TITLE: SPRI bead mix
AUTHORS: Philippe Jolivet, Joseph W. Foley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the preparation of stocks and buffers for inexpensive, convenient, and scalable DNA and RNA purification from aqueous solutions by solid-phase reversible immobilization (SPRI) on carboxylated paramagnetic beads. It also describes how to validate the effectiveness of the mixes before use.</div><div class = "text-block">The bead mixes described in this protocol are drop-in substitutes for AMPure XP and RNAClean XP beads (Beckman Coulter), but at about 1/100 of the cost (~$0.55/mL vs. $15–$70/mL at current Canadian prices).</div></div>

[STEPS]
?. [Preparing stock solutions]
In 50 mL volumetric flasks, prepare a separate 50 mL stock solution for each of the following components with the specified weights of solids.Some gentle heating may be necessary. Ensure the solution comes back to room temperature before completing the volume to the mark on the flask. Store in 50 mL conical tubes. Optional: filter the stocks with the syringes and filters to remove undissolved solids. It is strongly recommended to filter the solutions used for making RNA mix for sterilization.
?. [Preparing stock solutions]
Making 50 mL of 10% (v/v) Tween 20 stock
?. [Preparing stock solutions]
Place a labeled 50 mL conical tube on the balance and tare it.
?. [Preparing stock solutions]
With a new disposable Pasteur pipette, aspirate  to   of .
1 mL
5 mL
?. [Preparing stock solutions]
Slowly dispense the  into the 50 mL conical tube to reach .
5.475 g
?. [Preparing stock solutions]
Remove the tube from the balance and add  of  with a 25 mL serological pipette.
45 mL
?. [Preparing stock solutions]
Cap the tube and mix on a rotary mixer for  to dissolve the viscous liquid.
?. [Preparing stock solutions]
Making 25 mL of 50% (w/v) PEG 8000 stock
This recipe can be scaled up with larger cylinders to make mixing  easier, for example making 50 mL of solution in a 100 mL cylinder.
?. [Preparing stock solutions]
Place the 25 mL graduated cylinder on the balance and tare it.
?. [Preparing stock solutions]
Weigh  of powder directly into the cylinder. It is recommended to use a fresh pair of gloves to reduce static charges that make the powder fly off the spatula.
12.5 g
?. [Preparing stock solutions]
Add no more than  of   with a serological pipette on top of the PEG powder in the cylinder. The water level will reach over the 25 mL mark as the cylinder already contains about 20 mL of dry powder. Be sure not to fill the cylinder completely, as some air is required to make mixing possible. If the cylinder is too small, use a 50 mL one.
14 mL
?. [Preparing stock solutions]
Seal the cylinder with a double layer of Parafilm.
?. [Preparing stock solutions]
Shake vigorously to suspend the powder in the water until there are no more lumps of dry solid sticking to the cylinder wall. It will be very viscous and clumpy.
?. [Preparing stock solutions]
Let the suspension stand at room temperature for at least  to allow the solids to dissolve and the air bubbles to rise.
?. [Preparing stock solutions]
Remove the Parafilm and complete the volume with nuclease-free water to the  mark.
25 mL
?. [Preparing stock solutions]
Seal the cylinder again and mix well by inverting. The solution is very viscous and homogenizing it can take a while.
?. [Preparing stock solutions]
Transfer the solution to a 50 mL conical tube for storage. There  will be some loss inside the cylinder but you need only  for one batch of bead mix.
20 mL
?. [Buffer recipes]
Nucleic acid elution and storage buffersThese solutions are used for preparing the beads before adding them to the mix. They are also useful for DNA and RNA elution and storage. It is possible to make concentrates of these solutions for convenience. Keep them refrigerated.
?. [Buffer recipes]
Nucleic acid binding bead mixes
?. [Buffer recipes]
Mix the  very well to resuspend.
?. [Buffer recipes]
Quickly transfer  to a 1.5 mL microcentrifuge tube (the beads settle quickly).
1 mL
?. [Buffer recipes]
Place the tube on a magnet stand until the supernatant is clear, about .
?. [Buffer recipes]
Remove and discard the supernatant.
?. [Buffer recipes]
Add  of previously prepared "DNA buffer" or "RNA buffer",  depending on the kind of bead binding mix you are preparing, to the bead  pellet and close the tube.
1 mL
?. [Buffer recipes]
Remove the tube from the magnet and resuspend the beads by vortexing  for at least . Spin down the liquid with a microcentrifuge.
?. [Buffer recipes]
Put the tube back on the magnet until the beads clear.
?. [Buffer recipes]
Remove and discard the supernatant.
?. [Buffer recipes]
, for a total of 3 washes with the appropriate buffer, leaving the supernatant in the tube after the last wash.
[twice]
?. [Buffer recipes]
In a new 50 mL conical tube, combine the ,  and . For DNA, also add the  and . For RNA, add only the  instead. Cap and mix well.
?. [Buffer recipes]
Remove the buffer supernatant from the bead tube still on the magnet.
?. [Buffer recipes]
Add  of incomplete binding buffer (prepared at step 6.10) to the bead tube on the magnet.
1 mL
?. [Buffer recipes]
Remove the bead tube from the magnet and resuspend by vortexing for . Briefly spin down the liquid without pelleting the beads.
?. [Buffer recipes]
Add the washed beads to the incomplete binding buffer. Cap and vortex for .
?. [Buffer recipes]
With a 25 mL serological pipette, add  of .  Dispense slowly and allow the viscous liquid to slide down the inside  walls of the pipette to ensure an accurate volume is added.
20 mL
?. [Buffer recipes]
Add the .
?. [Buffer recipes]
Cap the tube and mix by inversion gently but thoroughly, until the color appears homogeneous.
?. [Buffer recipes]
The bead binding mix is ready to be used or validated. Store at .
4 °C
?. [Preparing stock solutions]
Prepare at least in a glass bottle from available concentrated stock.
10 mL
?. [Validation]
It is recommended to validate the bead mixes before use to ensure  their effectiveness. They can be compared to AMPure XP or RNAClean XP,  or to a previous batch of homemade mix. Validation can be done with DNA or RNA that is representative of a typical usage scenario, a DNA ladder (note that NEB ladders may contain modifications that make their SPRI behaviour unrepresentative of normal DNA), fragmented DNA across a range of sizes, or an RNA standard.