuclease-free water 5M NaCl 1N HCl 1M Trisodium citrateRemove the supernatant from the beads, add  of base buffer to the bead tube and resuspend by vortexing for . Briefly spin down the liquid without pelleting the beads.Add the washed beads to base buffer in 50ml tube. Cap and vortex for .Add  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.Add  10% Tween 20
50 ml
25 °C
4.672 ml
25 ml
28 µl
50 µl
1 ml
20 ml
250 µl
The SPRI Buffer is ready to be used, and can be stored at   for at least two weeks (probably months). Verify pH before use.
?. [SPRI buffer preparation]
Prepare 25 ml 50% PEG 8000 solutionWeigh  of PEG 8000 in a sterile 50 ml tubeAdd no more than  of DDWRotate for about an hour until all the PEG is dissolved and the solution is homogeneousMake up the volume to 25ml and allow the tube to rotate for another 10 minutes or so until a homogeneous solution is attained
12.5 g
14 ml
The 50% PEG solution is very viscous and takes a while to prepare