Product Class: Kit

Monarch® RNA Cleanup Kit (500 μg)

Effective March 25, 2024, component/product name changed to Monarch® Buffer BX.

Effective March 25, 2024, component/product name changed to Monarch® Buffer WX.

Product Introduction

The Monarch RNA Cleanup Kit (500 µg) enables fast and simple purification and concentration of up to 500 µg of RNA from in vitro transcription (IVT) and other enzymatic reactions.

  • Ideal for purification of synthesized RNA following high-yield in vitro transcription reactions (alternative to MEGAclear™)
  • Optimized for the cleanup of RNA after enzymatic treatments including DNase I, Proteinase K, labeling and capping
  • Efficiently purify RNA ≥ 25 nt (a simple modification enables purification of RNA ≥ 15 nt)
  • Elute in ≥ 50 µl for concentrated RNA 70-100% RNA recovery
  • Unique column design prevents buffer carryover and elution of silica particulates
  • Simplified workflow with a single wash buffer
  • Columns and buffers are available separately
  • Purified RNA is ready for use in a wide variety of downstream applications, including microinjection and transfection.

Check out our Technical Note containing comprehensive insights into measuring and analyzing nucleic acids.

Catalog # Size Concentration
T2050S 10.0 preps
T2050L 100.0 preps

Product Information

Description

The Monarch RNA Cleanup Kit (500 µg) reliably purifies up to 500 μg of concentrated, high-quality RNA (> 25 nt) from enzymatic reactions and in vitro transcription (IVT) reactions. The kit utilizes a bind/wash/elute workflow with minimal incubation and spin times. The columns ensure zero buffer retention and no carryover of contaminants, enabling elution of sample in volumes as low as 50 μl. Unwanted NTPs and short RNA fragments are removed, ensuring highly pure RNA transcripts following IVT/RNA synthesis. Eluted RNA is ready for use in a variety of downstream applications including RT-PCR, RNA Library Prep for NGS and RNA labelling. The protocol can also be modified to enable the purification of smaller RNA fragments (≥ 15 nt).

Designed with sustainability in mind, Monarch kits use significantly less plastic and responsibly-sourced, recyclable packaging.

Monarch RNA Cleanup Kits are also available for 10 µg (NEB #T2030) and 50 µg (NEB #T2040) binding capacities. Columns and buffers are also available separately for convenience.

 

Figure 1: Monarch RNA Cleanup Kit workflow

 

Specifications and Applications:

SPECIFICATIONS
RNA Sample Type Cleanup of RNA from large-scale in vitro transcription reactions
Binding Capacity 500 µg
RNA Size Range ≥ 25 nt ( ≥ 15 nt with modified protocol)
Typical Recovery 70–100%
Elution Volume 50–100 µl
Purity A260/280 > 1.8 and A260/230 > 1.8
Protocol Time 10–15 minutes of spin and incubation time
Compatible Downstream Applications RNA Labeling, RNAi, Microinjections, RT-PCR, RNA library prep for NGS, transfection

APPLICATIONS
RNA Cleanup and Concentration (including from the TRIzol aqueous phase) RNA purified by other methods can be further purified
Enzymatic Reaction Cleanup Enzymes such as RNA polymerases, DNase I, Proteinase K and phosphatases are removed allowing efficient desalting
In vitro Transcription Cleanup Enzymes and excess NTPs are removed to yield highly pure synthesized RNA
RNA Gel Extraction Purification of RNA from agarose gels
RNA Fractionation Fractionation of RNA into small and large RNA pools

 

Figure 2: The Monarch RNA Cleanup Kit (500 µg) is suitable for cleaning up large quantities (>250 µg) of RNA from in vitro transcription reactions

A. RNA transcripts of varying sizes (0.6-8 kb) were synthesized using the HiScribe®; T7 Quick High Yield RNA Synthesis Kit (NEB #E2050) using 1.5-1.8 µg of DNA template for two hours at 37°C. 40 µl of each in vitro transcription IVT) reaction was cleaned up using the Monarch RNA Cleanup Kit (500 µg, T2050) and eluted in 200 µl. RNA yields were calculated from the resulting A260, measured using a Nanodrop® spectrophotometer and ranged from 268-425 µg of RNA per IVT reaction.

B. RNA integrity (200 ng/lane) was assessed on a 1% agarose-TBE gel stained with SYBR® Gold.
Figure 3: The Monarch RNA Cleanup Kit (500 µg) cleans up large-scale in vitro transcription reactions and generates yields consistent with other large-scale cleanup kits

0.3 and 1.8 kb fragments were in vitro transcribed at 37°C (overnight and 2 hours, respectively) using the HiScribe®; T7 Quick High Yield RNA Synthesis Kit (NEB #E2050). Following DNase I treatment (4 U DNase I, 37°C, 15 minutes), transcription reactions were pooled and 200 µl cleaned up using either the NEB Monarch RNA Cleanup Kit (500 µg) or the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). In vitro transcribed RNA was eluted twice with 100 µl of nuclease-free water following a 5-minute on-column incubation (room temperature for Monarch and 65°C for MEGAclear. Recovery of the synthesized RNA transcript was calculated from the resulting A260 using a Trinean Dropsense™ 16. The Monarch RNA Cleanup Kit (500 µg) produces similar RNA yields as the MEGAclear Kit for large-scale in vitro transcription reactions.  
Figure 4: RNA recovery is increased by incubating the column with the elution buffer (nuclease-free water) prior to the elution spin

