New England Biolabs, T2030S, Monarch® Spin RNA Cleanup Kit (10 μg)

Effective September 24, 2024, product name modified to Monarch Related Categories RNA Cleanup,, RNA Extraction and Purification,, Nucleic Acid Purification FAQ Q: What is the composition of each buffer provided with the Monarch Spin RNA Cleanup Kits? A: The compositions of the buffers are proprietary. We can, however, share the following: Monarch Buffer BX....................Guanidine-based binding buffer Monarch Buffer WX.......................Ethanol-based wash buffer Q: What is the maximum binding capacity of the column for RNA Cleanup Column provided with the Monarch Spin RNA Cleanup Kit? A: Although in some cases greater yields have been observed, the matrix supplied in each column is sufficient to bind the following quantities of RNA: T2030/T2037: 10 µg of RNA T2040/T2047: 50 µg of RNA T2050/T2057: 500 µg of RNA It is important to note that enzymatic reactions introduce components such as detergents, dyes, protein and nucleotides, all of which can compete for binding to the membrane during cleanup and affect RNA recovery. Q: What is the smallest volume of nuclease-free water that can be used for elution with the Monarch Spin Columns S1A, S2A, S2B for RNA Cleanup? A: T2030/T2037: 6 µl; typical elution volumes are 6-20 µl T2040/T2047: 20 µl; typical elution volumes are 20-50 µl T2050/T2057: 50 µl; typical elution volumes are 50-100 µl Using lower elution volumes will likely result in variance due to inconsistent wetting of the matrix. Yield may slightly increase if a larger volume is used, but the RNA will be less concentrated. *A second elution can be performed to maximize RNA recovery. This second elution can be performed using a fresh aliquot of nuclease-free water or the eluent from the first elution to maximize recovery while minimizing volume. Recovery of RNA from Monarch Spin 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 Spin 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 A260 as measured using a Trinean DropSense 16. ~80% of RNA can be efficiently recovered in 6 µl from the Monarch Spin RNA Cleanup Kit (10 µg, NEB #T2030), 20 µl from the Monarch Spin RNA Cleanup Kit (50 µg, NEB #T2040), and 50 µl from the Monarch Spin RNA Cleanup Kit (500 µg, NEB #T2050). Q: Can I get better recovery with the Monarch Spin RNA Cleanup Kits if I do a second elution with my eluent from the first elution? A: A second elution can be performed to maximize RNA recovery. This second elution can be performed using a fresh aliquot of nuclease-free water or the eluent from the first elution to maximize recovery while minimizing volume. Q: What factors affect my (A260/A230) when using the Monarch Spin RNA Cleanup Kits? A: Guanidine and ethanol, both introduced during the prep, can reduce the A260/A230 ratio. Following the protocol, especially the wash steps, will ensure these components are removed. Q: What size RNA can be purified with the Monarch Spin RNA Cleanup Kit? A: Using the standard protocol, RNAs ≥ 25 nt can be purified. With a slight modification in the protocol (adding 2 volumes of ethanol instead of 1 volume after addition of the binding buffer), RNA > 15 nt can be purified. Recovery of Small RNAs using the Monarch Spin RNA Cleanup Kit. Synthesized RNA oligonucleotides (1 μg in 50 μl H2O) of varying lengths (15–100 nt) were purified using the Monarch Spin RNA Cleanup Kit (NEB #T2040) and eluted in 50 μl nuclease-free water. The percent recovery of Spin RNA was calculated from the resulting A260, measured using a Trinean DropSense™ 16. The Monarch Spin RNA Cleanup Kit standard protocol results in >70% recovery and cleanup of RNA ≥ 25 nt, while use of the Monarch Spin RNA Cleanup Kit with the modified protocol (addition of 2 volumes of ethanol in step 2) results in >70% recovery and cleanup of even smaller RNAs (as low as 15 nt). Please note that recovery of small RNAs (< 45 nt) can be affected by