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The Monarch RNA Cleanup Kits provide a fast and simple silica spin column-based solution for RNA cleanup and concentration after any enzymatic reaction (including
in vitro transcription, DNase I treatment, capping and labeling) and after other purification methods such as phenol/chloroform extraction. The Monarch RNA Cleanup Kits are available in 3 different binding capacities: 10 μg ( NEB #T2030), 50 μg ( NEB #T2040) and 500 μg ( NEB #T2050). Each kit contains unique columns, all designed to prevent buffer retention and ensure no carryover of contaminants, enabling low-volume elution of highly-pure RNA. Following the standard protocol, RNA ≥ 25 nt is purified with this kit; however, a modified protocol is available to enable the binding of RNA as small as 15 nt (including miRNAs). Reasons to choose Monarch for RNA Cleanup
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
Flexibility in Binding Capacity and Elution Volume
Monarch RNA Cleanup Kit
NEB #T2030 (10 µg)
NEB #T2040 (50 µg)
NEB #T2050 (500 µg)
Binding Capacity: 10 μg
RNA Size Range: ≥ 25 nt ( ≥ 15 nt with modified protocol)
Typical Recovery: 70–100%
Elution Volume: 6–20 µl
260/280 > 1.8 and A 260/230 > 1.8
Protocol Time: 5 minutes of spin and incubation time
10–15 minutes of spin and incubation time
Common Downstream Applications: RT-PCR, RNA library prep for NGS, Small RNA library prep for NGS, RNA labeling
RT-PCR, RNA library prep for NGS, formation of RNP complexes for genome editing, microinjection, RNA labeling, transfection
RT-PCR, RNA library prep for NGS, RNA labeling, RNAi, microinjection, transfection
Recovery of RNA from Monarch RNA Cleanup Kits with Varying Elution Volumes rRNA (10, 50 or 500 µg, respectively of 16S and 23S Ribosomal Standard from E. coli, Sigma) was purified using a Monarch RNA Cleanup Kit (10 µg, NEB #T2030) (50 µg, NEB #T2040) (500 µg, NEB #T2050). Nuclease-free water was used to elute the RNA. The percent recovery of the RNA was calculated from the resulting A 260 as measured using a Trinean DropSense 16. ~80% of RNA can be efficiently recovered in 6 µl from the Monarch RNA Cleanup Kit (10 µg, NEB #T2030), 20 µl from the Monarch RNA Cleanup Kit (50 µg, NEB #T2040), and 50 µl from the Monarch RNA Cleanup Kit (500 µg, NEB #T2050).
Purify Large Amounts of RNA from IVT Reactions The Monarch RNA Cleanup Kit (500 µg) is suitable for cleaning up large quantities (>250 µg) of RNA from
in vitro transcription reactions
1. 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 A 260, measured using a Nanodrop® spectrophotometer and ranged from 268-425 µg of RNA per IVT reaction.
2. RNA integrity (200 ng/lane) was assessed on a 1% agarose-TBE gel stained with SYBR ® Gold.
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.
Efficiently Remove Unincorporated Nucleotides
The Monarch RNA Cleanup Kit (50 µg) produces sgRNA yields consistent with other competitor RNA cleanup kits and with lower residual NTP contamination
Six different sgRNA synthesis reactions from the EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322) were cleaned up using either the Monarch RNA Cleanup Kit (50 µg, NEB #T2040) or a competitor kit (according to manufacturer’s recommendations) and eluted with 50 µl of nuclease-free water. sgRNA yield was calculated from the resulting A 260, as measured using a Trinean Dropsense™ 16. The Monarch Cleanup Kit produced sgRNA yields consistent with other commercially available RNA cleanup kits.
Following cleanup, residual nucleotides (NTPs) were measured by LC-MS and are reported as (percent area NTPs (rATP+rCTP+rGTP+rUTP)/percent area sgRNA). The Monarch RNA Cleanup Kit consistently outperforms other commercially available RNA cleanup kits in the removal of residual NTPs from sgRNA synthesis reactions.