Oct 28, 2020
SPRI bead mix
- Philippe Jolivet1,
- Joseph W. Foley2
- 1Université de Montréal;
- 2Stanford University
Protocol Citation: Philippe Jolivet, Joseph W. Foley 2020. SPRI bead mix. protocols.io https://dx.doi.org/10.17504/protocols.io.bnz4mf8w
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: October 26, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 43804
Keywords: SPRI, AMPure, SPRIselect, RNAclean, Sera-Mag,
Abstract
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.
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).
Guidelines
Chelating agents
EDTA and citrate may interfere with some enzymatic reactions by sequestering divalent cations such as Mg2+ and Mn2+. These ions may damage nucleic acids or activate contaminating nucleases. On the other hand, sequestering these ions may interfere with downstream reactions that require them; if so, you can compensate by adding ions equimolar to the EDTA or citrate.
pH
The pH titrations for the buffers and bead mixes were calculated with the Python package ionize 0.8.0. They may be inaccurate for the bead mixes due to the very high ionic strengths of those solutions. Colour-change pH indicators will also be inaccurate for the same reason. A properly calibrated pH meter may be able to measure these solutions correctly. Keep in mind that the bead mix will be diluted during use when added to the sample to be purified, which will change the ionic strength and thus the pH.
Tween 20
Adding Tween 20 to the solutions described in the protocol is optional but provides multiple benefits. It reduces adhesion of nucleic acids to plastics, which is increased during SPRI due to the high ionic strength. This improves sample recovery. Tween 20 also reduces surface tension, which can pull beads off the pellet during supernatant removal. This effect becomes very useful if the pellet is very small. If Tween 20 is not compatible with your downstream processes or if foaming is a problem, replace its volume with nuclease-free water when mixing the solutions.
Materials
Beads
- Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic), 15 mLGe HealthcareCatalog #65152105050250
Chemicals (molecular biology grade)
Common:
- Sodium chloride (NaCl)
- Poly(ethylene glycol) avg. mol. wt. 8000 (PEG 8000)
- Polysorbate 20 (Tween 20)
- Hydrochloric acid (HCl), concentrated
- Water, nuclease-free
For DNA mix only:
- Tris(hydroxymethyl)aminomethane (Tris base)
- Disodium ethylenediaminetetraacetate dihydrate (EDTA)
For RNA mix only:
- Trisodium citrate dihydrate (sodium citrate)
Consumables
- 50 mL conical tubes
- 1.5 mL microcentrifuge tubes
- Disposable weighing vessels
- Disposable Pasteur pipettes
- Parafilm
- 0.22 μm syringe filters
- 10 mL disposable syringes
- 25 mL, 10 mL, 5 mL serological pipettes
- 1000 μL, 200 μL micropipette tips
Equipment
- Milligram-range balance
- Funnels
- Spatulas
- Heating plate
- Rotary mixer
- Microcentrifuge
- 25 mL graduated cylinder
- 50 mL volumetric flasks and stoppers
- 1000 μL, 200 μL adjustable-volume micropipettes
- Squirt bottle
- Magnetic separation block for 1.5 mL microcentrifuge tubes
Stock solutions prepared in this protocol
Common solutions:
- 1 N HCl
- 5 M NaCl
- 10% (v/v) Tween 20
- 50% (w/v) PEG 8000
DNA solutions:
- 1 M Tris base
- 0.1 M EDTA
RNA solution:
- 1 M trisodium citrate
Protocol materials
Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic), 15 mLGE HealthcareCatalog #65152105050250
Sodium chloride (NaCl)
Poly(ethylene glycol) avg. mol. wt. 8000 (PEG 8000)
Hydrochloric acid (HCl), concentrated
Tris(hydroxymethyl)aminomethane (Tris base)
Trisodium citrate dihydrate (sodium citrate)
Polysorbate 20 (Tween 20)
Water, nuclease-free
Disodium ethylenediaminetetraacetate dihydrate (EDTA)
Polysorbate 20 (Tween 20)
Polysorbate 20 (Tween 20)
Water, nuclease-free
Poly(ethylene glycol) avg. mol. wt. 8000 (PEG 8000)
Water, nuclease-free
Sodium chloride (NaCl)
Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic), 15 mLGE HealthcareCatalog #65152105050250
Water, nuclease-free
Sodium chloride (NaCl)
Hydrochloric acid (HCl), concentrated
Tris(hydroxymethyl)aminomethane (Tris base)
Disodium ethylenediaminetetraacetate dihydrate (EDTA)
Trisodium citrate dihydrate (sodium citrate)
Poly(ethylene glycol) avg. mol. wt. 8000 (PEG 8000)
Polysorbate 20 (Tween 20)
Hydrochloric acid (HCl), concentrated
Safety warnings
The Sera-Mag bead suspension contains 0.05% sodium azide.