sequence, interactions with other nucleic acids and/or secondary structure. Secondary structure affects recovery of small RNAs (<45 nt) Synthesized RNA oligonucleotides (1 μg in 50 μl H2O) of varying lengths (25-40 nt) and varying predicted secondary structure* were purified using the Monarch Spin RNA Cleanup Kit (NEB #T2040) and eluted in 50 μl nuclease-free water. The percent recovery of the RNA was calculated from the resulting A260 as measured using a Trinean DropSense™ 16. Small RNAs (25–40 nt) with low predicted secondary structure were recovered efficiently using the Spin Monarch RNA Cleanup Kit standard protocol. However, increased levels of secondary structure decrease the recovery of small RNAs (25–40 nt in length). Recovery can typically be increased to >70% (orange line) using the Spin Monarch RNA Cleanup Kit with the modified protocol (addition of 2 volumes of ethanol in step 2). *Secondary structure was predicted using RNAstructure Web Server: Reuter, J. S., & Mathews, D. H. (2010). RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 11,129 If poor yield of a small RNA is observed using the standard RNA Cleanup Protocol (adding 1 volume of ethanol prior to binding to the column), we recommend using the modified protocol (adding 2 volumes of ethanol prior to binding to the column), which uses a higher ethanol-to-sample ratio to shift the exclusion cut-off and enable the capture of smaller RNAs. Q: Can I use the Monarch Spin RNA Cleanup Kit to cleanup up my DNase I-treated RNA? A: Yes, the Monarch Spin RNA Cleanup Kits will remove residual DNase I (NEB #M0303) activity from RNA samples. On-column treatment with DNase I can often result in residual DNase I activity, therefore, we do not recommend on-column DNase I treatment with the Monarch Spin RNA Cleanup Kit, but rather in-solution DNase I treatment prior to RNA cleanup. Q: Do you have a protocol for separating small and large RNAs into separate fractions? A: Yes, the Monarch Spin RNA Cleanup Kit manual contains a protocol which will separate RNA into RNAs > 200 nt and < 200 nt. The protocol can also be accessed online. Following this protocol enriches RNA below 200 nt into the “small RNA” fraction, while RNA above 200 nt is enriched in the “large RNA” fraction. No fractionation protocol allows precise separation of similarly sized RNA based on length alone. We find RNA between 100–200 nt will preferentially partition to the different fractions based on various factors, including predicted secondary structure and interactions with other RNAs within the sample. Highly structured molecules between 100–200 nt typically fall into the small RNA fraction while unstructured RNA between 100–200 nt fall into the large RNA fraction. The Monarch Spin RNA Cleanup Kit can be used to separate RNA into large and small RNA fractions HeLa Total RNA (5.2 μg, A1 and B1) was separated into large RNA (A2 and B2) and small RNA (A3 and B3) fractions following the Purification of Small and Large RNAs into Separate Fractions Protocol and running on an Agilent Bioanalyzer® RNA Pico chip. Over 96% (5 μg) of the RNA input was recovered in the separate RNA fractions. Samples 1 and 2 were diluted 1:20 prior to analysis. Q: Can I use the Monarch Spin RNA Cleanup Kits to purify RNA from agarose gels? A: Yes, the Monarch Spin RNA Cleanup Kit manual contains a protocol which will purify RNA from agarose gels, though this is not their primary application. Recoveries from extraction of RNA from agarose range from 40-70%, which is less than expected recovery for RNA Cleanup from enzymatic reactions. The protocol can also be accessed online. Q: Can I use the Monarch Spin RNA Cleanup Kits to cleanup RNA after a TRIzol®/chloroform extraction? A: Yes, the Monarch Spin RNA Cleanup Kit can be used to clean up the aqueous phase from any guanidinium thiocyanate-phenol-chloroform extraction eliminating the need for tedious RNA precipitation steps. The protocol can be