Description
The Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) provides a reliable, microcentrifuge-scale solution for purifying large amounts of RNA, up to 3 mg per column, in a convenient silica spin column format. This kit is optimized for the efficient cleanup of RNA generated from high-yield in vitro transcription (IVT) reactions, including RNA synthesized with modified nucleotides.
The streamlined bind–wash–elute workflow removes enzymes and unincorporated nucleotides using a single binding buffer and a single wash buffer, enabling benchtop purification without the need for complex equipment or lengthy protocols. The high 3 mg binding capacity supports purification of RNA following enzymatic reactions such as labeling, capping, and DNase I treatment, delivering high‑quality RNA suitable for downstream applications.
The Monarch spin columns in this kit are engineered specifically for the highest‑capacity RNA cleanup using standard microcentrifuges, with a design that minimizes buffer retention and contaminant carryover. This enables efficient recovery of highly pure RNA in elution volumes as low as 250 µl. RNA purified with this kit is suitable for a wide range of downstream applications, including RT‑PCR, transfection, RNA labeling, RNA library preparation for NGS and more. In line with NEB’s sustainability efforts, Monarch kits are designed to use significantly less plastic and are packaged using responsibly sourced, recyclable materials.
Monarch Spin RNA Cleanup Kits are also available with 10 µg (NEB #T2030), 50 µg (NEB #T2040), and 500 µg (NEB #T2050) binding capacities. Columns are available separately for added flexibility and convenience.
| Properties |
| Purification Format |
Spin Column |
| RNA Sample Type Compatibility |
RNA from various sources including in vitro transcription (IVT) samples, IVT samples synthesized with modified nucleotides and RNA extracted by other methods |
| Typical Recovery |
70-100% |
| RNA Purity |
A260/280 > 2.0 and A260/230 > 1.8 |
| Binding Capacity |
up to 3 mg |
| RNA Size Range |
≥ 25 nt |
| Elution Volume |
250-1000 μl |
| Compatible Downstream Applications |
RT-PCR, RNA library prep for NGS, RNA labeling, RNAi, microinjection, transfection |
| Equipment Compatibility |
Centrifuge |
Figure 1: High-capacity RNA purification in an easy spin column format
The Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) efficiently purifies large amounts of RNA following in vitro transcription (IVT) and other enzymatic reactions using a silica spin column at the microcentrifuge scale.
Figure 2: Monarch Spin High-Capacity RNA Cleanup Kit is suitable for cleaning up large quantities (3 mg) of RNA synthesized by in vitro transcription (IVT)
Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) was used to purify different sizes of RNA generating yields of up to
3 mg and resulting in high RNA integrity and purity metrics (A260/280 and A260/230> 2.0).
(A) 0.3–9 kb size RNA transcripts were synthesized through scaled-up in vitro transcription (IVT) using the HiScribe® T7 High Yield RNA Synthesis Kit (NEB #E2040). After DNase I-XT (NEB #M0570) treatment (2 U DNase I-XT per 20 µl IVT reaction, 37°C, 15 minutes), the following IVT reaction volumes were used as RNA input for cleanup of each RNA size with six replicates: 400 µl for 0.3 kb, 300 µl for 2 kb, 250 µl for 5 kb, 200 µl for 9 kb. A total of 500 µl of nuclease-free water was used in two elution steps for 0.3 kb, 2 kb, and 5 kb sizes of RNA. A total of 1000 µl of nuclease-free water was used in three elution steps for 9 kb size of RNA. Yield was calculated from A260 measured using a Lunatic® (Unchained Labs®).
(B) Corresponding RNA integrity (600 ng/lane) was assessed on 1.2% agarose-TBE gel stained with ethidium bromide. RNA was denatured by incubating samples with RNA loading dye (NEB #B0363) at 70°C for 10 minutes. LR – Low Range ssRNA Ladder (NEB #N0364). L – ssRNA Ladder (NEB #N0362). Mean absorbance ratios from six replicates
(A260/280 and A260/230) measured using a Lunatic (Unchained Labs) for each RNA size are reported.
Figure 3: Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) enables efficient recovery of up to 3 mg of RNA in as little as 250 µl
Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) was used to purify different input amounts of rRNA (16S and 23S ribosomal RNA extracted from E. coli). Over 90% of rRNA was recovered from 1–3 mg inputs in 250 µl of nuclease-free water. Eight replicates were used for each input amount of 1 mg, 2 mg and 3 mg of rRNA. Percent recovery was calculated from A260 measured using a Lunatic (Unchained Labs).
Figure 4: DNase I is effectively removed using the Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) demonstrating protein removal following enzymatic reactions
Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) was used to clean up 1 mg of RNA following DNase I treatment (2 units, RNase-free, NEB #M0303) and eluted in nuclease-free water. Purified RNA samples (four replicates) and their
DNase I-treated RNA input sample without cleanup (I) were tested for residual DNase I activity. Samples (2 µl) were incubated with 1 µg plasmid, pBR322 (NEB #N3033), in 1X DNase I Reaction Buffer at 37°C for 10 minutes. No digestion of the plasmid was observed in purified RNA samples compared to the digested plasmid in the RNA input sample without cleanup.
Figure 5: Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) purifies RNA synthesized with modified nucleotides and after capping reactions, producing high-quality RNA suitable for downstream applications
1) eGFP mRNA was synthesized using HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040) and modified nucleotide, N1-Methyl-Pseudo-UTP (m1ΨTP, NEB #N0431), in 1 mL reactions for 1.5 hours at 37°C. These 1 ml IVT reactions were treated with 100 µl DNase I-XT (NEB #M0570) for 15 minutes at 37°C to remove DNA template.
2) 300 µl of eGFP (m1Ψ) mRNA from step 1 was purified using the Monarch Spin High-Capacity RNA Cleanup Kit (3 mg). RNA yield was calculated from A260 measured using a Lunatic (Unchained Labs).
3) A portion of purified eGFP (m1Ψ) mRNA from step 2 was capped and 2´-O-methylated using the one-pot Cap-1 synthesis protocol with Faustovirus Capping Enzyme (NEB #M2081) and Cap 2´-O-Methyltransferase (NEB #M0366). These single-tube capping reactions were scaled up to 5 x 250 µl reactions using 250 µg of purified RNA each and run for 1 hour at 37°C.
4) A portion, 600 µg, of the Cap-1 eGFP (m1Ψ) mRNA from step 3 was purified using the Monarch Spin High-Capacity RNA Cleanup Kit (3 mg).
5) 0.5 µg of purified RNA from step 4 was used to transfect 293T cells (ATCC CRL-3216) using jetMessenger® mRNA transfection protocol for 24-well plate. Cells were viewed and imaged at 4X magnification with brightfield (top panel) and with fluorescence (bottom panel) using the GFP filter (482/524 nm) (EVOS® M7000 Imaging System). 95% viability was measured using Luna-II™ Automated Cell Counter.
Figure 6: Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) workflow
The Monarch Spin High-Capacity RNA Cleanup Kit (3 mg) is designed for efficient purification of large amounts of RNA synthesized from IVT and other enzymatic reactions, using a silica spin column format with the highest RNA binding capacity of up to 3 mg.
- This product is related to the following categories:
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RNA Cleanup,