Jan 05, 2023
RNA cleanup with magnetic beads
- 1University of Duisburg-Essen, Aquatic Ecosystem Research
Protocol Citation: Dominik Buchner 2023. RNA cleanup with magnetic beads. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3e17l47/v1
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: January 04, 2023
Last Modified: January 05, 2023
Protocol Integer ID: 74730
Keywords: RNA, cleanup, magnetic beads, PEG, NaCl
Abstract
This protocol describes cleaning up RNA extracts with carboxylated magnetic beads and a PEG-NaCl buffer. It can also be used for volume reduction of a sample or buffer exchange after enzymatic reactions (e.g. DNAse treatment).
Guidelines
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.
Materials
Materials required:
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable depending on the supply situation.
Chemicals:
Ethanol absolute Ethanol absolute 99.8%Fisher ScientificCatalog #11994041
Hydrochloric acid fuming 37% Hydrochloric acid fuming 37%Sigma AldrichCatalog #1003171011
Tris ultrapure 99.9% Tris ultrapure 99.9%DiagonalCatalog #A1086.1000
EDTA disodium salt EDTA disodium salt SigmEDTA disodium saltSigma AldrichCatalog #E5134-50G
Tween 20 Tween 20Carl RothCatalog #9127.1
Sera-Mag SpeedsBeads Sera-Mag SpeedBeads carboxylate modified particlesSigma AldrichCatalog #GE45152105050350
Tri-Sodium citrate tri-Sodium citrateSigma AldrichCatalog #1110371000
Citric acid Citric acidSigma AldrichCatalog #251275-100G
Sodium chloride Sodium Chloride Fisher BioReagents™Fisher ScientificCatalog #BP358-1
PEG 8000 Polyethylene Glycol 8000 (PEG)Fisher ScientificCatalog #10407773
Labware:
125 mL Nalgene Wide-Mouth Bottle Thermo Scientific Nalgene Wide-Mouth LDPE Bottle with ClosureFisher ScientificCatalog #10044180
Large magnet Neodyme magnetMagnethandelCatalog #3935
96-well plate magnet MM-Seperator M96Carl RothCatalog #2141.1
Hard-Shell PCR Plate Hard-Shell 96-well PCR plateBioRad SciencesCatalog #HSP9601
1.2 mL Square Deep Well Storage Microplate 96 Square Deep Well Storage Plate U shaped basesAzenta Life SciencesCatalog #4ti-0126
Stock solutions:
1 L Tris stock solution 1 Molarity (M) 8.5
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8.5 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Molarity (M) 8
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Molarity (M) 7.5
- Add 121.14 g Tris stock solution to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 7.5 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL trisodium citrate stock solution 300 millimolar (mM) 5
- Add 38.7 g tri-Sodium citrate to a beaker
- Adjust volume to 400 mL with ddH2O
- Adjust pH to 5 with citric acid
- Sterilize by filtering and store at Room temperature
1 L wash buffer stock solution (50 millimolar (mM) Tris ) 7.5
- Add 50 mL Tris stock solution 7.5 to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L PEG-NaCl buffer (2.5 Molarity (M) NaCl , 20 Mass / % volume PEG 8000 , 1 millimolar (mM) tri-Sodium citrate 5 , 0.05 % (v/v) Tween 20 ) 5
- Add 200 g PEG 8000 to a sterile glass bottle
- Add 146.1 g NaCl
- Add 3.33 mL trisodium citrate stock solution 5
- Add 250 µL Tween 20
- Adjust volume to 1 L with ddH2O
- Dissolve the PEG and NaCl by stirring and heating to 80 °C . The solution will become cloudy at this point.
- Let the solution cool down to Room temperature . A water bath may help speeding this up.
- Sterilize by filtering and store at 4 °C
Working solutions:
1 L TE minimum buffer (10 millimolar (mM) Tris , 1 millimolar (mM) EDTA )8
- Add 10 mL Tris stock solution 8 to a beaker
- Add 200 µL EDTA stock solution 8
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L wash buffer ( 10 millimolar (mM) Tris , 80 % (v/v) Ethanol ) 7.5
- Add 200 mL wash buffer stock solution to a beaker
- Adjust volume to 1 L with Ethanol absolute
- Sterilize by filtering and store at Room temperature
1 L elution buffer ( 10 millimolar (mM) Tris ) 8.5
- Add 10 mL Tris stock solution 8.5 to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
100 mL RNA cleanup solution 5
- Add 2 mL Sera-Mag SpeedBeads barboxylate modified to a clean 125 mL Nalgene bottle
- Add 25 mL TE minimum buffer
- Shake the bottle to wash the beads
- Place the bottle on a large magnet for 00:05:00 to pellet the beads
- Discard the supernatant
- Add 25 mL TE minimum buffer
- Shake the bottle to wash the beads
- Place the bottle on a large magnet for 00:05:00 to pellet the beads
- Discard the supernatant
- Add 100 mL PEG-NaCl buffer
- Shake well to resuspend the beads
- Store at 4 °C
Safety warnings
Reagents are potentially damaging to the environment. Dispose waste responsibly.
Before start
Make sure all buffers are prepared before starting.
For more effortless pipetting let the bead solution adjust to Room temperature
Note
The protocol described here is designed for the use of 1.2 mL Square Deep Well Storage Microplates, but can also be done in tubes, PCR plates, strips, or any sufficient reaction vessel. The recommended shaking speeds are adjusted to the plates mentioned in the materials.
Shake the RNA cleanup solution until the beads are homogeneously resuspended
Note
The protocol described here is designed to clean up 100 µL of RNA sample. The ratio of sample to RNA cleanup solution used is 1:2. When cleaning up a different sample volume the amount of RNA cleanup solution should be adjusted to maintain the same ratio.
To 100 µL RNA sample add 200 µL RNA cleanup solution in a 1.2 mL Deep Well Storage Plate
To bind the RNA to the beads shake at 900 rpm, Room temperature , 00:05:00
Note
If the protocol is not done in a plate, mixing can also be accomplished by pipetting or vortexing.
Place the plate on a magnet to pellet the beads for 00:05:00 or until the mixture appears clear
Note
Depending on the magnet and volume used separation times may vary and have to be adjusted accordingly.
5m
Discard the supernatant by pipetting
With the plate still on the magnet, add 100 µL wash buffer to each sample
Incubate for at least 00:00:30
30s
Discard the supernatant by pipetting
go to step #6 and repeat once for a total of 2 washes
With the plate still on the magnet, incubate the plate for 00:05:00 at Room temperature to dry off residuals of wash buffer
5m
Add 100 µL of elution buffer to each sample
900 rpm, Room temperature , 00:05:00 to elute the RNA from the beads
Place the plate on a magnet to pellet the beads for 00:05:00
5m
Transfer 100 µL of the eluted RNA to a new storage plate. Store at -80 °C
Note
If bead-carryover is a concern, only 95 µL can be transferred for storage.
Note that not all sealing films are suitable for storage at -80 °C . If in doubt transfer the RNA to tubes for long-term storage.