Jun 22, 2020
Version 1
Purification of RNA from the Aqueous Phase Following TRIzol®/Chloroform Extraction using the Monarch® RNA Cleanup Kits V.1
This protocol is a draft, published without a DOI.
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2020. Purification of RNA from the Aqueous Phase Following TRIzol®/Chloroform Extraction using the Monarch® RNA Cleanup Kits. protocols.io https://protocols.io/view/purification-of-rna-from-the-aqueous-phase-followi-7rxhm7n
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: September 27, 2019
Last Modified: March 29, 2021
Protocol Integer ID: 28183
Keywords: trizol cleanup, RNA cleanup, RNA clean up, trizol clean up
Abstract
RNA isolation reagents containing guanidine thiocyanate and phenol (e.g., TRIzol, RNAzol®, QIAzol®, etc) combined with chloroform extraction, are often used for sample lysis and RNA purification. The aqueous phase from any guanidinium thiocyanate-phenol-chloroform extraction can be cleaned up using the Monarch RNA Cleanup Kits (NEB #T2030, T2040, T2050), thereby eliminating the need for tedious RNA precipitation step
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
- Add 4 volumes of ethanol (≥ 95 %) to the Monarch RNA Wash Buffer before use, as directed on the bottle.
- All centrifugation steps should be carried out at 16,000 x g. (~ 13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.
Following guanidinium-thiocyanate-phenol-chloroform extraction, carefully transfer the upper aqueous phase into an RNase-free tube (not provided).
Add 1 volume of ethanol (≥ 95 %).
Mix well by pipetting up and down or flicking the tube. Do not vortex.
Insert an RNA cleanup column into a collection tube, load sample onto the column and close the cap.
Spin for 00:01:00 , then discard flow-through.
For diluted samples ≥ 900 μl, load a portion of the sample, spin, and then repeat as necessary.
Note
To save time, spin for 30 seconds, instead of 1 minute.
Re-insert the column into the collection tube. Add 500 µL RNA Cleanup Wash Buffer and spin for 00:01:00 . Discard the flow-through.
Note
To save time, spin for 30 seconds, instead of 1 minute.
Repeat wash (Step 6).
Transfer the column to an RNase-free 1.5 ml microfuge tube (not provided). Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to next step.
Elute in nuclease-free water according to the table below. The eluted RNA can be used immediately or stored at -70 °C .
Note
Care should be used to ensure the elution buffer is delivered onto the center of the matrix and not the wall of the column to maximize elution efficiency.
| KIT | ELUTION VOLUME | INCUBATION TIME | SPIN TIME | |
| T2030 | 6 – 20 µl | N/A | 1 minute | |
| T2040 | 20 – 100 µl | N/A | 1 minute | |
| T2050** | 50 – 100 µl | 5 minutes (Room temp.) | 1 minute |
* When cleaning up large amounts of RNA (> 100 μg, NEB #T2050), some precipitation may occur following the addition of the Monarch RNA Cleanup Binding Buffer and ethanol to the sample (Steps 1– 3). A pellet containing the RNA of interest may form on the side of the column following the first binding spin (Steps 4 and 5). To maximize recovery of this RNA, a second elution is recommended.
** Yield may slightly increase if a larger volume is used, but the RNA will be less concentrated.
Note
To save time, spin for 30 seconds, instead of 1 minute.