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:
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SPECIFICATIONS
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RNA Sample Type
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Cleanup of RNA from large-scale in vitro transcription reactions
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Binding Capacity
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500 µg
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RNA Size Range
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≥ 25 nt ( ≥ 15 nt with modified protocol)
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Typical Recovery
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70-100%
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Elution Volume
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50-100 µl
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Purity
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A260/280 > 1.8 and A260/230 > 1.8
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Protocol Time
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10-15 minutes of spin and incubation time
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Compatible Downstream Applications
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RNA Labeling, RNAi, Microinjections, RT-PCR, RNA library prep for NGS, transfection
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APPLICATIONS
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RNA Cleanup and Concentration (including from the TRIzol aqueous phase)
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RNA purified by other methods can be further purified
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Enzymatic Reaction Cleanup
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Enzymes such as RNA polymerases, DNase I, Proteinase K and phosphatases are removed allowing efficient desalting
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In vitro Transcription Cleanup
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Enzymes and excess NTPs are removed to yield highly pure synthesized RNA
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RNA Gel Extraction
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Purification of RNA from agarose gels
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RNA Fractionation
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Fractionation of RNA into small and large RNA pools
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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.
Properties & Usage
Protocols
Manuals
The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.
Quality Control Assays
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.
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]
Specifications & Change Notifications
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]
The following link is to a PDF Safety Data Sheet (SDS) that applies 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 busdev@neb.com.
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.