Preparing stock solutions
Preparing stock solutions
Prepare at least 10 mL 1 Molarity (M) Hydrochloric acid (HCl), concentrated in a glass bottle from available concentrated stock.
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.
Common solutionsFrom 17 to 37 steps
| 5 M NaCl | 14.610 g |
Making 50 mL of 10% (v/v) Tween 20 stock
Place a labeled 50 mL conical tube on the balance and tare it.
With a new disposable Pasteur pipette, aspirate 1 mL to 5 mL of Polysorbate 20 (Tween 20) .
Slowly dispense the Polysorbate 20 (Tween 20) into the 50 mL conical tube to reach 5.475 g .
Remove the tube from the balance and add 45.0 mL of Water, nuclease-free with a 25 mL serological pipette.
Cap the tube and mix on a rotary mixer for 01:00:00 to dissolve the viscous liquid.
1h
Making 25 mL of 50% (w/v) PEG 8000 stock
Note
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.
Place the 25 mL graduated cylinder on the balance and tare it.
Weigh 12.5 g of Poly(ethylene glycol) avg. mol. wt. 8000 (PEG 8000) 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.
Add no more than 14 mL of Water, nuclease-free Sodium chloride (NaCl) 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.
Seal the cylinder with a double layer of Parafilm.
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.
Let the suspension stand at room temperature for at least 01:00:00 to allow the solids to dissolve and the air bubbles to rise.
1h
Remove the Parafilm and complete the volume with nuclease-free water to the 25 mL mark.
Seal the cylinder again and mix well by inverting. The solution is very viscous and homogenizing it can take a while.
Transfer the solution to a 50 mL conical tube for storage. There will be some loss inside the cylinder but you need only 20 mL for one batch of bead mix.
Nucleic acid elution and storage buffers
These 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.
DNA bufferFrom 19 to 20 steps
TE+Tween (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 8.0 @ 25 °C)
| Nuclease-free water | 48.564 mL | |
| Tris base, 1 M | 0.500 mL | |
| Disodium EDTA, 0.1 M | 0.500 mL | |
| Tween 20, 10% (v/v) | 0.250 mL | |
| HCl, 1 M | 0.186 mL |
Ingredients for 50 mL
Nucleic acid binding bead mixes
Mix the Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic), 15 mLGe HealthcareCatalog #65152105050250 very well to resuspend.
Quickly transfer 1 mL to a 1.5 mL microcentrifuge tube (the beads settle quickly).
Place the tube on a magnet stand until the supernatant is clear, about 00:00:30 .
30s
Remove and discard the supernatant.
Add 1 mL 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.
Remove the tube from the magnet and resuspend the beads by vortexing for at least 00:00:15 . Spin down the liquid with a microcentrifuge.
15s
Put the tube back on the magnet until the beads clear.
Remove and discard the supernatant.
go to step #6.5 twice , for a total of 3 washes with the appropriate buffer, leaving the supernatant in the tube after the last wash.
In a new 50 mL conical tube, combine the Water, nuclease-free , Sodium chloride (NaCl) and Hydrochloric acid (HCl), concentrated . For DNA, also add the Tris(hydroxymethyl)aminomethane (Tris base) and Disodium ethylenediaminetetraacetate dihydrate (EDTA) . For RNA, add only the Trisodium citrate dihydrate (sodium citrate) instead. Cap and mix well.
Remove the buffer supernatant from the bead tube still on the magnet.
Add 1 mL of incomplete binding buffer (prepared at step 6.10) to the bead tube on the magnet.
Remove the bead tube from the magnet and resuspend by vortexing for 00:00:15 . Briefly spin down the liquid without pelleting the beads.
15s
Add the washed beads to the incomplete binding buffer. Cap and vortex for 00:00:30 .
30s
With a 25 mL serological pipette, add 20 mL of Poly(ethylene glycol) avg. mol. wt. 8000 (PEG 8000) . Dispense slowly and allow the viscous liquid to slide down the inside walls of the pipette to ensure an accurate volume is added.
Add the Polysorbate 20 (Tween 20) .
Cap the tube and mix by inversion gently but thoroughly, until the color appears homogeneous.
The bead binding mix is ready to be used or validated. Store at 4 °C .
DNA binding bead mix1 step
10 mM Tris base, 1 mM EDTA, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, pH 8.0 @ 25 °C
| NaCl, 5 M | 25.000 mL | |
| Nuclease-free water | 3.582 mL | |
| Tris base, 1 M | 0.500 mL | |
| Disodium EDTA, 0.1 M | 0.500 mL | |
| HCl, 1 M | 0.168 mL | |
| PEG 8000, 50% (w/v) | 20.000 mL | |
| Tween 20, 10% (v/v) | 0.250 mL | |
| Sera-Mag bead suspension | 1.000 mL |
Ingredients for 50 mL
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.