Jun 22, 2020
Version 1
NEB Monarch® RNA Cleanup Kit 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. NEB Monarch® RNA Cleanup Kit. protocols.io https://protocols.io/view/neb-monarch-rna-cleanup-kit-74dhqs6
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 10, 2019
Last Modified: March 29, 2021
Protocol Integer ID: 28517
Keywords: RNA, RNA cleanup, RNA clean, T2030, T2040, T2050, NEB, RNA cleanup, RNA clean up, RNA concentration, IVT cleanup, IVT clean up
Abstract
The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 2 can allow for the purification of RNA as small as 15 nt.
Materials
MATERIALS
CW Microfuge Tubes, 1.5ml, 50/pkPromegaCatalog #AS8201
Monarch RNA CleanupCatalog #T2030
Monarch RNA Cleanup Kit (50 ug)Catalog #T2040
Monarch RNA Cleanup Kit New England BiolabsCatalog #T2050
Safety warnings
For information regarding the composition of buffers, please consult the Safety Data Sheets available on the NEB website (www.neb.com). Proper laboratory safety practices should be employed, including the use of lab coats, gloves and eye protection.
Before start
Add 4 volumes of ethanol (≥ 95%) to the Monarch RNA Wash Buffer before use, as directed on the bottle.
For the 10-prep kit, add 10 ml of ethanol to 2.5 ml of Monarch RNA Cleanup Wash Buffer. For the 100-prep kit, add 80 ml of ethanol to 20 ml of Monarch RNA Cleanup Wash Buffer.
If a precipitate has formed in the Monarch RNA Cleanup Binding Buffer, warm to room temperature to re-dissolve before use.
Add 100 µL RNA Cleanup Binding Buffer to the 50 μl sample.
A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. For samples larger than 50 μl, scale buffer volumes accordingly. Samples with a starting volume > 150 μl will require reloading of the column during Step 3.
Note
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 and 2). A pellet containing the RNA of interest may form on the side of the column following the first binding spin (Step 3). To maximize recovery of this RNA, a second elution (Step 7) is recommended.
Add 150 µL ethanol (≥95%) to your sample and mix by pipetting or flicking the tube. Do not vortex.
This will enable the binding of RNA ≥ 25 nt. If you wish to bind RNA as small as 15 nt, add 2 volumes (300 μl) of ethanol to your sample instead of 1 volume (150 μl). The addition of 2 volumes of ethanol shifts the cutoff size of RNA binding from 25 nt down to 15 nt.
Insert column into collection tube, load sample onto column and close the cap. Spin for 00:01:00 at 16000 rpm , 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 column into collection tube. Add 500 µL RNA Cleanup Wash Buffer and spin for 00:01:00 at 16000 rpm . Discard the flow-through.
Note
To save time, spin for 30 seconds, instead of 1 minute.
Repeat wash: Re-insert column into collection tube. Add 500 µL RNA Cleanup Wash Buffer and spin for 00:01:00 at 16000 rpm . Discard the flow-through.
Note
To save time, spin for 30 seconds, instead of 1 minute.
Transfer 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 at 16000 rpm according to the table below.
The eluted RNA can be used immediately or stored at -70°C. Care should be used to ensure the elution buffer is delivered onto 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 |
** 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.
Recovery of RNA from Monarch RNA Cleanup Kits with Varying Elution Volumes