rRNA (16S and 23S Ribosomal Standard from E.coli, Sigma) was purified using the Monarch RNA Cleanup Kit (500 µg, NEB #T2050). 100 µl of nuclease-free water was added to the column and incubated for either 0,1,3 or 5 minutes at room temperature before spinning to elute the RNA. The percent recovery of RNA was calculated from the resulting A260 as measured using a Trinean Dropsense™ 16. A five-minute incubation period produced the maximum yield.
This product is related to the following categories:
RNA Cleanup Products,
RNA Extraction and Purification,
Nucleic Acid Purification Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Monarch® RNA Cleanup Kit Protocol
  2. Purification of RNA from the Aqueous Phase Following TRIzol®/Chloroform Extraction using the Monarch® RNA Cleanup Kits
  3. Separation of Large and Small RNA into Fractions using the Monarch® RNA Cleanup Kits
  4. Extraction of RNA from Agarose Gels using the Monarch® RNA Cleanup Kits
  5. RNA Extraction from Cells Using the Monarch RNA Cleanup Kits

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Usage & Guidelines

Application Notes

FAQs & Troubleshooting

FAQs

  1. What is the composition of each buffer provided with the Monarch RNA Cleanup Kits?
  2. What is the maximum binding capacity of the Monarch RNA Cleanup Column provided with the Monarch RNA Cleanup Kit?
  3. What is the smallest volume of nuclease-free water that can be used for elution with the Monarch RNA Cleanup Columns?
  4. Can I get better recovery with the Monarch RNA Cleanup Kits if I do a second elution with my eluent from the first elution?
  5. What factors affect my (A260/A230) when using the Monarch RNA Cleanup Kits?
  6. What size RNA can be purified with the Monarch RNA Cleanup Kit?
  7. Can I use the Monarch RNA Cleanup Kit to cleanup up my DNase I-treated RNA?
  8. Do you have a protocol for separating small and large RNAs into separate fractions?
  9. Can I use the Monarch RNA Cleanup Kits to purify RNA from agarose gels?
  10. Can I use the Monarch RNA Cleanup Kits to cleanup RNA after a TRIzol®/chloroform extraction?
  11. Can I use the Monarch RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction?
  12. Are the Monarch RNA Cleanup Kits compatible with Luna RT-qPCR reagents?
  13. Are the Monarch RNA Cleanup Kits compatible with NEBNext reagents for RNA library prep?
  14. Why do I need to incubate my column for 5 minutes with elution buffer (nuclease-free water)?
  15. My sample turned cloudy after adding the Monarch RNA Cleanup Binding Buffer and ethanol. Is this normal?
  16. How can I assess RNA integrity and purity?
  17. Are the columns in the Monarch RNA Cleanup Kits the same as those in the Monarch Total RNA Miniprep Kit (NEB #T2010)?
  18. Can I purchase Monarch® buffers and columns separately?
  19. Can I do an on-column DNase I treatment with the Monarch RNA Cleanup Columns?
  20. Can the Monarch RNA Cleanup Kits (NEB #T2030, #T2040, #T2050) also be used to purify DNA?
  21. I would like to clean up  ~150 µg of RNA. Would it be better to use multiple 50 µg columns (NEB #T2040/#T2047) or a single 500 µg column (NEB #T2050/#T2057)?
  22. I noticed the buffer names have changed in the RNA Cleanup Kits. Are these buffers the same formulation as before?

Troubleshooting

Problem Cause Solution
Low RNA Yield Reagents added incorrectly Check protocol to ensure correct buffer reconstitution, order of addition of buffers and ethanol, and proper handling of column flow-through and eluents.
Insufficient mixing of reagents Ensure the ethanol is thoroughly mixed with RNA sample and RNA Cleanup Binding Buffer before applying the sample to the RNA Cleanup Column.
Incomplete elution during prep Ensure the nuclease-free water used for elution is delivered directly to the center of the column so that the matrix is completely saturated. Larger elution volumes, multiple elutions, and longer incubation times can increase yield of RNA, but will dilute the sample and may increase processing times. For typical RNA samples, the recommended elution volumes and incubation times should be sufficient.
High degree of RNA secondary structure Binding and elution of smaller RNAs (< 45 nt) can be affected by secondary structure of the RNA molecules. If poor yield of a small RNA is observed, we recommend diluting your sample with 2 volumes of ethanol instead of one volume in Step 2 of the protocol.
Purified RNA is Degraded RNase contamination In order to avoid RNase contamination during RNA cleanup, make sure to work on a clean lab bench, wear gloves and use disposable RNase-free pipet tips and microfuge tubes (not provided). Keep all kit components tightly sealed when not in use.
Improper storage of RNA Purified RNA should be used immediately in downstream applications or stored at -70°C.
Low A260/230  Ratios Residual guanidine salt carry-over   Ensure wash steps are carried out prior to eluting sample. Use care to ensure the tip of the column does not contact the flow-through. If unsure, repeat centrifugation. When reusing collection tubes, blot the rim of the tube on a Kimwipe prior to reattachment to the column to remove any residual wash buffer.
Low Performance of RNA in Downstream Steps Salt and/or ethanol carry-over Ethanol and salt remaining after the washes may inhibit downstream applications. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-centrifuge for 1 minute to ensure traces of salt and ethanol are not carried over in the eluted RNA.
DNA contamination DNA removal may be necessary for certain applications. Incubate RNA sample with DNase I (NEB #M0303) and cleanup RNA using the Monarch RNA Cleanup Protocol.

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specification Change Notifications

Effective March 25, 2024, component/product name changed to Monarch® Buffer BX. Product specifications have been updated.

Effective March 25, 2024, component/product name changed to Monarch® Buffer WX. Product specifications have been updated.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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