accessed online or in the product manual. Q: Are the Monarch Spin RNA Cleanup Kits compatible with Luna RT-qPCR reagents? A: Yes. RNA purified with these kits can be used for RT-qPCR with all of NEB’s Luna One-Step RT-qPCR Kits and the LunaScript® RT products. Q: Are the Monarch Spin RNA Cleanup Kits compatible with NEBNext reagents for RNA library prep? A: Yes. RNA purified with this kit can be used for RNA library preparation using NEB's NEBNext RNA Library Prep reagents. Q: Are the columns in the Monarch Spin RNA Cleanup Kits the same as those in the Monarch Total RNA Miniprep Kit (NEB #T2010)? A: No, the columns in the Monarch RNA Cleanup Kits (NEB #T2030, #T2040 and #T2050) are not the same as the ones in the Monarch Total RNA Miniprep Kit (NEB #T2010). Q: Can I purchase Monarch® buffers and columns separately? A: Monarch spin columns and several Monarch buffers and reagents are also offered separately. Q: Can I do an on column DNase I treatment with the columns for RNA Cleanup? A: DNase I will not be completely removed if used for on-column digestion with the Monarch columns for RNA Cleanup (Monarch Spin Columns S1A, S2A, S2B). In-solution DNase I treatment is recommended, followed by RNA cleanup. Q: Can the Monarch Spin RNA Cleanup Kits (NEB #T2030, #T2040, #T2050) also be used to purify DNA? A: We have successfully used the Monarch Spin RNA Cleanup Kits for the purification of RNA, ssDNA and dsDNA. However, for optimal cleanup of DNA, we recommend the use of our Monarch Spin PCR & DNA Cleanup Kit (NEB #T1130). Q: Can the Monarch Spin RNA Cleanup Kits be used for RNA extraction? A: Yes. Although originally developed for RNA cleanup applications, these kits can also be used for RNA extraction from cells, saliva, and swab samples. When used for viral extraction, the Monarch DNA/RNA Protection Reagent (sold separately, NEB #T2011) must be used for viral inactivation. Visit our online protocols for more information: RNA Extraction from Cells Using the Monarch Spin RNA Cleanup Kits RNA Extraction from Buccal/Nasopharyngeal Swabs Using the Monarch Spin RNA Cleanup Kits RNA Extraction from Saliva Using the Monarch Spin RNA Cleanup Kits Automation of Viral RNA Extraction from Saliva Using the Monarch Spin RNA Cleanup Kit Q: I noticed the buffer names have changed in the RNA Cleanup Kits. Are these buffers the same formulation as before? A: Yes NEB is standardizing Monarch buffer naming nomenclature. The buffer names are changing, but the buffer formulations are the same. The Monarch® RNA Cleanup Binding Buffer is now named Monarch® Buffer BX. The Monarch® RNA Cleanup Wash Buffer is now named Monarch® Buffer WX. Q: I notice that I'm applying force to fit the column into a collection tube. Should I be doing this? What collection tubes are compatible with columns? A: Matching collection tubes are provided in all Monarch® kits for the spin columns. If you use collection tubes outside of a given kit, please use the Monarch® Spin Collection Tubes (NEB #T2118). Do not force any columns into non-recommended collection tubes that have a very tight fit to avoid damaging the column. Q: I see some silica residue in my Monarch column. Is this a problem? A: No, this is not an issue. The presence of some silica residue or particles in the nucleic acid purification column does not impact nucleic acid binding or purity. Our columns are designed to retain silica fibers, helping ensure they do not end up in the final eluate. Q: How can I assess RNA integrity and purity? A: The purity of eluted RNA samples can be quickly assessed by reviewing OD ratios collected during routine spectrophotometry. Pure RNA typically has an A260/280 of 1.9–2.1, and an A260/230 of 2.0–2.2. Many factors can influence these values such as the use of a proper reference blank solution, the buffer pH, and contaminants such as protein, buffer salts, ethanol, etc. For further information on this topic, please see Assessing RNA.