Effective March 25, 2024, component/product name changed to Monarch® Buffer BX.
Effective March 25, 2024, component/product name changed to Monarch® Buffer WX.
The Monarch RNA Cleanup Kit (10 µg) enables fast and simple purification and concentration of up to 10 µg of RNA from enzymatic reactions.
Ideal for cleanup and concentration of RNA after enzymatic treatments including DNase I, Proteinase K, labeling, capping or in vitro transcription (IVT)
Elute in ≥ 6 µl for concentrated RNA
70-100% RNA recovery, even with inputs < 20 ng
Efficiently purify RNA ≥ 25 nt (a simple modification enables purification of RNA ≥ 15 nt)
Can be used to purify RNA from the aqueous phase following TRIzol® or similar extractions
Simplified workflow with a single wash buffer
Unique column design prevents buffer carryover and elution of silica particulates
The Monarch RNA Cleanup Kit (10 µg) rapidly and reliably purifies up to 10 μg of concentrated, high-quality RNA (> 25 nt) from enzymatic reactions including labeling, capping, in vitro transcription (IVT) and DNase I treatment. This kit utilizes a bind/wash/elute workflow with minimal incubation and spin times. Our unique column design ensures zero buffer retention and no carryover of contaminants, enabling elution of sample in volumes as low as 6 μl. 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 nts).
Designed with sustainability in mind, Monarch kits use significantly less plastic and responsibly-sourced, recyclable packaging.
Monarch RNA Cleanup kits are also available for 50 µg (NEB #T2040) and 500 µg (NEB #T2050) 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 and concentration of RNA from enzymatic reactions (labeling, capping, in vitro
transcription reactions, DNase I treatment)
Binding Capacity
10 μg
RNA Size Range
≥ 25 nt ( ≥ 15 nt with modified protocol)
Typical Recovery
70–100%
Elution Volume
6–20 µl
Purity
A260/280 > 1.8 and A260/230 > 1.8
Protocol Time
5 minutes of spin and incubation time
Compatible Downstream Applications
RT-PCR, Small RNA
library prep for NGS, RNA Library Prep for NGS
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 enables efficient recovery of RNA in as little as 6 µl
rRNA (10 µg of 16S and 23S Ribosomal Standard from E. coli, Sigma) was purified using the Monarch RNA Cleanup Kit (10 µg, NEB #2030) and eluted with nuclease-free water using the elution volumes indicated. The percent recovery of RNA was calculated from the resulting A260 as measured using a Trinean DropSense 16. ~80% of RNA can be efficiently recovered in as little as 6 µl.
Figure 3: The Monarch RNA Cleanup Kit enables efficient recovery of <20 ng of RNA in as little as 6 µl
A 2-fold dilution series (from 1000 to 15.6 ng) of rRNA (16S and 23S Ribosomal Standard from E.coli, Sigma) was purified using the Monarch RNA Cleanup Kit (10 µg, NEB #1030) and eluted in 6 µl of nuclease-free water. The percent recovery of RNA was calculated from the resulting A260, measured using a Trinean DropSense 16. Even low RNA inputs (<20 ng) are efficiently cleaned up and recovered (>80%) in as little as 6 µl.
Figure 4: The Monarch RNA Cleanup Kit (10 µg) offers efficient recovery of sample volume
The Monarch RNA Cleanup Columns (10 µg, NEB #T2037) ensure zero buffer retention and no carryover of contaminants, enabling sample elution in volumes as low as 6 µl. Moreover, ~90% (~5.4 µl) of the minimal elution volume is recovered from the Monarch RNA Cleanup Columns, compared to other competitor kits.
This product is related to the following categories:
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 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.
Specifications & 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.
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 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]
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.
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